Note: Descriptions are shown in the official language in which they were submitted.
DEMANDES OU BREVETS VOLUMINEUX
LA PRESENTE PARTIE I)E CETTE DEMANDE OU CE BREVETS
COMPRI~:ND PLUS D'UN TOME.
CECI EST ~.E TOME 1 DE 2
NOTE: Pour les tomes additionels, veillez contacter 1e Bureau Canadien des
Brevets.
JUMBO APPLICATIONS / PATENTS
THIS SECTION OF THE APPLICATION / PATENT CONTAINS MORE
THAN ONE VOLUME.
THIS IS VOLUME 1 OF 2
NOTE: For additional vohxmes please contact the Canadian Patent Oi~ice.
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
Human Tumor Necrosis Factor Receptors TR13 and TR14
s
Background of the Invention
l0 Field of the Invention
The present invention relates to two novel members of the tumor necrosis
factor family
of receptors. More specifically, isolated nucleic acid molecules are provided
encoding the novel
human tumor necrosis factor receptors, TR13 and TR14. TR13 and TR14
polypeptides are also
provided, as are vectors, host cells, and recombinant methods for producing
the same. The
15 invention further relates to screening methods for identifying agonists and
antagonists of TR13
and/or TR14 activity.
Related Art
Many biological actions, for instance, response to certain stimuli and natural
biological
2o processes, are controlled by factors, such as cytokines. Many cytokines act
through receptors
by engaging the receptor and producing an infra-cellular response.
For example, tumor necrosis factors (TNF) alpha and beta are cytokines, which
act
through TNF receptors to regulate numerous biological processes, including
protection against
infection and induction of shock and inflammatory disease. The TNF molecules
belong to the
2s "TNF-ligand" superfamily, and act together with their receptors or counter-
ligands, the "TNF-
receptor" superfamily. So far, nine members of the TNF ligand superfamily have
been
identified and ten members of the TNF-receptor superfamily have been
characterized.
Among the ligands there are included TNF-«, lymphotoxin-« (LT-« , also known
as
TNF-(3), LT-[3 (found in complex heterotrimer LT-2-~), FasL, CD40L, CD27L,
CD30L, 4-
30 1BBL, OX40L and nerve growth factor (NGF). The superfamily of TNF receptors
includes
the p55TNF receptor, p75TNF receptor, TNF receptor-related protein, FAS
antigen or APO-1,
CD40, CD27, CD30, 4-1BB, OX40, low affinity p75 and NGF-receptor (A. Meager,
Biologicals 22:291-295 ( 1994)).
Many members of the TNF-ligand superfamily are expressed by activated T-cells,
3s implying that they are necessary for T-cell interactions with other cell
types which underlie cell
ontogeny and functions. (A. Meager, supra).
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
2
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 (R. Watanabe-Fukunaga 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 (R.C. Allen et al., Science 259:990 (1993)). Targeted mutations of
the low affinity
nerve growth factor receptor cause a disorder characterized by faulty sensory
innovation of
to peripheral structures (K.F. Lee et al., Cell 69:737 (1992)).
TNF- « and LT- « 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- «,
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-« are
involved in the pathogenesis of a wide range of diseases, including endotoxic
shock, cerebral
malaria, tumors, autoimmune disease, AIDS and graft-host rejection (B. Beutler
and C. Von
Huffel, Science 264:667-668 (1994)). Mutations in the p55 receptor cause
increased
susceptibility to microbial infection.
2o 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)).
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); 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
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
3
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 81:505-512 (1995); M. P. Boldin et al., J. 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 13:151-153 (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)).
Recently, a new apoptosis inducing TNF ligand has been discovered. S.R. Wiley
et
al., Immunity 3:673-682 (1995), named the new molecule, "TNF-related apoptosis-
inducing
ligand" or "TRAIL." R.M. Pitti et al., J. Biol. Chem. 271:12687-12690 (1996),
named the
molecule "Apo-2ligand" or "Apo-2L." This molecule was also disclosed in co-
pending U.S.
2o provisional patent application no. 60/013405. For convenience, this
molecule will be referred
to herein as TRAIL.
Unlike FAS ligand, whose transcripts appear to be largely restricted to
stimulated T-
cells, significant levels of TRAIL are detected in many human tissues (e.g.,
spleen, lung,
prostate, thymus, ovary, small intestine, colon, peripheral blood lymphocytes,
placenta,
kidney), and it is constitutively transcribed by some cell lines. It has been
shown that TRAIL
acts independently from the FAS ligand (5.R. Wiley et al., supra). It has also
been shown that
TRAIL activates apoptosis rapidly, within a time frame that is similar to
death signaling by
Fas/Apo-1L, but much faster than TNF-induced apoptosis. S.A. Marsters et al.,
Current
Biology 6:750-752 (1996). The inability of TRAIL to bind TNFR-1, Fas, or the
recently
3o identified DR3, suggests that TRAIL may interact with a unique receptor(s).
Work to date suggests that there are several unique TNF receptors for TRAIL.
In co-
pending U.S. provisional patent application no. 60/035,722, one novel death
domain
containing receptor for TRAIL, DR4, was disclosed. See, Pan et al., Science
276:111-113
(April 1997). In co-pending U.S. provisional patent application no.
60/040,846, a novel death
domain containing receptor, DRS (TR7), was disclosed. This receptor has now
been shown to
bind TRAIL. In co-pending U.S. provisional patent application no. 60/035,496,
another
receptor, TRS, was disclosed. This receptor has also now been shown to bind
TRAIL,
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
4
however, TRS has been shown to be a non-signaling decoy receptor which
antagonizes
apoptos~s.
The effects of TNF family ligands and receptors are varied and influence
numerous
functions, both normal and abnormal, in the biological processes of the
mammalian system.
There is a clear need, therefore, for identification and characterization of
such receptors and
ligands that influence biological activity, both normally and in disease
states. In particular,
there is a need to isolate and characterize additional novel receptors that
bind TRAIL.
Summary of the Invention
l0 The present invention provides isolated nucleic acid molecules comprising a
polynucleotide encoding the TR13 receptor having the amino acid sequence shown
in SEQ ID
N0:2 (Figures lA-C), amino acid sequence shown in SEQ ID N0:40 (Figures 7A-D)
or the
amino acid sequence encoded by the cDNA clone deposited as American Type
Culture
Collection ("ATCC") Deposit No. PTA-349 (HWLHM70) on July 13, 1999, and/or the
amino
~5 acid sequence encoded by the cDNA clone deposited as American Type Culture
Collection
("ATCC") Deposit No. PTA-507 (HWLHN83) on August 12, 1999. The ATCC is located
at
10801 University Boulevard, Manassas, Virginia 20110-2209. It would be
apparent to the '
skilled artisan that the various methods of use, including but not limited to
diagnostic and
therapeutic uses described herein, for the TR13 receptor polynucleotides and
polypeptides
2o would apply equally to all variants and fragments thereof (e.g., fragments
of the TR13 receptor
disclosed and described herein in Figures lA-C, Figure 7A-D, SEQ ID NO:1, SEQ
ID N0:2,
SEQ ID N0:39, SEQ ID N0:40 and/or contained or encoded by one or both of the
deposited
cDNA clones HWLHM70 and HWLHN83).
The present invention also relates to recombinant vectors, which include the
isolated
25 nucleic acid molecules of the present invention, and to host cells
containing the recombinant
vectors, as well as to methods of making such vectors and host cells and for
using them for
production of TR13 polypeptides (e.g., the TR13 polypeptide sequence shown in
Figures lA
C and/or Figures 7A-D, or a fragment thereof) by recombinant techniques.
The invention further provides an isolated TR13 polypeptide (e.g., the TR13
30 polypeptide sequence shown in Figures lA-C and/or Figures 7A-D or fragments
thereof)
having an amino acid sequence encoded by a polynucleotide described herein
(e.g., the
polynucleotide sequence shown in SEQ ID NO:1 and/or SEQ ID N0:39, or a
fragment
thereof).
The present invention also provides diagnostic assays such as quantitative and
35 diagnostic assays for detecting levels of TR13 polynucleotide and/or
protein (e.g., the TR13
protein shown in Figures lA-C and/or Figures 7A-D, or fragments thereof).
Thus, for
instance, a diagnostic assay in accordance with the invention for detecting
over-expression of
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
TR13, or soluble form thereof, compared to normal control tissue samples may
be used to
detect the presence of tumors.
The present invention provides isolated nucleic acid molecules comprising a
polynucleotide encoding the TR14 receptor having the amino acid sequence shown
in SEQ ~
5 N0:61 (Figures l0A-H), and/or the amino acid sequence encoded by the cDNA
clone
deposited as American Type Culture Collection ("ATCC") Deposit No. PTA-348
(HMSHK47)
on July 13, 1999. While the sequence of SEQ ID N0:61 and Figures l0A-H are
preferred
embodiments of TR14 receptor protein, the present invention provides
alternative isolated
nucleic acid molecule embodiments comprising a polynucleotide encoding the
TR14 receptor
having the amino acid sequence shown in SEQ ID NO:S (Figures 4A-D). The
sequence of
amino acid residues T-78 to M-231 of SEQ ID N0:61 is identical to the sequence
of amino acid
residues T-73 to M-226 of SEQ ID N0:5. It would be apparent to the skilled
artisan that the
various methods of use, including, but not limited to, diagnostic and
therapeutic uses described
herein, for the TR13 receptor polynucleotides and polypeptides would apply
equally to all
variants and fragments thereof (e.g., fragments of the TR14 receptor disclosed
and described in
Figures l0A-H and SEQ ID NOS:60 and 61, or, alternatively, Figures 4A-D and
SEQ ID
N0:4, SEQ ID N0:5 and/or contained or encoded by the deposited cDNA clone
(HMSHK47)).
The present invention also relates to recombinant vectors, which include the
isolated
nucleic acid molecules of the present invention, and to host cells containing
the recombinant
2o vectors, as well as to methods of making such vectors and host cells and
for using them for
production of TR14 polypeptides by recombinant techniques.
The invention further provides an isolated TR14 polypeptide (e.g., the TR14
polypeptide sequence shown in Figures l0A-H or, alternatively, Figures 4A-D,
or fragments
thereof) having an amino acid sequence encoded by a polynucleotide described
herein (e.g., the
polynucleotide sequence shown in SEQ ID N0:60, or, alternatively SEQ ID N0:4,
or
fragments thereof).
The present invention also provides diagnostic assays such as quantitative and
diagnostic assays for detecting levels of TR14 polynucleotide and/or protein
(e.g., the TR14
polypeptide sequence disclosed in Figures l0A-H or 4A-D, or fragments
thereof). Thus, for
instance, a diagnostic assay in accordance with the invention for detecting
over-expression of
TR14, or soluble form thereof, compared to normal control tissue samples may
be used to
detect the presence of tumors.
Tumor Necrosis Factor (TNF) family ligands are known to be among the most
pleiotropic cytokines, inducing a large number of cellular responses,
including cell
proliferation, cytotoxicity, anti-viral activity, immunoregulatory activities,
hematopoiesis, and
the transcriptional regulation of several genes. Cellular response to TNF-
family ligands include
not only normal physiological responses, but also diseases associated with
increased apoptosis
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
6
or the inhibition of apoptosis. Apoptosis-programmed cell death is a
physiological mechanism
involved in the deletion of peripheral T lymphocytes of the immune system, and
its
dysregulation can lead to a number of different pathogenic processes. Diseases
associated with
increased cell survival, unregulated cell proliferation, or the inhibition of
apoptosis, include
cancers, autoimmune disorders, viral infections, inflammation, graft vs. host
disease, acute
graft rejection, and chronic graft rejection. Diseases associated with
increased apoptosis
include AIDS, neurodegenerative disorders, myelodysplastic syndromes, ischemic
injury,
toxin-induced liver disease, septic shock, cachexia, and anorexia.
Thus, the invention further provides a method for inhibiting TR13 mediated
signaling
and/or apoptosis induced by a TNF-family ligand, which involves administering
to a cell which
expresses the TR13 polypeptide (i.e., the TR13 polypeptide shown in Figures lA-
C and/or
Figures 7A-D, or a fragment thereof) an effective amount of a TR13 antagonist
capable of
decreasing TR13 mediated apoptosis and/or decreasing TR13 mediated signaling.
Preferably,
TR13 mediated signaling is decreased to treat a disease wherein increased
apoptosis is
exhibited.
Thus, the invention further provides a method for promoting TR13 mediated
signalling
and/or apoptosis induced by a TNF-family ligand, which involves administering
to a cell which
expresses the TR13 polypeptide (e.g., the TR13 polypeptide shown in Figures lA-
C and/or
Figures 7A-D, or a fragment thereof) an effective amount of a TR13 agonist
capable of
2o increasing TR13 mediated apoptosis and/or increasing TR13 mediated
signaling. Preferably,
TR13 mediated signaling is increased to treat a disease wherein decreased
apoptosis is
exhibited.
Thus, the invention further provides a method for inhibiting TR14 mediated
signaling
and/or apoptosis induced by a TNF-family ligand, which involves administering
to a cell which
expresses the TR14 polypeptide an effective amount of a TR14 antagonist
capable of
decreasing TR14 mediated apoptosis and/or capable of decreasing TR14 mediated
signaling.
Preferably, TR14 mediated signaling is decreased to treat a disease wherein
increased apoptosis
is exhibited.
Thus, the invention further provides a method for promoting TR14 mediated
signaling
3o and/or apoptosis induced by a TNF-family ligand, which involves
administering to a cell which
expresses the TR14 polypeptide an effective amount of a TR14 agonist capable
of increasing
TR14 mediated apoptosis and/or capable of increasing TR14 mediated signaling.
Preferably,
TR14 mediated signaling is increased to treat a disease wherein decreased
apoptosis is
exhibited.
In a further aspect, the present invention is directed to a method for
enhancing TR13
mediated signaling induced by a TNF-family ligand (e.g., APRIL and Neutrokine-
alpha
(International Application Publication No. WO 98/18921), which involves
administering to a
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
7
cell which expresses the TR13 polypeptide (e.g., the polypeptide shown in
Figures lA-C
and/or Figures 7A-D, or a fragment thereof) an effective amount of an agonist
capable of
increasing TR13 mediated activity. Preferably, TR13 mediated activity is
increased to treat a
disease wherein decreased apoptosis is exhibited.
Whether any candidate "agonist" or "antagonist" of the present invention can
enhance or
inhibit TR13 mediated signaling can be determined using art-known TNF-family
ligand/receptor cellular response assays, including those described in more
detail below. Thus,
in a further aspect, a screening method is provided for determining whether a
candidate agonist
or antagonist is capable of enhancing or inhibiting a cellular response to a
TR13 TNF-family
to ligand. The method involves contacting cells which express the TR13
polypeptide (e.g., the
polypeptide shown in Figures lA-C and/or Figures 7A-D, or a fragment thereof)
with a
candidate compound and a TNF-family ligand (e.g., Neutrokine-alpha or APRIL),
assaying a
cellular response, and comparing the cellular response to a standard cellular
response, the
standard being assayed when contact is made with the ligand in absence of the
candidate
compound, whereby an increased cellular response over the standard indicates
that the
candidate compound is an agonist of the ligand/receptor signaling pathway and
a decreased
cellular response compared to the standard indicates that the candidate
compound is an
antagonist of the ligand/receptor signaling pathway. By the invention, a cell
expressing a TR13
polypeptide (e.g., the polypeptide shown in Figures lA-C and/or Figures 7A-D,
or a fragment
thereof) can be contacted with either an endogenous or exogenously
administered TNF-family
ligand.
In a further aspect, the present invention is directed to a method for
enhancing apoptosis
TR14 mediated signaling induced by a TNF-family ligand, which involves
administering to a
cell which expresses the TR14 polypeptide (e.g., the polypeptide shown in
Figures l0A-H, or,
alternatively 4A-D, or a fragment thereof) an effective amount of an agonist
capable of
increasing TR14 mediated activity. Preferably, TR14 mediated activity is
increased to treat a
disease wherein decreased apoptosis is exhibited.
In specific, preferred embodiments, TR14 polynucleotides and polypeptides, as
well as
antibodies that agonize TR14 receptor (as described in the section on
Antibodies, above),
stimulate epithelial cell proliferation and/or development to ameliorate the
diseases and
disorders described in this section. Members of the TNF family of proteins are
known to
signal through the NF-KB singaling pathway. NF-KB is a transcription factor
activated by a
wide certain agents to stimulate cell activation and differentiation. It is
believed that the TR14
receptor of the instant invention signals through the NF-xB pathway to
activate proliferation
and development of cells. Thus, TR14 polynucleotides and polypeptides of the
invention as
well as antibodies and peptides that agonize TR14 may be used in accordance
with the
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
8
invention to stimulate NF-xB-mediated epithelial cell proliferation, including
but not limited to
ectodermal dysplasia.
Whether any candidate "agonist" or "antagonist" of the present invention can
enhance or
inhibit TR14 mediated signaling can be determined using art-known TR14 TNF-
family
ligand/receptor cellular response assays, including those described in more
detail below. Thus,
in a further aspect, a screening method is provided for determining whether a
candidate agonist
or antagonist is capable of enhancing or inhibiting a cellular response to a
TNF-family ligand.
The method involves contacting cells which express the TR14 polypeptide with a
candidate
compound and a TNF-family ligand, assaying a cellular response, and comparing
the cellular
response to a standard cellular response, the standard being assayed when
contact is made with
the ligand in absence of the candidate compound, whereby an increased cellular
response over
the standard indicates that the candidate compound is an agonist of the
ligand/receptor signaling
pathway and a decreased cellular response compared to the standard indicates
that the candidate
compound is an antagonist of the ligand/receptor signaling pathway. By the
invention, a cell
expressing the TR14 polypeptide (e.g., the polypeptide shown in Figures l0A-H,
or,
alternatively 4A-D) can be contacted with either an endogenous or exogenously
administered
TNF-family ligand.
Brief Description of the Figures
2o Figures lA-C shows the nucleotide (SEQ ID NO:1) and deduced amino acid
sequence
(SEQ ID N0:2) of the TR13 receptor. Predicted amino acids from about 105 to
about 170,
about 251 to about 265, about 331 to about 410, and about 580 to about 610
constitute the
cysteine-rich domains (amino acid residues from about 105 to about 170, about
251 to about
265, about 331 to about 410, and about 580 to about 610 in SEQ ID N0:2) and
are represented
by the underlined amino acid regions; amino acids from about 139 to about 142,
about 140 to
about 143, about 153 to about 156, about 293 to about 296, about 325 to about
328, about 421
to about 424, about 466 to about 469, about 696 to about 699, and about 728 to
about 731
constitute potential sites of N-glycosylation (amino acid residues from about
139 to about 142,
about 140 to about 143, about 153 to about 156, about 293 to about 296, about
325 to about
328, about 421 to about 424, about 466 to about 469, about 696 to about 699,
and about 728
to about 731 in SEQ ID N0:2) which are represented by the bolded amino acids;
amino acids
from about 312 to about 315, and about 458 to about 461, constitute potential
cAMP
phosphorylation sites (amino acid residues from about from about 312 to about
315, and about
458 to about 461 in SEQ ID N0:2) and are represented by asterisks (*) above
the amino acid
residues; amino acids from about 50 to about 53, about 66 to about 69, about
80 to about 83,
about 276 to about 279, about 311 to about 314, about 438 to about 441, about
559 to about
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
9
562, about 564 to about 567, about 698 to about 701, and about 725 to about
728 constitute
potential sites of protein kinase C (PKC) phosphorylation (amino acid residues
from about 50
to about 53, about 66 to about 69, about 80 to about 83, about 276 to about
279, about 311 to
about 314, about 438 to about 441, about 559 to about 562, about 564 to about
567, about 698
to about 701, and about 725 to about 728 in SEQ ID N0:2) and are represented
by the italicized
amino acid residues; amino acids from about 80 to about 83, about 89 to about
92, about 180 to
about 183, about 198 to about 201, about 214 to about 217, about 272 to about
275, about 306
to about 309, about 510 to about 513, about 529 to about 532, about 584 to
about 587, about
609 to about 312, about 642 to about 645, and about 698 to about 701 casein
kinase II
1o phosphorylation sites (amino acid residues from about 80 to about 83, about
89 to about 92,
about 180 to about 183, about 198 to about 201, about 214 to about 217, about
272 to about
275, about 306 to about 309, about 510 to about 513, about 529 to about 532,
about 584 to
about 587, about 609 to about 312, about 642 to about 645, and about 698 to
about 701 in
SEQ ID N0:2) and are represented by the double underlined amino acids; amino
acids from
about 69 to about 74, about 149 to about 154, about 154 to about 159, about
163 to about 168,
about 212 to about 217, about 248 to about 253, about 365 to about 370, about
383 to about
388, about 393 to about 398, about 588 to about 593, about 623 to about 628,
about 661 to
about 666, and about 665 to about 670 N-myristoylation sites (amino acids from
about 69 to
about 74, about 149 to about 154, about 154 to about 159, about 163 to about
168, about 212
2o to about 217, about 248 to about 253, about 365 to about 370, about 383 to
about 388, about
393 to about 398, about 588 to about 593, about 623 to about 628, about 661 to
about 666,
and about 665 to about 670 in SEQ ID N0:2) and are represented by the
strikethrough amino
acids (e.g. ~); and amino acids from about 456 to about 459 constitute a
potential amidylation
site (amino acid residues from about 456 to about 459 of SEQ ID N0:5) and is
represented by
the lowercase amino acids.
Figures 2A-C show the regions of similarity between the amino acid sequences
of the
TR13 receptor protein (SEQ ID N0:2), and the OX40 protein (SEQ ID N0:3).
Figure 3 shows an analysis of the TR13 amino acid sequence (SEQ ID N0:2).
Alpha,
beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic
regions; flexible
regions; antigenic index and surface probability are shown. In the "Antigenic
Index - Jameson-
Wolf" graph, amino acid residues from about M1 to about A9, about K12 to about
L20, about
N47 to about T55, about H58 to about S66, about D63 to about S71, about P77 to
about F85,
about A90 to about Q98, about F136 to about Q144, about S152 to about C160,
about 8159 to
about A167, about A211 to about M219, about M235 to about V243, about V266 to
about
V274, about W277 to about S285, about I290 to about F298, about A310 to about
V318,
about E343 to about C351, about I360 to about H368, about 6391 to about I399,
about F409
to about T417, about S436 to about Y444, about C453 to about S461, about I472
to about
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
S480, about Y548 to about S556, about C557 to about 1565, about V567 to about
V575, about
T584 to about 6592, about 8632 to about 6640, about W680 to about Y688, about
Q684 to
about K692, about T698 to about A706, about 5726 to about S734, and about S734
to about
L742 of SEQ ID N0:2 (Figures lA-C) correspond to the highly antigenic regions
of the TR13
5 protein, predicted using the Jameson-Wolf antigenic index (See Figure 3 and
Table I). These
highly antigenic fragments correspond to the amino acid residues illustrated
in Figures lA-C
and in SEQ ID N0:2.
Figures 4A-D shows the nucleotide (SEQ ID N0:4) and deduced amino acid
sequence
(SEQ ID N0:5) of the TR14 receptor. The predicted extracellular domain
constitutes amino
10 acids from about 1 to about 133 (amino acid residues from 1 to 133 of SEQ
ID N0:5) and are
represented by the underlined amino acids; amino acids from about 65 to about
85 constitute a
conserved cysteine-rich domain (amino acid residues from about 65 to about 85
of SEQ ID
N0:5) and is represented by the italized amino acid residues; amino acids from
about I34 to
about 150 constitute the predicted transmembrane domain (amino acid residues
from about 134
~5 to about 150 in SEQ ID N0:5) which are represented by the double underlined
amino acid
residues; amino acid residues from about 151 to about 226 constitutes the
predicted
intracellular domain (amino acid residues from about 151 to about 226 of SEQ
ID N0:5) and
are represented by the lower case amino acid residues; amino acids from about
178 to about
180 constitute potential protein kinase C (PKC) phosphorylation sites (amino
acid residues
2o from about 178 to about 180 of SEQ ID N0:5) and are represented by
asterisks (*) above the
amino acid residues; amino acids from about 5 to about 8, about 118 to about
121, about 178 to
about 181, and about 193 to about 196 constitute potential sites of casein
kinase II
phosphorylation (amino acid residues from about 5 to about 8, about 118 to
about 121, about
178 to about 181, about 193 to about 196 of SEQ ID N0:5) and are represented
by the
25 strikethrough amino acid residues; and amino acids from about 9 to about 14
contitutes a
potential N-myristoylation site (amino acid residues from about 9 to about 14
of SEQ ID N0:5)
and is represented by the bold amino acids.
Figures 5A-B show the regions of similarity between the amino acid sequences
of the
TR14 receptor protein (SEQ ID N0:5), and the Tumor Necrosis Factor Receptor
protein (SEQ
3o ID N0:6).
Figure 6 shows an analysis of the TR14 amino acid sequence (SEQ ID N0:5).
Alpha,
beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic
regions; flexible
regions; antigenic index and surface probability are shown. In the "Antigenic
Index - Jameson-
Wolf" graph, amino acid residues from about T3 to about S 11, from about V 16
to about R24,
35 from about Q44 to about M52, from about F85 to about G93, from about T103
to about V 111,
from about F161 to about 6169, from about V 187 to about A195, from about P218
to about
M226 of SEQ ID N0:5 (Figures 4A-D) correspond to the highly antigenic regions
of the TR14
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
11
protein, predicted using the Jameson-Wolf antigenic index (See Figure 6 and
Table II). These
highly antigenic fragments correspond to the amino acid residues illustrated
in Figures 4A-D
and in SEQ ID N0:5.
Figures 7A-D shows the nucleotide (SEQ ID N0:39) and deduced amino acid
sequence
(SEQ ID N0:40) of the full-length TR13 receptor. The predicted signal sequence
constitutes
amino acids from about 1 to about 41 (amino acid residues from about 1 to
about 41 of SEQ 117
N0:40) and are represented by the dotted underlined amino acids; amino acids
from about 42 to
about 906 constitutes the predicted extracellular domain (amino acid residues
from 42 to 906 of
SEQ ID N0:40) and are represented by the single underlined amino acids; amino
acids from
about 271 to about 421 and from about 585 to about 595 constitute conserved
cysteine-rich
domains (amino acid residues from about 271 to about 421 and from about 585 to
about 595 of
SEQ ID N0:40) and is represented by the italized amino acid residues; amino
acids from about
907 to about 931 constitute the predicted transmembrane domain (amino acid
residues from
about 907 to about 931 in SEQ ID N0:40) which are represented by the double
underlined
amino acid residues; amino acid residues from about 932 to about 1001
constitutes the
predicted intracellular domain (amino acid residues from about 932 to about
1001 of SEQ ID
N0:40) and are represented by the lower case amino acid residues; amino acids
from about 11
to about 13, about 18 to about 20, 107 to about 109, about 156 to about 158,
about 224 to
about 226, about 301 to about 303, about 317 to about 319, about 331 to about
333, about 527
2o to about 529, about 562 to about 564, about 689 to about 691, about 810 to
about 812, about
815 to about 817, about 949 to about 951, and about 976 to about 978
constitute potential
protein kinase C (PKC) phosphorylation sites (amino acid residues from about
11 to about 13,
about 18 to about 20, 107 to about 109, about 156 to about 158, about 224 to
about 226, about
301 to about 303, about 317 to about 319, about 331 to about 333, about 527 to
about 529,
about 562 to about 564, about 689 to about 691, about 810 to about 812, about
815 to about
817, about 949 to about 951, and about 976 to about 978 of SEQ ID N0:40) and
are
represented by asterisks (*) above the amino acid residues; amino acids from
about 42 to about
45, about 59 to about 62, about 81 to about 84, about 146 to about 149, about
282 to about
285, about 331 to about 334, about 340 to about 343, about 431 to about 434,
about 449 to
about 452, about 465 to about 468, about 523 to about 526, about 557 to about
560, about 761
to about 764, about 780 to about 783, about 780 to about 783, about 835 to
about 838, about
860 to about 863, about 893 to about 896, and about 949 to about 952
constitute potential sites
of casein kinase II phosphorylation (amino acid residues from about 42 to
about 45, about 59
to about 62, about 81 to about 84, about 146 to about 149, about 282 to about
285, about 331
to about 334, about 340 to about 343, about 431 to about 434, about 449 to
about 452, about
465 to about 468, about 523 to about 526, about 557 to about 560, about 761 to
about 764,
about 780 to about 783, about 780 to about 783, about 835 to about 838, about
860 to about
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
12
863, about 893 to about 896, and about 949 to about 952 of SEQ ID N0:40) and
are
represented by the strikethrough amino acid residues; amino acids from about
77 to about 82,
about 88 to about 93, about 152 to about 157, about 268 to about 273, about
288 to about 293,
about 320 to about 325, about 400 to about 405, about 414 to about 419, about
463 to about
468, about 599 to about 604, about 616 to about 621, about 634 to about 639,
about 644 to
about 649, about 839 to about 844, about 874 to about 879, about 912 to about
917, and about
916 to about 921 contitute potential N-myristoylation sites (amino acid
residues from about 77
to about 82, about 88 to about 93, about 152 to about 157, about 268 to about
273, about 288
to about 293, about 320 to about 325, about 400 to about 405, about 414 to
about 419, about
463 to about 468, about 599 to about 604, about 616 to about 621, about 634 to
about 639,
about 644 to about 649, about 839 to about 844, about 874 to about 879, about
912 to about
917, and about 916 to about 921 of SEQ ID N0:40) and are represented by a plus
sign ("+")
above the amino acids; amino acids from about 50 to about 56, and 109 to about
116 constitute
potential tyrosine phosphorylation sites (amino acids from about 50 to about
56, and about 109
to about 116 of SEQ ID N0:40) are represented by the double strikethrough
amino acids; and
amino acids from about 153 to about 156, 390 to about 393, 391 to about 394,
about 404 to
about 407, about 544 to about 547, about 576 to about 579, about 672 to about
675, about 717
to about 720, about 947 to about 950, and about 979 to about 982 constitute
potential N-
glycosylation sites (amino acids from about 153 to about 156, 390 to about
393, 391 to about
394, about 404 to about 407, about 544 to about 547, about 576 to about 579,
about 672 to
about 675, about 717 to about 720, about 947 to about 950, and about 979 to
about 982 of
SEQ ID N0:40) which are represented by the shaded amino acids.
Figures 8A-B show the regions of similarity between the amino acid sequences
of the
full-length TR13 receptor protein (SEQ ID N0:40), and the Tumor Necrosis
Factor Receptor II
homolog (gbIAAB94382.1) (SEQ ID NO: 41).
Figure 9 shows an analysis of the full-length TR13 amino acid sequence
disclosed in
Figures 7A-D (SEQ ID N0:40). Alpha, beta, turn and coil regions;
hydrophilicity and
hydrophobicity; amphipathic regions; flexible regions; antigenic index and
surface probability
are shown. In the "Antigenic Index - Jameson-Wolf" graph, amino acid residues
from about
3o M1 to about H9, about V14 to about I22, about H47 to about H55, about C61
to about R69,
about L82 to about E90, about D102 to about P110, about K109 to about 5117,
about F124 to
about H132, about M141 to about E149, about S146 to about C154, about S157 to
about
W 165, about F168 to about T 176, about N 182 to about N 190, about Q207 to
about A215,
about P213 to about M221, about M221 to about E229, about V233 to about V241,
about
T253 to about V261, about T282 to about S290, about N298 to about T306, about
C308 to
about Y316, about K315 to about S323, about P328 to about F336, about A341 to
about
Q349, about F387 to about Q395, about 5403 to about C411, about T409 to about
P417, about
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
13
F443 to about N451, about W451 to about Y459, about A462 to about M470, about
6478 to
about M486, about A487 to about A495, about V517 to about V525, about T527 to
about
Q535, about I541 to about F549, about A561 to about V569, about E594 to about
C602, about
I611 to about H619, about 6643 to about I650, about P686 to about K694, about
C704 to
about S712, about 8722 to about I730, about E727 to about T735, about P746 to
about 6754,
about D776 to about L784, about Y799 to about S807, about C808 to about I816,
about V818
to about V826, about T835 to about 6843, about 8883 to about 6891, about K932
to about
K940, about Q935 to about K943, about T949 to about A957, about S977 to about
S985,
about S981 to about P989, and about N986 to about L994 of SEQ ID N0:40
(Figures 7A-D)
correspond to the highly antigenic regions of the TR13 protein, predicted
using the Jameson-
Wolf antigenic index (See Figure 9 and Table III). These highly antigenic
fragments
correspond to the amino acid residues illustrated in Figures 7A-D and in SEQ
ID N0:40.
Figures l0A-H show a preferred nucleotide (SEQ ID N0:60) and deduced amino
acid
sequence (SEQ ID N0:61) of the TR14 receptor. The transmembrane domain from
amino
acids L-139 to L-155 is underlined.
Figure 11 shows an analysis of the full-length TR14 amino acid sequence
disclosed in
Figures l0A-H (SEQ ID N0:61). Alpha, beta, turn and coil regions;
hydrophilicity and
hydrophobicity; amphipathic regions; flexible regions; antigenic index and
surface probability
are shown. The data are presented in tabular form, amino acid by amino acid,
in Table IV,
2o below.
Detailed Description of the Preferred Embodiments
The present invention provides isolated nucleic acid molecules comprising a
polynucleotide encoding a TR13 polypeptide having the amino acid sequence
shown in Figures
lA-C (SEQ ID N0:2) and/or Figures 7A-D (SEQ ID N0:40) and/or fragments or
variants
thereof. The TR13 polypeptide of the present invention shares sequence
homology with the
human OX40 homologue (Figures 2A-C) and the tumor necrosis factor receptor II
homolog
(Figures 8A-B). The nucleotide sequence shown in Figures lA-C (SEQ ID NO:1)
was
obtained by sequencing a cDNA clone (HWLHM70), which was deposited on July 13,
1999 at
3o the American Type Culture Collection, and given Accession Number PTA-349.
The nucleotide
sequence shown in Figures 7A-D (SEQ ID N0:39) was obtained, in part, by
sequencing a
cDNA clone (HWLHN83), which was deposited on August 12, 1999 at the American
Type
Culture Collection, and given Accession Number PTA-507. The deposited clone is
inserted in
the pSportl clone (Life Technologies, Rockville, MD) using the SaII and NotI
restriction
endonuclease cleavage sites.
The present invention provides isolated nucleic acid molecules comprising a
polynucleotide encoding a TR14 polypeptide having the amino acid sequence
shown in Figures
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
14
l0A-H (SEQ ID NO:51), or, alternatively 4A-D (SEQ ID NO:S) and/or fragments or
variants
thereof, which were determined by sequencing a cloned cDNA. The TR14
polypeptide of the
present invention shares sequence homology with the Tumor Necrosis Factor
Receptor
(Figures SA-B). The nucleotide sequence shown in Figures l0A-H (SEQ ID N0:60)
was
obtained by sequencing a cDNA clone (HMSHK47), which was deposited on July 13,
1999 at
the American Type Culture Collection, and given Accession Number PTA-348. The
deposited
clone is inserted in the pBluescript clone (Life Technologies, Rockville, MD)
using the EcoRI
restriction endonuclease cleavage sites. While SEQ ID N0:60 is a preferred
sequence for
TR14, an alternative TR14 related sequence is shown in Figures 4A-D (SEQ ID
N0:4).
to
Nucleic Acid Molecules
Unless otherwise indicated, all nucleotide sequences determined by sequencing
a DNA
molecule herein were determined using an automated DNA sequencer (such as the
Model 3700
and Model 373 from Applied Biosystems, Inc.), and all amino acid sequences of
polypeptides
encoded by DNA molecules determined herein were predicted by translation of a
DNA
sequence determined as above. Therefore, as is known in the art for any DNA
sequence
determined by this automated approach, any nucleotide sequence determined
herein may
contain some errors. Nucleotide sequences determined by automation are
typically at least
about 90% identical, more typically at least about 95% to at least about 99.9%
identical to the
2o actual nucleotide sequence of the sequenced DNA molecule. The actual
sequence can be more
precisely determined by other approaches including manual DNA sequencing
methods well
known in the art. As is also known in the art, a single insertion or deletion
in a determined
nucleotide sequence compared to the actual sequence will cause a frame shift
in translation of
the nucleotide sequence such that the predicted amino acid sequence encoded by
a determined
nucleotide sequence will be completely different from the amino acid sequence
actually encoded
by the sequenced DNA molecule, beginning at the point of such an insertion or
deletion.
Using the information provided herein, such as the nucleic acid sequence set
out in SEQ
ID NO:1 and/or SEQ ID N0:39, a nucleic acid molecule of the present invention
encoding a
TR13 polypeptide may be obtained using standard cloning and screening
procedures, such as
those for cloning cDNAs using mRNA as starting material. Illustrative of the
invention, the
nucleic acid molecule described in SEQ ID NO:1 was discovered in a cDNA
library derived
from activated monocytes. Further, illustrative of the invention, the nucleic
acid molecule
described in SEQ ID N0:39 was discovered in a cDNA library derived from normal
human
colon. TR13 polynucleotides of the invention have also been identified in cDNA
libraries from
the following tissues: pancreas tumor, endometrial tumor, adult small
intestine, colon cancer,
breast cancer cell line, resting T-cell, amygdala, rectum, T-cell helper,
pineal gland, apoptotic
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
T-cell, epididymus; greater omentum, prostate BPH, osteoclastoma, endometrial
stromal cells,
stromal cell, substantia nigra, activated T-cell, tonsil, and testes tissue.
The determined TR13 nucleotide sequence of SEQ ID NO:1 contains an open
reading
frame encoding a protein of about 750 amino acid residues, and a deduced
molecular weight of
5 about 82 kDa. The amino acid sequence of the predicted TR13 receptor is
shown in SEQ >D
N0:2 from amino acid residue about 1 to residue about 750. OF known members of
the TNF
receptor family, this TR13 polypeptide shares the greatest degree of homology
with human
OX40 (See Figures 2A-C), including significant sequence homology over multiple
cysteine
rich domains.
to The determined TR13 nucleotide sequence of SEQ ID N0:39 contains an open
reading
frame encoding a protein of about 1001 amino acid residues, with a predicted
signal
encompassing amino acids about 1 to about 41, a predicted extracellular domain
encompassing
amino acids from about 42 to about 906, a transmembrane domain encompassing
amino acids
from about 907 to about 931, and an intracellular domain encompassing amino
acids from
15 about 932 to 1001, of SEQ ID N0:40, and a deduced molecular weight of about
110 kDa. The
amino acid sequence of the predicted TR13 receptor is shown in SEQ ID N0:40
from amino
acid residue about 1 to residue about 1001. Of known members of the TNF
receptor family,
this TR13 polypeptide shares the greatest degree of homology with the tumor
necrosis factor
receptor II homolog (See Figures 8A-B), including significant sequence
homology over
multiple cysteine rich domains.
Using the information provided herein, such as the nucleic acid sequence set
out,
preferably, in SEQ ID N0:60, or, alternatively SEQ ID N0:4, a nucleic acid
molecule of the
present invention encoding a TR14 polypeptide may be obtained using standard
cloning and
screening procedures, such as those for cloning cDNAs using mRNA as starting
material.
Illustrative of the invention, the nucleic acid molecule contained in
deposited clone HMSHK47
(described in SEQ ID N0:60) was discovered in a cDNA library derived from
colon. The gene
of the present invention has also been identified in cDNA libraries from the
following tissues:
activated T-cell, endometrial tumor, thymus, and 12 week early stage human
tissue.
The determined nucleotide sequence of the TR14 cDNA of SEQ ID N0:60 contains
an
open reading frame encoding a protein of about 231 amino acid residues, with a
predicted
extracellular domain encompassing amino acids from about 1 to about 138, a
transmembrane
domain encompassing amino acids from about 139 to about 155, and an
intracellular domain
encompassing amino acids from about 156 to about 231 of SEQ ID N0:61 and a
deduced
molecular weight of about 25 kDa.
The TR14 nucleotide sequence of SEQ ID N0:4 contains an open reading frame
encoding a protein of about 226 amino acid residues, with a predicted
extracellular domain
encompassing amino acids from about 1 to about 133, a transmembrane domain
encompassing
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
16
amino acids from about 134 to about 150 (from about 139 to about 155 of SEQ ID
N0:61 ),
and an intracellular domain encompassing amino acids from about 151 to about
226 of SEQ 117
N0:4 (acids from about 156 to about 231 of SEQ ID N0:61) and a deduced
molecular weight
of about 24.5 kDa. Of known members of the TNF receptor family, the TR14
polypeptide of
the SEQ ID NO:S shares the greatest degree of homology with tumor necrosis
factor receptor
(See Figures SA-B).
As indicated, the present invention also encompasses mature forms) of the TR13
and/or TR14 polypeptides of the present invention. According to the signal
hypothesis,
proteins secreted by mammalian cells have a signal or secretory leader
sequence which is
to cleaved from the mature protein once export of the growing protein chain
across the rough
endoplasmic reticulum has been initiated. Most mammalian cells and even insect
cells cleave
secreted proteins with the same specificity. However, in some cases, cleavage
of a secreted
protein is not entirely uniform, which results in two or more mature species
on the protein.
Further, it has long been known that the cleavage specificity of a secreted
protein is ultimately
determined by the primary structure of the complete protein, that is, it is
inherent in the amino
acid sequence of the polypeptide.
Therefore, the present invention provides a nucleotide sequence encoding the
mature
form of the TR13 polypeptide having the amino acid sequence encoded by the
cDNA clone
identified as ATCC Deposit No. PTA-349 (HWLHM70), and/or of the amino acid
sequence
shown in Figures lA-C (SEQ ID N0:2). By the mature form of TR13 polypeptide
having the
amino acid sequence encoded by, for example, the cDNA clone identified as ATCC
Deposit
No. PTA-349 (HWLHM70) is meant, the mature forms) of the TR13 receptor
produced by
expression in a mammalian cell (e.g., COS cells, as described below) of the
complete open
reading frame encoded by the human DNA sequence of the clone contained in the
deposited
vector. As indicated herein, the mature form of the TR13 polypeptide having
the amino acid
sequence encoded by the cDNA clone contained in ATCC Deposit No. PTA-349
(HWLHM70), may or may not differ from the predicted mature TR13 protein shown
in SEQ
ID N0:2 (amino acids from about 1 to about 750) depending on the accuracy of
the predicted
cleavage site based on computer analysis. Polypeptides encoded by the
nucleotide sequences
are also encompassed by the invention.
Therefore, the present invention provides a nucleotide sequence encoding the
mature
form of the TR13 polypeptide having the amino acid sequence encoded by the
cDNA clone
identified as ATCC Deposit No. PTA-507 (HWLHN83), and/or of the amino acid
sequence as
shown in Figures 7A-D (SEQ ID N0:40). By the mature form of the TR13
polypeptide having
the amino acid sequence encoded by, for example, the cDNA clone identified as
ATCC Deposit
No. PTA-507 (HWLHN83), is meant, the mature forms) of the TR13 receptor
produced by
expression in a mammalian cell (e.g., COS cells, as described below) of the
complete open
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
17
reading frame encoded by the human DNA sequence of the clone contained in the
deposited
vector. As indicated herein, the mature form of the TR13 polypeptide having
the amino acid
sequence encoded by the cDNA clone contained in ATCC Deposit No. PTA-507
(HWLHN83), may or may not differ from the predicted mature TR13 protein shown
in SEQ
ID N0:40 (amino acids from about 42 to about 1001 ) depending on the accuracy
of the
predicted cleavage site based on computer analysis. Polypeptides encoded by
these nucleotide
sequences are also encompassed by the invention.
Methods for predicting whether a protein has a secretory leader as well as the
cleavage
point for that leader sequence are available. For instance, the method of
McGeoch (Virus Res.
l0 3:271-286 (1985)) and von Heinje (Nucleic Acids Res. 14:4683-4690 (1986))
can be used.
The accuracy of predicting the cleavage points of known mammalian secretory
proteins for each
of these methods is in the range of 75-80%. von Heinje, supra. However, the
two methods
do not always produce the same predicted cleavage points) for a given protein.
1n the present case, the predicted amino acid sequence of the TR13 polypeptide
of the
present invention was analyzed by a computer program ("PSORT"). See K. Nakai
and M.
Kanehisa, Genomics 14:897-911 (1992). PSORT is an expert system for predicting
the cellular
location of a protein based on the amino acid sequence. As part of this
computational
prediction of localization, the methods of McGeoch and von Heinje are
incorporated.
Thereafter, the complete amino acid sequences were further analyzed by visual
inspection,
applying a simple form of the (-1,-3) rule of von Heinje. von Heinje, supra.
Thus, the TR13
protein is predicted to consist of residues from about 1-750 in SEQ ID N0:2,
and/or 1-1001 in
SEQ ID N0:40. The mature form of the polypeptide sequence disclosed in SEQ ID
N0:40 is
predicted to consist of residues from about 42 to 1001.
As one of ordinary skill would appreciate, due to the possibilities of
sequencing errors,
as well as the variability of cleavage sites for leaders in different known
proteins, the predicted
full-length TR13 polypeptide encoded by the deposited cDNA clones comprises
about 1001
amino acids, but may be anywhere in the range of about 700 to about 1200 amino
acids. It will
further be appreciated that, the domains described herein have been predicted
by computer
analysis, and accordingly, depending on the analytical criteria used for
identifying various
3o functional domains, the exact "address" of, for example, the extracelluar
domain, intracelluar
domain, cysteine-rich domains, and transmembrane domain of TR13 may differ
slightly (e.g.,
the address may "shift" by about 1 to about 20 residues, more likely about 1
to about 5
residues). For example, the exact location of the TR13 cysteine-rich domains
in Figures lA-C
(SEQ ID N0:2) and/or Figures 7A-D (SEQ ID N0:40) may vary slightly (e.g., the
address
may "shift" by about 1 to about 20 residues, more likely about 1 to about 5
residues) depending
on the criteria used to define the motifs. In any event, as discussed further
below, the invention
further provides polypeptides having various residues deleted from the N-
terminus and/or C-
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
18
terminus of the full-length TR13, including polypeptides lacking one or more
amino acids from
the N-termini of the extracellular domain described herein, which constitute
soluble forms of
the extracellular domain of the TR13 polypeptides.
As one of ordinary skill would appreciate, due to the possibilities of
sequencing errors,
the preferred predicted full-length TR14 polypeptide encoded by the deposited
cDNA clone
comprises about 231 amino acids as shown in SEQ ID N0:61, but may be anywhere
in the
range of 175-275 amino acids. In an alternative embodiment, predicted full-
length TR14
polypeptide comprises about 226 amino acids, but may be anywhere in the range
of 175-275
amino acids, but may be anywhere in the range of about 45 to about 200 amino
acids. It will
to further be appreciated that, the domains described herein have been
predicted by computer
analysis, and accordingly, that depending on the analytical criteria used for
identifying various
functional domains, the exact "address" of, for example, the extracelluar
domain, intracelluar
domain, cysteine-rich domains, and transmembrane domain of TR14 may differ
slightly (e.g.,
the address may "shift" by about 1 to about 20 residues, more likely about 1
to about 5
residues). For example, the exact location of the TR14 extracellular domain
and/or cysteine-
rich domains in Figures l0A-H (SEQ ID N0:61) or, alternatively Figures 4A-D
(SEQ ID
NO:S) may vary slightly (e.g., the address may "shift" by about 1 to about 20
residues, more
likely about 1 to about 5 residues) depending on the criteria used to define
the domain.
Additionally, in the event the polypeptide sequence of TR14 is longer than the
sequence
2o depicted in Figures l0A-H or, alternatively Figures 4A-D, the skilled
artisan would appreciate
that the sequence could affect the ultimate location of the extracellular,
transmembrane, or
intracellular domain. In any event, as discussed further below, the invention
further provides
polypeptides having various residues deleted from the N-terminus and/or C-
terminus of the
full-length TR14, including polypeptides lacking one or more amino acids from
the N-termini
of the extracellular domain described herein, which constitute soluble forms
of the extracellular
domain of the TR14 polypeptides.
As indicated, nucleic acid molecules of the present invention may be in the
form of
RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and
genomic
DNA obtained by cloning or produced synthetically. The DNA may be double-
stranded or
single-stranded. Single-stranded DNA may be the coding strand, also known as
the sense
strand, or it may be the non-coding strand, also referred to as the anti-sense
strand.
By "isolated" nucleic acid molecules) is intended a nucleic acid molecule, DNA
or
RNA, which has been removed from its native environment For example,
recombinant DNA
molecules contained in a vector are considered isolated for the purposes of
the present
invention. Further examples of isolated DNA molecules include recombinant DNA
molecules
maintained in heterologous host cells or purified (partially or substantially)
DNA molecules in
solution. Isolated RNA molecules include in vivo or ire vitro RNA transcripts
of the DNA
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
19
molecules of the present invention. Isolated nucleic acid molecules according
to the present
invention further include such molecules produced synthetically. However, a
nucleic acid
molecule contained in a clone that is a member of a mixed clone library (e.g.,
a genomic or
cDNA library) and that has not been isolated from other clones of the library
(e.g., in the form
of a homogeneous solution containing the clone without other members of the
library) or a
chromosome isolated or removed from a cell or a cell lysate (e.g., a
"chromosome spread", as
in a karyotype), is not "isolated" for the purposes of this invention.
Isolated nucleic acid molecules of the present invention include, for example,
DNA
molecules comprising, or alternatively consisting of, an open reading frame
(ORF) shown in
Figures lA-C (SEQ ID NO:1), Figures 7A-D (SEQ ID N0:39) and/or contained in a
deposited
cDNA clone (e.g., HWLHM70 and HWLHN83); DNA molecules comprising, or
alternatively
consisting of, the coding sequence for the mature TR13 protein shown in
Figures lA-C (SEQ
ID NO:1) and/or Figures 7A-D (SEQ ID N0:39) and/or contained in a deposited
cDNA clone
(e.g., HWLHM70 and HWLHN83); DNA molecules comprising, or alternatively
consisting
of, a fragment of the coding sequence for the full-length TR13 protein
disclosed in Figures lA
C and/or Figures 7A-D and/or encoded by a deposited cDNA clone; and DNA
molecules which
comprise, or alternatively consist of, a sequence substantially different from
those described
above, but which, due to the degeneracy of the genetic code, still encode TR13
polypeptides
(including fragments of variants thereof). Of course, the genetic code is well
known in the art.
Thus, it would be routine for one skilled in the art to generate such
degenerate variants.
Isolated nucleic acid molecules of the present invention include, for example,
DNA
molecules comprising, or alternatively consisting of, an open reading frame
(ORF) shown
preferably in Figures l0A-H (SEQ ID N0:60) or, alternatively, in Figures 4A-D
(SEQ ID
N0:4) and/or contained in the deposited cDNA clone (HMSHK47); DNA molecules
comprising, or alternatively consisting of, the coding sequence for the mature
TR14 protein
shown preferably in Figures l0A-H (amino acids 1-164 of SEQ ID N0:61), or
alternatively, in
Figures 7A-D (SEQ ID N0:4) and/or contained in the deposited cDNA clone
(HMSHK47);
DNA molecules comprising, or alternatively consisting of, a fragment of the
coding sequence
for the full-length TR14 protein disclosed in preferably in Figures l0A-H or,
alternatively, in
3o Figures 4A-D and/or encoded by the deposited cDNA clone (HMSHK47); and DNA
molecules
which comprise a sequence substantially different from those described above,
but which, due
to the degeneracy of the genetic code, still encode TR14 polypeptides
(including fragments or
variants thereof). Of course, the genetic code is well known in the art. Thus,
it would be
routine for one skilled in the art to generate such degenerate variants.
In another aspect, the invention provides isolated nucleic acid molecules
encoding the
TR13 polypeptide having an amino acid sequence encoded by the cDNA clone
contained in
ATCC Deposit No. PTA-349 (HWLHM70). In a further embodiment, nucleic acid
molecules
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
are provided that encode the mature form of the TR13 polypeptide disclosed in
Figures lA-C
and/or encoded by the cDNA contained in ATCC Deposit No. PTA-349. In a further
embodiment, nucleic acids are provided that the full-length TR13 polypeptide
disclosed in
Figures lA-C and/or encoded by the deposited cDNA clone, but lacking the N-
terminal
5 methionine. In a further embodiment, nucleic acid molecules are provided
that encode The
invention further provides an isolated nucleic acid molecule having the
nucleotide sequence
shown in SEQ ID NO:1 or the nucleotide sequence of the TR I 3 cDNA contained
in the above-
described deposited cDNA clone, or a nucleic acid molecule having a sequence
complementary
to one of the above sequences. Such isolated molecules, particularly DNA
molecules, are
1o useful, for example, as probes for gene mapping by in situ hybridization
with chromosomes,
and for detecting expression of the TR13 gene in human tissue, for instance,
by Northern blot
analysis.
In another aspect, the invention provides isolated nucleic acid molecules
encoding the
TR13 polypeptide having an amino acid sequence as encoded by the cDNA clone
contained in
15 ATCC Deposit No. PTA-507 (HWLHN83). In a further embodiment, nucleic acid
molecules
are provided that encode the mature form of the TR13 polypeptide disclosed in
Figures 7A-D,
and/or encoded by the cDNA contained in ATCC Deposit No. PTA-507. In a further
embodiment, nucleic acid molecules are provided that encode the full-length
TR13 polypeptide
disclosed in Figures 7A-D, and/or encoded by the deposited cDNA clone, but
lacking the N-
20 terminal methionine. The invention further provides an isolated nucleic
acid molecule having
the nucleotide sequence shown in SEQ ID N0:39 or the nucleotide sequence of
the TR13
cDNA contained in the above-described deposited cDNA clone, or a nucleic acid
molecule
having a sequence complementary to one of the above sequences. Such isolated
molecules,
particularly DNA molecules, are useful, for example, as probes for gene
mapping by in situ
hybridization with chromosomes, and for detecting expression of the TR13 gene
in human
tissue, for instance, by Northern blot analysis.
In another aspect, the invention provides isolated nucleic acid molecules
encoding the
TR14 polypeptide having an amino acid sequence encoded by the cDNA clone
contained in
ATCC Deposit No. PTA-348 (HMSHK47). In a further embodiment, nucleic acid
molecules
are provided that encode the full-length TR14 polypeptide disclosed in Figures
l0A-H or,
alternatively, in Figures 4A-D, and/or encoded by the deposited cDNA clone,
but lacking the
N-terminal methionine. The invention further provides an isolated nucleic acid
molecule having
the nucleotide sequence shown preferably in SEQ ID N0:60 or, alternatively, in
SEQ ID N0:4
or the nucleotide sequence of the TR14 cDNA contained in the above-described
deposited
cDNA clone, or a nucleic acid molecule having a sequence complementary to one
of the above
sequences. Such isolated molecules, particularly DNA molecules, are useful,
for example, as
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
21
probes for gene mapping by in situ hybridization with chromosomes, and for
detecting
expression of the TR14 gene in human tissue, for instance, by Northern blot
analysis.
In addition, the invention provides nucleic acid molecules having nucleotide
sequences
related to extensive portions of SEQ ID NO:I which have been determined from
the following
related cDNA clones: HETAQ12R (SEQ ID N0:8), HETAK82R (SEQ ID N0:9), HETBH18R
(SEQ ID NO:10), HEPAB26R (SEQ ID NO:11), HETAN38R (SEQ ID N0:12), HPWDD30R
(SEQ ID N0:13), HETATOSR (SEQ ID N0:14), HETDQ39R (SEQ ID NO:15), HETEM84R
(SEQ ID N0:16), and HSIDV42R (SEQ ID N0:17).
In addition, the invention provides nucleic acid molecules having nucleotide
sequences
l0 related to extensive portions of SEQ ID N0:39 which have been determined
from the following
related cDNA clones: HETAQ12R (SEQ ID N0:48), HETAK82R (SEQ ID N0:49),
HETBM71 R (SEQ ID NO:50), HETBH 18R (SEQ ID NO:51 ), HEPAB26R (SEQ ID N0:52),
HETAN38R (SEQ ID N0:53), HPWDD30R (SEQ ID N0:54), HETATOSR (SEQ ID NO:55),
HETDQ39R (SEQ ID N0:56), HPWBL93R (SEQ ID N0:57), HETEM84R (SEQ ID N0:58),
and HSIDV42R (SEQ ID N0:59).
In addition, the invention provides nucleic acid molecules having nucleotide
sequences
related to extensive portions of SEQ ID NOS:60 and 4 which have been
determined from the
following related cDNA clones: HSABDSOR (SEQ ID N0:18), HTXMX53R (SEQ
N0:19), HE20R74R (SEQ ID N0:20), HMSHK47R (SEQ ID N0:21 ), and HMSHK59R
(SEQ ID N0:22).
The present invention is further directed to fragments of the isolated TR13
nucleic acid
molecules described herein. By a fragment of an isolated DNA molecule having
the nucleotide
sequence of a deposited cDNA clone (e.g., HWLHN83 and/or HWLHM70), or the
nucleotide
sequence shown in Figures lA-C (SEQ ID NO:1) and/or Figures 7A-D (SEQ ID
N0:39), or
the complementary strand thereto, is intended DNA fragments at least about
l5nt, and more
preferably at least about 20 nt, or at least 25 nt, still more preferably at
least about 30 nt, or at
least 35 nt, and even more preferably, at least about 40 nt, or at least about
50 nt in length
which are useful, for example, as diagnostic probes and primers as discussed
herein. Of
course, larger fragments 50-1500 nt in length are also useful according to the
present invention,
3o as are fragments corresponding to most, if not all, of the nucleotide
sequence of the deposited
cDNA or as shown in SEQ ID NO:1 and/or SEQ ID N0:39. By a fragment at least 20
nt in
length, for example, is intended fragments which include 20 or more contiguous
bases from the
nucleotide sequence of a deposited cDNA clone or the nucleotide sequence as
shown in Figures
lA-C (SEQ ID NO:1) and/or Figures 7A-D (SEQ ID N0:39). In this context "about"
includes
the particularly recited size, or may be larger or smaller by several (5, 4,
3, 2, or 1)
nucleotides, at either terminus or at both termini.
The present invention is further directed to fragments of the isolated TR14
nucleic acid
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
22
molecules described herein. By a fragment of an isolated DNA molecule having
the nucleotide
sequence of the deposited cDNA clone (HMSHK47), or the nucleotide sequence
shown
preferably in Figures l0A-H (SEQ ID N0:60) or, alternatively in Figures 4A-D
(SEQ ID
N0:4), or the complementary strand thereto, is intended DNA fragments at least
about l5nt,
and more preferably at least about 20 nt, or at least 25 nt, still more
preferably at least about 30
nt, or at least 35 nt, and even more preferably, at least about 40 nt, or at
least 50 nt, in length
which are useful, for example, as diagnostic probes and primers as discussed
herein. Of
course, larger fragments 50-1500 nt in length are also useful according to the
present invention,
as are fragments corresponding to most, if not all, of the nucleotide sequence
of the deposited
cDNA or as shown preferably in Figures l0A-H (SEQ ID N0:60) or, alternatively
in Figures
4A-D (SEQ ID N0:4). By a fragment at least 20 nt in length, for example, is
intended
fragments which include 20 or more contiguous bases from the nucleotide
sequence of the
deposited cDNA or the nucleotide sequence as shown in preferably in SEQ ID
N0:60, or,
alternatively, in SEQ ID N0:4. In this context "about" includes the
particularly recited size, or
1_5 may be larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at
either terminus or at both
termini.
Representative examples of TR13 polynucleotide fragments of the invention
include, -
for example, fragments that comprise, or alternatively, consist of, a sequence
from about
nucleotide 1 to about 50, from about 51 to about 108, from about 109 to about
159, from about
160 to about 210, from about 211 to about 261, from about 262 to about 273,
from about 274
to about 324, from about 325 to about 375, from about~376 to about 426, from
about 427 to
about 477, from about 478 to about 528, from about 529 to about 579, from
about 580 to
about 630, from about 631 to about 681, from about 682 to about 732, from
about 733 to
about 744, from about 745 to about 798, from about 799 to about 849, from
about 850 to
about 900, from about 901 to about 951, from about 952 to about 1002, from
about 1003 to
about 1053, from about 1054 to about 1104, from about 1105 to about 1155, from
about 1156
to about 1164, from about 1165 to about 1197, from about 1198 to about 1248,
from about
1249 to about 1266, from about 1267 to about 1317, from about 1318 to about
1368, from
about 1369 to about 1419, from about 1420 to about 1470, from about 1471 to
about 1521,
3o from about 1522 to about 1572, from about 1573 to about 1623, from about
1624 to about
1674, from about 1675 to about 1725, from about 1726 to about 1776, from about
1777 to
about 1827, from about 1828 to about 1878, from about 1879 to about 1929, from
about 1930
to about 1980, from about 1981 to about 2031, from about 2032 to about 2082,
from about
2083 to about 2133, from about 2134 to about 2184, from about 2185 to about
2235, from
about 2236 to about 2286, from about 2287 to about 2337, from about 2338 to
about 2388,
from about 2389 to about 2489, from about 2490 to about 2540, from about 2451
to about
2501, from about 2502 to about 2554 of the polynucleotide sequence shown in
Figures lA-C
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
23
(SEQ ID NO: l ), or the complementary strand thereto, or the cDNA contained in
the deposited
clone (HWLHM70). Other representative examples of polynucleotide fragments of
the
invention include, for example, fragments that comprise, or alternatively
consist of, a sequence
from about nucleotide 1 to about 362, from about 705 to about 830, from about
31 to about
2280, from about 343 to 414, from about 415 to about 459, from about 460 to
about 540, 343
to about 540, from about 781 to about 804, from about 805 to about 830, from
about 781 to
about 822, from about 1021 to about 1260, from about 1768 to about 1812, from
about 1813
to about 1866, from about 1768 to about 1866, from about 31 to about 540, from
about 660 to
about 984, from about 1057 to about 1470, from about 1672 to about 1806,
and/or from about
1924 to about 2256 of the polynucleotide sequence shown in Figures lA-C (SEQ
ID NO:1 ), or
the complementary strand thereto. 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. Polynucleotides which hybridize to any l, 2, 3, 4, 5 or more of these
polynucleotide
fragments are also encompassed by the invention. Moreover, polypeptides
encoded by these
polynucleotides and/or polynucleotide fragments are also encompassed by the
invention.
Additional representative examples of TR13 polynucleotide fragments of the
invention
include, for example, fragments that comprise, or alternatively, consist of, a
sequence from
about nucleotide 1 to about 50, from about 51 to about 108, from about 109 to
about 159, from
about 160 to about 210, from about 211 to about 261, from about 262 to about
273, from
2o about 274 to about 324, from about 325 to about 375, from about 376 to
about 426, from
about 427 to about 477, from about 478 to about 528, from about 529 to about
579, from
about 580 to about 630, from about 631 to about 681, from about 682 to about
732, from
about 733 to about 744, from about 745 to about 798, from about 799 to about
849, from
about 850 to about 900, from about 901 to about 951, from about 952 to about
1002, from
about 1003 to about 1053, from about 1054 to about 1104, from about 1105 to
about 1155,
from about 1156 to about 1164, from about 1165 to about 1197, from about 1198
to about
1248, from about 1249 to about 1266, from about 1267 to about 1317, from about
1318 to
about 1368, from about 1369 to about 1419, from about 1420 to about 1470, from
about 1471
to about 1521, from about 1522 to about 1572, from about 1573 to about 1623,
from about
1624 to about 1674, from about 1675 to about 1725, from about 1726 to about
1776, from
about 1777 to about 1827, from about 1828 to about 1878, from about 1879 to
about 1929,
from about 1930 to about 1980, from about 1981 to about 2031, from about 2032
to about
2082, from about 2083 to about 2133, from about 2134 to about 2184, from about
2185 to
about 2235, from about 2236 to about 2286, from about 2287 to about 2337, from
about 2338
to about 2388, from about 2389 to about 2489, from about 2490 to about 2540,
from about
2451 to about 2501, from about 2502 to about 2554, about 2600 to about 2650,
about 2651 to
about 2700, about 2701 to about 2750, about 2751 to about 2800, about 2801 to
about 2850,
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
24
about 2851 to about 2900, about 2901 to about 2950, about 2951 to about 3000,
about 3001 to
about 3050, about 3051 to about 3100, about 3101 to about 3150, about 3151 to
about 3200,
about 3201 to about 3250, about 3251 to about 3300, and about 3301 to about
3334 of the
polynucleotide sequence shown in Figures 7A-D (SEQ ID N0:39), or the
complementary
strand thereto, or the cDNA contained in the deposited clone (HWLHN83). Other
representative examples of polynucleotide fragments of the invention include,
for example,
fragments that comprise, or alternatively consist of, a sequence from about
nucleotide 1 to
about 42, from about 181 to about 2775, from about 984 to about 1142, from
about 1485 to
about 1610, from about 2361 to about 2718, from about 61 to about 3060, from
about 58 to
to about 3060, from about 58 to about 183, from about 58 to about 2775, from
about 2776 to
about 2850, from about 2851 to about 3060, from about 868 to about 1320, from
about 868 to
about 915, from about 925 to about 957, about 960 to about 1017, about 1042 to
about 1140,
about 1267 to about 1320, about 870 to about 1320, about 1810 to about 1842,
about 2038 to
about 2079, about 2185 to about 2289, about 2995 to about 3054, about 190 to
about 237,
about 418 to about 462, about 58 to about 843, about 847 to about 1326, about
1366 to about
2424, about 1812 to about 1842, about 2776 to about 2850, about 2428 to about
3060, about
2851 to about 3060, and/or from about 490 to about 537 of the polynucleotide
sequence shown
in Figures 7A-D (SEQ ID N0:39), or the complementary strand thereto. In this
context
"about" includes the particularly recited ranges, larger or smaller by several
(5, 4, 3, 2, or 1)
2o nucleotides, at either terminus or at both termini. Polynucleotides which
hybridize to any 1, 2,
3, 4, 5 or more of these polynucleotide fragments are also encompassed by the
invention.
Moreover, polypeptides encoded by these polynucleotides and/or polynucleotide
fragments are
also encompassed by the invention. Moreover, polypeptides encoded by the
polynucleotides
and/or polynucleotide fragments are also encompassed by the invention.
Preferably, the polynucleotide fragments of the invention encode a polypeptide
which
demonstrates a TR13 functional activity. By a polypeptide demonstrating a TR13
"functional
activity" is meant, a polypeptide capable of displaying one or more known
functional activities
associated with a full-length (complete) TR13 protein. Such functional
activities include, but
are not limited to, biological activity (e.g., cell proliferation activity),
antigenicity (ability to
3o bind (or compete with a TR13 polypeptide for binding) to an anti-TR13
antibody),
immunogenicity (ability to generate antibody which binds to a TR 13
polypeptide), ability to
form multimers with TR13 polypeptides of the invention, and ability to bind to
a receptor or
ligand for a TR13 polypeptide.
In one embodiment where one is assaying for the ability to bind or compete
with full-
length TR13 polypeptide for binding to anti-TR13 antibody, various
immunoassays known in
the art can be used, including but not limited to, competitive and non-
competitive assay systems
using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent
assay),
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
"sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitation
reactions,
immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or
radioisotope
labels, for example), western blots, precipitation reactions, agglutination
assays (e.g., gel
agglutination assays, hemagglutination assays), complement fixation assays,
5 immunofluorescence assays, protein A assays, and immunoelectrophoresis
assays, etc. In one
embodiment, antibody binding is detected by detecting a label on the primary
antibody. In
another embodiment, the primary antibody is detected by detecting binding of a
secondary
antibody or reagent to the primary antibody. In a further embodiment, the
secondary antibody
is labeled. Many means are known in the art for detecting binding in an
immunoassay and are
1o within the scope of the present invention.
In another embodiment, where a TR13 ligand is identified, or the ability of a
polypeptide fragment, variant or derivative of the invention to multimerize is
being evaluated,
binding can be assayed, e.g., by means well-known in the art, such as, for
example, reducing
and non-reducing gel chromatography, protein affinity chromatography, and
affinity blotting.
15 See generally, Phizicky, E., et al., Microbiol. Rev. 59:94-123 (1995). In
another
embodiment, physiological correlates of TR13 binding to its substrates (signal
transduction)
can be assayed.
In addition, assays described herein (see, for example, Examples 5, and 16-21)
and
those otherwise known in the art may routinely be applied to measure the
ability of TR13
2o polypeptides and fragments, variants derivatives and analogs thereof to
elicit a particular
biological activity (e.g., to inhibit TRAIL induced apoptosis, to regulate
(e.g., inhibit) B cell
proliferation (see, e.g., Example 33), to regulate proliferation of other
cells, and/or to inhibit
hematopoiesis in vitro or in vivo). For example, techniques known in the art
(such as for
example assaying for thymidine incorporation), may be applied or routinely
modified to assay
25 for the ability of the compositions of the invention to regulate (e.g.,
inhibit apoptosis) and/or to
regulate (e.g., inhibit) proliferation of hematopoietic cells. Additionally,
assays desribed herein
(see e.g., Example 15 and Example 33) and otherwise known in the art may be
applied or
routinely modified to assay for the ability of the compositions of the
invention to inhibit or
stimulate B cell proliferation.
3o Representative examples of TR14 polynucleotide fragments of the invention
include,
for example, fragments that comprise, or alternatively, consist of, a sequence
from about
nucleotide 1 to about 50, from about 51 to about 108, from about 109 to about
159, from about
160 to about 210, from about 211 to about 261, from about 262 to about 273,
from about 274
to about 324, from about 325 to about 375, from about 376 to about 426, from
about 427 to
about 477, from about 478 to about 528, from about 529 to about 579, from
about 580 to
about 630, from about 631 to about 681, from about 682 to about 732, from
about 733 to
about 744, from about 745 to about 798, from about 799 to about 849, from
about 850 to
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
26
about 900, from about 901 to about 951, from about 952 to about 1002, from
about 1003 to
about 1053, from about 1054 to about 1104, from about 1105 to about 1155, from
about 1156
to about 1164, from about 1165 to about 1197, from about 1198 to about 1248,
from about
1249 to about 1266, from about 1267 to about 1317, from about 1318 to about
1368, from
about 1369 to about 1419, from about 1420 to about 1470, from about 1471 to
about 1521,
from about 1522 to about 1572, from about 1573 to about 1623, from about 1624
to about
1674, from about 1675 to about 1725, from about 1726 to about 1776, from about
1777 to
about 1827, from about 1828 to about 1878, from about 1879 to about 1929, from
about 1930
to about 1980, from about 1981 to about 2031, from about 2032 to about 2082,
from about
2083 to about 2133, from about 2134 to about 2184, from about 2185 to about
2235, from
about 2236 to about 2286, from about 2287 to about 2337, from about 2338 to
about 2388,
from about 2389 to about 2489, from about 2490 to about 2540, from about 2451
to about
2501, from about 2502 to about 2552, from about 2553 to about 2603, from about
2604 to
about 2654, from about 2655 to about 2705, from about 2706 to about 2756, from
about 2806
to about 2856, from about 2857 to about 2907, from about 2908 to about 2958,
from about
2959 to about 3009, from about 3010 to about 3060, from about 3061 to about
3111, and/or
from about 3112 to about 3152 of the polynucleotide sequence shown in Figures
l0A-H (SEQ
ID N0:60), or the complementary strand thereto, or the cDNA contained in the
deposited clone
(HMSHK47). Other representative examples of polynucleotide fragments of the
invention
2o include, for example, fragments that comprise, or alternatively, consist
of, a sequence from
about nucleotide 1 to about 1451, from about 1761 to about 2251, from about 89
to about 766,
from about 89 to about 487, from about 488 to about 538, from about 539 to
about 766, from
about 92 to about 160, from about 212 to about 243, from about 281 to about
313, from about
314 to about 343, from about 281 to about 343, from about 325 to about 433,
and/or 550 to
about 766 of the polynucleotide sequence shown in Figures l0A-H (SEQ ID
N0:60), or the
complementary strand thereto. 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.
Polynucleotides which hybridize to any 1, 2, 3, 4, 5 or more of these
polynucleotide fragments
are also encompassed by the invention. Moreover, polypeptides encoded by these
polynucleotides and/or polynucleotide fragments are also encompassed by the
invention.
Alternative representative examples of TR14 polynucleotide fragments of the
invention
include, for example, fragments that comprise, or alternatively, consist of, a
sequence from
about nucleotide 1 to about 50, from about 51 to about 108, from about 109 to
about 159, from
about 160 to about 210, from about 211 to about 261, from about 262 to about
273 , from
about 274 to about 324, from about 325 to about 375, from about 376 to about
426, from
about 427 to about 477, from about 478 to about 528, from about 529 to about
579, from
about 580 to about 630, from about 631 to about 681, from about 682 to about
732, from
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
27
about 733 to about 744, from about 745 to about 798, from about 799 to about
849, from
about 850 to about 900, from about 901 to about 951, from about 952 to about
1002, from
about 1003 to about 1053, from about 1054 to about 1104, from about 1105 to
about 1155,
from about 1156 to about 1164, from about 1165 to about 1197, from about 1198
to about
1248, from about 1249 to about 1266, from about 1267 to about 1317, from about
1318 to
about 1368, from about 1369 to about 1419, from about 1420 to about 1470, from
about 1471
to about 1521, from about 1522 to about 1572, from about 1573 to about 1623,
from about
1624 to about 1674, from about 1675 to about 1725, from about 1726 to about
1776, from
about 1777 to about 1827, from about 1828 to about 1878, from about 1879 to
about 1929,
to from about 1930 to about 1980, from about 1981 to about 2031, from about
2032 to about
2082, from about 2083 to about 2133, from about 2134 to about 2184, from about
2185 to
about 2235, from about 2236 to about 2286, from about 2287 to about 2337, from
about 2338
to about 2388, from about 2389 to about 2489, from about 2490 to about 2540,
from about
2451 to about 2501, from about 2502 to about 2552, from about 2553 to about
2603, from
about 2604 to about 2654, from about 2655 to about 2705, from about 2706 to
about 2756,
from about 2806 to about 2856, from about 2857 to about 2907, from about 2908
to about
2958, from about 2959 to about 3009, from about 3010 to about 3060, from about
3061 to
about 3111, from about 3112 to about 3162, from about 3163 to about 3213, from
about 3214
to about 3264, from about 3265 to about 3315, from about 3316 to about 3366,
from about
3367 to about 3417, from about 3418 to about 3468, from about 3469 to about
3519, from
about 3520 to about 3566, from about 3567 to about 3599, from about 3600 to
about 3649,
from about 3650 to about 3699, from about 3700 to about 3749, from about 3750
to about
3799, and/or from 3800 to about 3861 of the polynucleotide sequence shown in
Figures 4A-D
(SEQ ID N0:4), or the complementary strand thereto, or the cDNA contained in
the deposited
clone (HMSHK47). Other representative examples . of polynucleotide fragments
of the
invention include, for example, fragments that comprise, or alternatively,
consist of, a
sequence from about nucleotide 1 to about 1451, from about 1761 to about 2251,
from about
3133 to about 3861, from about 89 to about 766, from about 89 to about 487,
from about 488
to about 538, from about 539 to about 766, from about 92 to about 160, from
about 212 to
3o about 243, from about 281 to about 313, from about 314 to about 343, from
about 281 to
about 343, from about 325 to about 433, and/or 550 to about 766 of the
polynucleotide
sequence shown in Figures 4A-D (SEQ ID N0:4), or the complementary strand
thereto. 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. Polynucleotides
which hybridize to any
1, 2, 3, 4, 5 or more of these polynucleotide fragments are also encompassed
by the invention.
Moreover, polypeptides encoded by these polynucleotides and/or polynucleotide
fragments are
also encompassed by the invention.
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
28
Preferably, the polynucleotide fragments of the invention encode a polypeptide
which
demonstrates a TR14 functional activity. By a polypeptide demonstrating a TR14
"functional
activity" is meant, a polypeptide capable of displaying one or more known
functional activities
associated with a full-length (complete) TR14 protein. Such functional
activities include, but
are not limited to, biological activity, antigenicity (ability to bind (or
compete with a TRI4
polypeptide for binding) to an anti-TR14 antibody), immunogenicity (ability to
generate
antibody which binds to a TR14 polypeptide), ability to form multimers with
TR14
polypeptides of the invention, and ability to bind to a receptor or ligand for
a TR14
polypeptide.
In one embodiment where one is assaying for the ability to bind or compete
with full-
length TR14 polypeptide for binding to anti-TRI4 antibody, various
immunoassays known in
the art can be used, including but not limited to, competitive and non-
competitive assay systems
using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent
assay),
"sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitation
reactions,
immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or
radioisotope
labels, for example), western blots, precipitation reactions, agglutination
assays (e.g., gel
agglutination assays, hemagglutination assays), complement fixation assays,
immunofluorescence assays, protein A assays, and immunoelectrophoresis assays,
etc. In one
embodiment, antibody binding is detected by. detecting a label on the primary
antibody. In
2o another embodiment, the primary antibody is detected by detecting binding
of a secondary
antibody or reagent to the primary antibody. In a further embodiment, the
secondary antibody
is labeled. Many means are known in the art for detecting binding in an
immunoassay and are
within the scope of the present invention.
In another embodiment, where a TRI4 ligand is identified, or the ability of a
polypeptide fragment, variant or derivative of the invention to multimerize is
being evaluated,
binding can be assayed, e.g., by means well-known in the art, such as, for
example, reducing
and non-reducing gel chromatography, protein affinity chromatography, and
affinity blotting.
See generally, Phizicky et al., Microbiol. Rev. 59:94-123 (1995). In another
embodiment,
physiological correlates of TR14 binding to its substrates (signal
transduction) can be assayed.
3o In addition, assays described herein (see, for example, Examples 5 and 15-
21 and 33
and those otherwise known in the art may routinely be applied to measure the
ability of TRI4
polypeptides and fragments, variants derivatives and analogs thereof to elicit
a particular
biological activity (e.g., to inhibit TRAIL induced apoptosis, to regulate
(e.g., inhibit) B cell
proliferation (see, e.g., Example 33), and/or to inhibit hematopoiesis in
vitro or in vivo). For
example, techniques known in the art (such as for example assaying for
thymidine
incorporation), may be applied or,routinely modified to assay for the ability
of the compositions
of the invention to regulate (e.g., inhibit apoptosis) and/or to regulate
(e.g., inhibit)
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
29
proliferation of hematopoietic cells. Additionally, assays desribed herein
(see e.g., Example 15
and Example 33) and otherwise known in the art may be applied or routinely
modified to assay
for the ability of the compositions of the invention to inhibit or stimulate B
cell proliferation.
Other methods will be known to the skilled artisan and are within the scope of
the
invention.
Preferred nucleic acid fragments of the present invention include nucleic acid
molecules
encoding a member selected from the group: a polypeptide comprising or
alternatively,
consisting of any combination of one, two, three or all four TR13 cysteine
rich domains (amino
acid residues from about 105 to about 170, from about 251 to about 264, from
about 331 to
about 410 and from about 580 to about 610 in Figures lA-C (amino acids from
about 105 to
about 170, from about 251 to about 265, from about 331 to about 410 and from
about 580 to
about 610 in SEQ ID NO:1). Since, as discussed above, the location of these
domains have
been predicted by computer analysis, one of ordinary skill would appreciate
that the amino acid
residues constituting these domains may be the particularly recited ranges for
each domain or
t5 may vary slightly (e.g., by about I , 2, 3, 4, 5, 10, or 15 residues at
either extreme or at both
extremes) depending on the criteria used to define each domain.
Additional preferred nucleic acid fragments of the present invention include
nucleic acid
molecules encoding a member selected from the group: a polypeptide comprising
or
alternatively, consisting of the TR13 receptor extracellular domain (amino
acids 1 to 906 in
2o Figures 7A-D); a polypeptide comprising or alternatively, consisting of,
the mature TR13
receptor extracellular domain (amino acids 42 to 906 in Figures 7A-D); a
polypeptide
comprising or alternatively, consisting of, one or more of the TR13 cysteine
rich domains
disclosed in Figures 7A-D (e.g., amino acid residues from about 271 to about
421, from about
271 to about 286, about 290 to about 300, about 301 to about 320, about 329 to
about 361,
25 about 404 to about 421, and from about 585 to about 595 in Figures 7A-D
(amino acid residues
from about 271 to about 421, from about 271 to about 286, about 290 to about
300, about 301
to about 320, about 329 to about 361, about 404 to about 421, and from about
585 to about
595 in SEQ ID N0:39 and SEQ ID N0:40); a polypeptide comprising, or
alternatively,
consisting of the TR13 transmembrane domain (amino acids 907 to 931 in Figures
7A-D); and
30 a polypeptide comprising, or alternatively consisting of the TR13
intracellular domain (amino
acid 932 to 1001 in Figures 7A-D). As above, since the location of these
domains have been
predicted by computer analysis, one of ordinary skill would appreciate that
the amino acid
residues constituting these domains may be the particularly recited ranges for
each domain or
may vary slightly (e.g., by about 1, 2, 3, 4, 5, 10, or 15 residues at either
extreme or at both
35 extremes) depending on the criteria used to define each domain.
It is believed that the cysteine rich motifs of TR13 disclosed in Figures lA-C
are
important for interactions between TR13 and its ligands. Accordingly, specific
embodiments of
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
the invention are directed to polynucleotides encoding polypeptides which
comprise, or
alternatively consist of, the amino acid sequence of amino acid residues from
about 105 to
about 170, from about 251 to about 265, from about 331 to about 410, and from
about 580 to
about 610 of SEQ ID NO:S (corresponding to amino acid residues from about 105
to about
5 170, from about 251 to about 265, from about 331 to about 410, and from
abour 580 to about
610 of Figures 4A-D). In a specific embodiment, the polynucleotides encoding
TR13
polypeptides of the invention comprise or alternatively consist of,
polynucleotide sequences
encoding any combination of 2, 3, or all four of the cysteine-rich motifs of
TR13. In this
context, "about" includes the particularly recited ranges, larger or smaller
by several (5, 4, 3, 2,
10 or 1) nucleotides, at either terminus or at both termini. Polypeptides
encoded by these
polynucleotides are also encompassed by the invention.
Further, specific embodiments of the invention are directed to polynucleotides
encoding
polypeptides which comprise, or alternatively consist of, the amino acid
sequence of amino
acid residues from about 271 to about 421, from about 271 to about 286, from
about 290 to
15 about 300, from about 301 to about 320, about 329 to about 361, about 404
to about 421, and
about 585 to about 595 of SEQ ID N0:40 (corresponding to amino acid residues
from about
271 to about 421, from about 271 to about 286, from about 290 to about 300,
from about 301
to about 320, about 329 to about 361, about 404 to about 421, and about 585 to
about 595 of
Figures 7A-D). In a specific embodiment, the polynucleotides encoding TR13
polypeptides of
2o the invention comprise or alternatively consist of, polynucleotide
sequences encoding any
combination of 2, 3, or all four of the cysteine-rich motifs of TR13 disclosed
in Figures 7A-D.
In this context, "about" includes the particularly recited ranges, larger or
smaller by several (S,
4, 3, 2, or 1) nucleotides, at either terminus or at both termini.
Polypeptides encoded by these
polynucleotides are also encompassed by the invention.
25 Preferred nucleic acid fragments of the invention encode a full-length TR13
polypeptide
lacking the nucleotides encoding the amino terminal methionine (e.g.,
nucleotides 34-750 in
SEQ ID NO:1), as it is known that the methionine is cleaved naturally and such
sequences may
be useful in genetically engineering TR13 expression vectors. Polypeptides
encoded by such
nucleic acids are also contemplated by the invention.
3o Preferred nucleic acid fragments of the invention encode a full-length TR13
polypeptide
lacking the nucleotides encoding the amino terminal methionine (e.g.,
nucleotides 61-1001 in
Figures 7A-D and SEQ ID N0:39), as it is known that the methionine is cleaved
naturally and
such sequences may be useful in genetically engineering TR13 expression
vectors.
Polypeptides encoded by such nucleic acids are also contemplated by the
invention.
Preferred nucleic acid fragments of the present invention further include
nucleic acid
molecules encoding epitope-bearing portions of the TR13 receptor protein. In
particular, such
nucleic acid fragments of the present invention include nucleic acid molecules
encoding: a
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
31
polypeptide comprising or alternatively consisting of, amino acid residues
from about 1 to
about 170 in Figures lA-C (corresponding to about amino acid 1 to about 170 in
SEQ 117
N0:2); a polypeptide comprising or alternatively consisting of, amino acid
residues from about
210 to about 318 in Figures lA-C (corresponding to about amino acid 210 to
about 318 in SEQ
ID N0:2); a polypeptide comprising or alternatively consisting of, amino acid
residues from
about 343 to about 480 in Figures lA-C (corresponding to about amino acid 343
to about 480
in SEQ ID N0:2); a polypeptide comprising or alternatively consisting of,
amino acid residues
from about 548 to about 592 in Figures lA-C (corresponding to about amino acid
548 to about
592 in SEQ ID N0:2); and a polypeptide comprising or alternatively consisting
of, amino acid
to residues from about 632 to about 742 in Figures lA-C (corresponding to
about amino acid 632
to about 742 in SEQ ID N0:2). The inventors have determined that the above
polypeptide
fragments are antigenic regions of the TR13 protein. Methods for determining
other such
epitope-bearing portions of the TR13 protein are described in detail below.
Preferred nucleic acid fragments of the present invention further include
nucleic acid
molecules encoding antigenic fragments of the TR13 receptor protein. In
particular, such
nucleic acid fragments of the present invention include nucleic acid molecules
encoding: a
polypeptide comprising or alternatively consisting of, amino acid residues
from about M1 to
about A9, about K12 to about L20, about N47 to about T55, about H58 to about
S66, about
D63 to about S71, about P77 to about F85, about A90 to about Q98, about F136
to about
Q 144, about S 152 to about C 160, about 8159 to about A 167, about A211 to
about M219,
about M235 to about V243, about V266 to about V274, about W277 to about S285,
about I290
to about F298, about A310 to about V318, about E343 to about C351, about I360
to about
H368, about 6391 to about I399, about F409 to about T417, about S436 to about
Y444, about
C453 to about S461, about I472 to about S480, about Y548 to about S556, about
C557 to
about I565, about V567 to about V575, about T584 to about 6592, about 8632 to
about
6640, about W680 to about Y688, about Q684 to about K692, about T698 to about
A706,
about S726 to about S734, and about S734 to about L742 of SEQ ID N0:2 (Figures
lA-C)
correspond to the highly antigenic regions of the TR13 protein, predicted
using the Jameson-
Wolf antigenic index (See Figure 3 and Table I). These highly antigenic
fragments correspond
to the amino acid residues illustrated in Figure lA-C and in SEQ ID N0:2. In
this context,
"about" includes the particularly recited ranges, larger or smaller by several
(5, 4, 3, 2, or 1)
nucleotides, at either terminus or at both termini. Methods for determining
other such antigenic
fragments of the TR13 protein are described in detail below.
Additional preferred nucleic acid fragments of the present invention further
include
nucleic acid fragments encoding: a polypeptide comprising or alternatively
consisting of, amino
acid residues from about 1 to about 262 in Figures 7A-D (corresponding to
about amino acid 1
to about 262 in SEQ ID N0:40); a polypeptide comprising or alternatively
consisting of, amino
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
32
acid residues from about 264 to about 423 in Figures 7A-D (corresponding to
about amino acid
264 to about 423 in SEQ ID N0:40); a polypeptide comprising or alternatively
consisting of,
amino acid residues from about 437 to about 789 in Figures 7A-D (corresponding
to about
amino acid 437 to about 789 in SEQ ID N0:40); and a polypeptide comprising or
alternatively
consisting of, amino acid residues from about 791 to about 1001 in Figures 7A-
D
(corresponding to about amino acid 791 to about 1001 in SEQ ID N0:40). The
inventors have
determined that the above polypeptide fragments are antigenic regions of the
TR13 protein.
Methods for determining other such epitope-bearing portions of the TR13
protein are described
in detail below.
Additional preferred nucleic acid fragments of the present invention encoding
antigenic
fragments of the TR13 receptor protein include nucleic acid molecules
encoding: a polypeptide
comprising or alternatively consisting of, amino acid residues from about M1
to about H9,
about V 14 to about I22, about H47 to about H55, about C61 to about R69, about
L82 to about
E90, about D102 to about P110, about K109 to about S117, about F124 to about
H132, about
M141 to about E149, about 5146 to about C154, about S157 to about W165, about
F168 to
about T 176, about N 182 to about N 190, about Q207 to about A215, about P213
to about
M221, about M221 to about E229, about V233 to about V241, about T253 to about
V261,
about T282 to about S290, about N298 to about T306, about C308 to about Y316,
about K315
to about S323, about P328 to about F336, about A341 to about Q349, about F387
to about
Q395, about S403 to about C411, about T409 to about P417, about F443 to about
N451,
about W451 to about Y459, about A462 to about M470, about 6478 to about M486,
about
A487 to about A495, about V517 to about V525, about T527 to about Q535, about
I541 to
about F549, about A561 to about V569, about E594 to about C602, about I611 to
about H619,
about 6643 to about I650, about P686 to about K694, about C704 to about S712,
about 8722
to about I730, about E727 to about T735, about P746 to about 6754, about D776
to about
L784, about Y799 to about S807, about C808 to about I816, about V818 to about
V826, about
T835 to about 6843, about 8883 to about 6891, about K932 to about K940, about
Q935 to
about K943, about T949 to about A957, about S977 to about S985, about S981 to
about P989,
and about N986 to about L994 of SEQ ID N0:40 (Figures 7A-D) correspond to the
highly
antigenic regions of the TR13 protein, predicted using the Jameson-Wolf
antigenic index (See
Figure 9 and Table III). These highly antigenic fragments correspond to the
amino acid
residues illustrated in Figure 7A-D and in SEQ ID N0:40. 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. Methods for determining other such antigenic
fragments of the
TR13 protein are described in detail below.
Additionally, it is believed that the extracellular cysteine rich motif of
TR14 disclosed in
Figures l0A-H or, alternatively, Figures 4A-D is important for interactions
between TR14 and
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
33
its ligands. Accordingly, specific embodiments of the invention are directed
to nucleic acid
molecules encoding polypeptides which comprise, or alternatively consist of,
preferably amino
acids Cys-31 to Cys-104 of Figures l0A-B and SEQ ID N0:61, or, alternatively,
the amino
acid sequence of amino acid residues from about 70 to about 90 of Figure 10A
and SEQ 1D
N0:61 (corresponding to amino acid residues from about 65 to about 85 of
Figures 4A-D or
SEQ ID NO:S). 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.
Polypeptides encoded by these polynucleotides are also encompassed by the
invention.
Preferred nucleic acid fragments of the present invention include nucleic acid
molecules
encoding a member selected from the group: a polypeptide comprising or
alternatively,
consisting of, the TR14 receptor extracellular domain (preferably amino acid
residues from
about 1 to 138 in Figures l0A-H or, alternatively, from about 1 to about 133
in Figures 4A-D);
a polypeptide comprising or alternatively, consisting of, the TR14 cysteine
rich domain
(preferably amino acid residues from about 31 to about 104 of Figures l0A-H,
or amino acid
residues from about 70 to 90 in Figures 10A, or, alternatively, from about 65
to about 85 in
Figures 4A-D); a polypeptide comprising or alternatively, consisting of the
TR14
transmembrane domain (preferably amino acid residues from about 139 to 155 in
Figures l0A-
H or, alternatively, 134 to about 150 in Figures 4A-D); and a polypeptide
comprising or
alternatively, consisting of, the TR14 intracellular domain (preferably amino
acid residues
from about 156 to about 231 in Figures l0A-H or, alternatively, amino acid
residues from
about 151 to about 226 in Figures 4A-D). Since the location of these domains
have been
predicted by computer analysis, one of ordinary skill would appreciate that
the amino acid
residues constituting these domains may be the particularly recited ranges for
each domain or
may vary slightly (e.g., by about 1, 2, 3, 4, 5, 10, or 15 residues at either
extreme or at both
extremes) depending on the criteria used to define each domain.
Preferred nucleic acid fragments of the invention encode a full-length TR14
polypeptide
lacking the nucleotides encoding the amino terminal methionine (e.g.,
nucleotides 70-759 of
Figures l0A-H or SEQ ID N0:60, or nucleotides 102-765 in SEQ ID N0:4), as it
is known
that the methionine is cleaved naturally and such sequences may be useful in
genetically
3o engineering TR14 expression vectors. Polypeptides encoded by such nucleic
acids are also
contemplated by the invention.
Preferred nucleic acid fragments of the present invention further include
nucleic acid
molecules encoding epitope-bearing portions of the TR14 receptor protein. In
particular,
preferred epitope-bearing polypeptides of the present invention comprise, or
alternatively
consist of, one, two, three, four, five, six, or all six of the immunogenic
epitopes of the TR14
protein shown in SEQ ID NO: 61 as residues: Asp-2 to Asp-10, Thr-17 to Asp-38,
Pro-45 to
Ser-52, Pro-88 to Arg-95, Thr-108 to Glu-115, Thr-131 to Glu-136, Phe-166 to
Gly-174,
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
34
Ala-180 to Ala-200, and Gln-224 to Met-231. Fragments and/or variants of these
polypeptides,
such as, for example, fragments and/or variants as described herein, are
encompassed by the
invention. Polynucleotides encoding these polypeptides (including fragments
and/or variants)
are also encompassed by the invention, as are antibodies that bind these
polypeptides.
Alternatively, such nucleic acid fragments of the present invention include
nucleic acid
molecules encoding: a polypeptide comprising or alternatively consisting of,
amino acid
residues from about 2 to about 24 in Figures 4A-D (corresponding to about
amino acid 2 to
about 24 in SEQ ID N0:5); a polypeptide comprising or alternatively consisting
of, amino acid
residues from about 42 to about 52 in Figures 4A-D (corresponding to about
amino acid 42 to
about 52 in SEQ ID NO:S); a polypeptide comprising or alternatively consisting
of, amino acid
residues from about 80 to about 115 in Figures 4A-D (corresponding to about
amino acid 80 to
about 115 in SEQ ID NO:S and about amino acid 85 to about 120 of SEQ ID
N0:61); and a
polypeptide comprising or alternatively consisting of, amino acid residues
from about 155 to
about 226 in Figures 4A-D (corresponding to about amino acid 155 to about 226
in SEQ ID
N0:5 and about amino acid 160 to about amino acid 231 of SEQ ID N0:61). The
inventors
have determined that the above polypeptide fragments are antigenic regions of
the TR14
protein. Methods for determining other such epitope-bearing portions of the
TR14 protein are
described in detail below.
Alternative nucleic acid fragments of the present invention further include
nucleic acid
molecules encoding antigenic fragments of the TR14 receptor protein. In
particular, such
nucleic acid fragments of the present invention include nucleic acid molecules
encoding: a
polypeptide comprising or alternatively consisting of, amino acid residues of
SEQ ID N0:5
(Figures 4A-D) from about T3 to about S 11, from about V 16 to about R24, from
about Q44 to
about M52, from about F85 to about G93 (about F90 to about G98 of SEQ ID
N0:61), from
about T103 to about V 111 (about T108 to about V 116 of SEQ ID N0:61), from
about F161 to
about 6169 (about F165 to about 6174 of SEQ ID N0:61 ), from about V 187 to
about A 195
(from about V 192 to about A200 of SEQ ID N0:61 ), from about P218 to about
M226 (about
P223 to about M231 of SEQ ID N0:61) correspond to the highly antigenic regions
of the TR14
protein, predicted using the Jameson-Wolf antigenic index (See Figure 11 and
Table IV).
3o These highly antigenic fragments correspond to the amino acid residues
illustrated in Figure
4A-D and in SEQ ID NO:S (or Figures l0A-H and SEQ ID N0:61, as indicated
above). 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. Methods for
determining other such
antigenic fragments of the TR14 protein are described in detail below.
The data presented in Figure 3 are also represented in tabular form in Table
I. The columns in
Table I are labeled with the headings "Res", "Position", and Roman Numerals I-
XIV. The column
headings refer to the following features of the amino acid sequence presented
in Figure 3 and Table I:
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
"Res": amino acid residue of SEQ ID N0:2 and Figures IA-1C; "Position":
position of the corresponding
residue within SEQ ID N0:2 and Figures IA-C; I: Alpha, Regions - Gamier-
Robson; II: Alpha, Regions
Chou-Fasman; III: Beta, Regions - Gamier-Robson; IV: Beta, Regions - Chou-
Fasman; V: Turn, Regiot
- Gamier-Robson; VI: Turn, Regions - Chou-Fasman; VII: Coil, Regions - Gamier-
Robson; VIII:
5 Hydrophilicity Plot - Kyte-Doolittle; IX: Hydrophobicity Plot - Hopp-Woods;
X: Alpha, Amphipathic
Regions - Eisenberg; XI: Beta, Amphipathic Regions - Eisenberg; XII: Flexible
Regions - Karplus-Schul
XIII: Antigenic Index - Jameson-Wolf; and XIV: Surface Probability Plot -
Emini.
The data presented in Figure 9 are also represented in tabular form in Table
III. The columns in
Table III are labeled with the headings "Res", "Position", and Roman Numerals
I-XIV . The column
t o headings refer to the following features of the amino acid sequence
presented in Figure 9 and Table III:
"Res": amino acid residue of SEQ ID N0:40 and Figures 7A-D; "Position":
position of the corresponding
residue within SEQ ID N0:40 and Figures 7A-D; I: Alpha, Regions - Garnier-
Robson; II: Alpha, Regior
- Chou-Fasman; III: Beta, Regions - Gamier-Robson; IV: Beta, Regions - Chou-
Fasman; V: Turn,
Regions - Garnier-Robson; VI: Turn, Regions - Chou-Fasman; VII: Coil, Regions -
Gamier-Robson;
15 VIII: Hydrophilicity Plot - Kyte-Doolittle; IX: Hydrophobicity Plot - Hopp-
Woods; X: Alpha,
Amphipathic Regions - Eisenberg; XI: Beta, Amphipathic Regions - Eisenberg;
XII: Flexible Regions -
Karplus-Schulz; XIII: Antigenic Index - Jameson-Wolf; and XIV: Surface
Probability Plot - Emini..
In additional embodiments, the nucleic acid molecule of the invention encodes
a
polypeptide comprising, or alternatively consisting of, a functional attribute
of TR13.
20 Preferred embodiments of the invention in this regard include fragments
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, flexible regions, surface-forming regions and high
antigenic index
25 regions of TR13.
The data representing the structural or functional attributes of TR13 (SEQ ID
N0:40)
set forth in Figure 9 and/or Table III, as described above, was generated
using the various
modules and algorithms of the DNA*STAR set on default parameters. In a
preferred
embodiment, the data presented in columns VIII, IX, XIII, and XIV of Table III
can be used to
30 determine regions of TR13 which exhibit a high degree of potential for
antigenicity. Regions
of high antigenicity are determined from the data presented in columns VIII,
IX, XIII, andlor
XIV by choosing values which represent regions of the polypeptide which are
likely to be
exposed on the surface of the polypeptide in an environment in which antigen
recognition may
occur in the process of initiation of an immune response.
35 Certain preferred regions in these regards are set out in Figure 3, but
may, as shown in
Table I, be represented or identified by using tabular representations of the
data presented in
Figure 3. The DNA*STAR computer algorithm used to generate Figure 3 (set on
the original
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
36
default parameters) was used to present the data in Figure 3 in a tabular
format (See Table I).
The tabular format of the data in Figure 3 may be used to easily determine
specific boundaries
of a preferred region.
Certain preferred regions in these regards are set out in Figure 9, but may,
as shown in
Table III, be represented or identified by using tabular representations of
the data presented in
Figure 9. The DNA*STAR computer algorithm used to generate Figure 9 (set on
the original
default parameters) was used to present the data in Figure 9, in a tabular
format (See Table I11).
The tabular format of the data in Figure 9 may be used to easily determine
specific boundaries
of a preferred region.
l0 The above-mentioned preferred regions set out in Figure 3 and in Table I
include, but
are not limited to, regions of the aforementioned types identified by analysis
of the amino acid
sequence set out in Figure 1. As set out in Figure 3 and in Table I, 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 and
~5 Hopp-Woods hydrophobic regions, Eisenberg alpha- and beta-amphipathic
regions, Karplus-
Schulz flexible regions, Jameson-Wolf regions of high antigenic index and
Emini surface-
forming regions.
The above-mentioned preferred regions set out in Figure 9 and in Table III
include, but
are not limited to, regions of the aforementioned types identified by analysis
of the amino acid
20 sequence set out in Figure 9. As set out in Figure 9 and in Table III, 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 and
Hopp-Woods hydrophobic regions, Eisenberg alpha- and beta-amphipathic regions,
Karplus-
Schulz flexible regions, Jameson-Wolf regions of high antigenic index and
Emini surface-
25 forming regions.
The data presented in Figure 11 are also represented in tabular form in Table
IV. The columns in
Table IV are labeled with the headings "Res", "Pos", and Roman Numerals I-XIV.
The column heading
refer to the following features of the amino acid sequence presented in
Figures l0A-H, and 11: "Res":
amino acid residue of SEQ ID N0:61 and Figures l0A-H; "Pos": position of the
corresponding residue
30 within SEQ ID N0:61 and Figures I OA-H; I: Alpha, Regions - Gamier-Robson;
II: Alpha, Regions -
Chou-Fasman; III: Beta, Regions - Gamier-Robson; IV: Beta, Regions - Chou-
Fasman; V: Turn, Region
- Garnier-Robson; VI: Turn, Regions - Chou-Fasman; VII: Coil, Regions - Gamier-
Robson; VIII:
Hydrophilicity Plot - Kyte-Doolittle; IX: Hydrophobicity Plot - Hopp-Woods; X:
Alpha, Amphipathic
Regions - Eisenberg; XI: Beta, Amphipathic Regions - Eisenberg; XII: Flexible
Regions - Karplus-Schul
35 XIII: Antigenic Index - Jameson-Wolf; and XIV: Surface Probability Plot -
Emini.
The data presented in Figure 6 are also represented in tabular form in Table
II. As above, the
columns in Table II are labeled with the headings "Res", "Position", and Roman
Numerals I-XIV. The
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
37
column headings refer to the following features of the amino acid sequence
presented in Figure 6 and
Table II: "Res": amino acid residue of SEQ ID NO:S and Figures 4A-D;
"Position": position of the
corresponding residue within SEQ ID NO:S and Figures 4A-D; I: Alpha, Regions -
Gamier-Robson; II:
Alpha, Regions - Chou-Fasman; III: Beta, Regions - Gamier-Robson; IV: Beta,
Regions - Chou-Fasmar
V: Turn, Regions - Gamier-Robson; VI: Turn, Regions - Chou-Fasman; VII: Coil,
Regions - Garnier-
Robson; VIII: Hydrophilicity Plot - Kyte-Doolittle; IX: Hydrophobicity Plot -
Hopp-Woods; X: Alpha,
Amphipathic Regions - Eisenberg; XI: Beta, Amphipathic Regions - Eisenberg;
XII: Flexible Regions -
Karplus-Schulz; XIII: Antigenic Index - Jameson-Wolf; and XIV: Surface
Probability Plot - Emini.
In additional embodiments, the polynucleotides of the invention encode
functional
l0 attributes of TR14. Preferred embodiments of the invention in this regard
include fragments
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, flexible regions, surface-
forming regions and
high antigenic index regions of TR14.
The data representing the structural or functional attributes of TR13 (SEQ ID
N0:2) set
forth in Figure 3 and/or Table I, as described above, was generated using the
various modules
and algorithms of the DNA*STAR set on default parameters. In a preferred
embodiment, the
data presented in columns VIII, IX, XIII, and XIV of Table I can be used to
determine regions
2o of TR13 which exhibit a high degree of potential for antigenicity. Regions
of high antigenicity
are determined from the data presented in columns VIII, IX, XIII, and/or XIV
by choosing
values which represent regions of the polypeptide which are likely to be
exposed on the surface
of the polypeptide in an environment in which antigen recognition may occur in
the process of
initiation of an immune response.
The data representing the structural or functional attributes of TR 14 (SEQ ID
N0:61, as
set forth in Figure 11 and/or Table IV; or, alternatively, SEQ ID N0:5, as set
forth in Figure 6
and/or Table II), as described above, were generated using the various modules
and algorithms
of the DNA*STAR set on default parameters. In a preferred embodiment, the data
presented in
columns VIII, IX, XIII, and XIV of Table II can be used to determine regions
of TR14 which
exhibit a high degree of potential for antigenicity. Regions of high
antigenicity are determined
from the data presented in columns VIII, 1X, XIII, and/or XIV by choosing
values which
represent regions of the polypeptide which are likely to be exposed on the
surface of the
polypeptide in an environment in which antigen recognition may occur in the
process of
initiation of an immune response.
Certain preferred regions in these regards are set out in Figure 6, but may,
as shown in
Table II, be represented or identified by using tabular representations of the
data presented in
Figure 6. The DNA*STAR computer algorithm used to generate Figure 6 (set on
the original
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
38
default parameters) was used to present the data in Figure 6 in a tabular
format (See Table II).
The tabular format of the data in Figure 6 may be used to easily determine
specific boundaries
of a preferred region.
Certain even more preferred regions in these regards are set out in Figure 11,
but may,
as shown in Table IV, be represented or identified by using tabular
representations of the data
presented in Figure 11. The DNA*STAR computer algorithm used to generate
Figure 11 (set
on the original default parameters) was used to present the data in Figure 11
in a tabular format
(See Table IV). The tabular format of the data in Figure 11 may be used to
easily determine
specific boundaries of a preferred region.
l0 The above-mentioned preferred regions set out in Figure 11 and in Table IV
include,
but are not limited to, regions of the aforementioned types identified by
analysis of the amino
acid sequence set out in Figures l0A-H. As set out in Figure 11 and in Table
IV, 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 and Hopp-Woods hydrophobic regions, Eisenberg alpha- and
beta-
amphipathic regions, Karplus-Schulz flexible regions, Jameson-Wolf regions of
high antigenic
index and Emini surface-forming regions.
The above-mentioned preferred regions set out in Figure 6 and in Table II
include, but
are not limited to, regions of the aforementioned types identified by analysis
of the amino acid
2o sequence set out in Figures 4A-D. As set out in Figure 6 and in Table II,
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 and Hopp-Woods hydrophobic regions, Eisenberg alpha- and beta-
amphipathic
regions, Karplus-Schulz flexible regions, Jameson-Wolf regions of high
antigenic index and
Emini surface-forming regions.
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
39
Table
I
Res Pos II III IV V VI VII VIII IX X XI XII XIII XIV
I
Met 1 . . B . . . . 0.73 -0.31. . . 1.40 1.44
Asp 2 . . . . T T . 1.12 -0.26. . . 2.25 1.62
Gln 3 . . . . T T . 0.92 -0.29* . . 2.50 2.20
Ser 4 . . . . . T C 0.64 -0.21. . . 2.05 2.24
Thr 5 . . B . . T . 0.44 -0.26. . F 1.60 0.72
Gln 6 . A B . . . . 0.70 0.24. . F 0.35 0.42
Ala 7 . A B . . . . 0.70 0.27. . . -0.050.31
Cys 8 . A . . T . . 0.74 -0.11. . . 0.70 0.37
Ala 9 . A . . T . . 1.01 -0.60. . . 1.00 0.43
Gly 10 . A . . T . . 0.66 -0.50. . F 0.85 0.58
Glu 11 . A . . T . . 0.62 -0.43. . F 0.85 0.58
Lys 12 . A . . T . . 1.21 -0.50. . F 1.10 0.78
His 13 . A . . T . . 1.99 -0.60. * . 1.65 1.27
Cys 14 . A . . T . . 2.23 -1.03. . . 1.90 1.43
His 15 . . . . T . . 2.23 -0.60. * . 2.20 0.71
Asn 16 . . . . T T . 1.42 -0.17. * F 2.50 0.52
Arg 17 . . . . T T . 1.34 0.01* * F 1.65 0.79
Gly 18 . . . . T T . 0.68 -0.06* * F 2.00 0.79
Gly 19 . . . . T T . 1.46 0.23* * F 1.15 0.43
Leu 20 . A . . . . C 0.89 -0.17* * . 0.75 0.43
His 21 . A B . . . . 0.08 0.44* * . -0.600.43
Phe 22 . A B . . . . -0.240.70. * . -0.600.36
Arg 23 . A B . . . . -0.710.70. * . -0.600.67
Met 24 . A B . . . . -0.370.70. * . -0.600.40
Leu 25 . A B . . . . 0.13 0.60. * . -0.600.81
Pro 26 . A . . . . C -0.120.30. * . -0.100.60
Leu 27 . . . B T . . 0.54 1.21* . . -0.200.63
Gln 28 . . . B T . . -0.421.10. . . -0.051.05
Thr 29 . . . B T . . -0.491.06* . . -0.200.50
Trp 30 . . B B . . . 0.43 1.20* . . -0.600.33
His 31 . . B B . . . 0.64 0.51* . . -0.600.37
Val 32 . . B B . . . 0.87 0.51* . . -0.600.44
Cys 33 . . B B . . . 0.52 0.53* . . -0.600.43
Arg 34 . . B B . . . 0.02 0.04* . . -0.300.31
Gln 35 . . . B T . . -0.500.23* . . 0.10 0.34
Ala 36 . . . B T . . -1.170.27* . . 0.10 0.53
Gly 37 . . . B T . . -1.120.49* . . -0.200.23
Leu 38 . . B B . . . -0.461.17* . . -0.600.11
Leu 39 . . B B . . . -0.881.17* . . -0.600.19
Phe 40 . . B B . . . -1.691.16* . . -0.600.28
Leu 41 . . B B . . . -1.311.41* . . -0.600.28
Gln 42 . . B B . . . -1.271.16. . . -0.600.52
Thr 43 . . B B . . . -0.460.86. . F -0.450.81
Leu 44 . . B B . . . 0.06 0.47. . F -0.301.57
Pro 45 . . . . . T C 0.51 0.17* . F 0.60 1.22
Ser 46 . . . . T T . 1.02 0.53. . F 0.50 1.32
Asn 47 . . . . T T . 1.02 0.43. . F 0.84 2.15
Ser 48 . . . . . T C 1.38 0.14. . F 1.28 2.24
Tyr 49 . . . . T . . 1.84 -0.29. . F 2.22 3.34
Ser 50 . . . . . . C 2.06 -0.24. . F 2.36 2.05
Asn 51 . . . . T T . 2.04 -0.64. . F 3.40 2.65
Lys 52 . . . . T T . 1.74 -0.54. . F 3.06 2.44
Gly 53 . . . . T T . 1.38 -0.91. * F 2.72 2.44
Glu 54 . . . . T T . 1.59 -0.73* * F 2.23 0.81
Thr 55 . . . . T T . 1.89 -0.63* . F 1.89 0.55
Ser 56 . . B . . T . 1.22 -0.23* . F 0.85 0.97
Cys 57 . . B . . T . 1.18 -0.09. * . 1.04 0.30
His 58 . . B . . T . 1.31 -0.09. * . 1.38 0.35
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
Gln 59 . . . . T . . 1.31 -0.14 . . 1.920.40
.
Cys 60 . . . . T . . 1.67 -0.53 . . 2.711.25
.
Asp 61 . . . . T T . 1 -1.10 . F 3.401.84
.72 .
Pro 62 . . . . T T . 2.09 -0.84 . F 3.061.66
.
5 Asp 63 . . . . T T . 2.12 -0.86 . F 3.064.15
.
Lys 64 . . . . T T . 2.17 -1.43 * F 3.064.31
*
Tyr 65 . . B . . T . 2.49 -1.43 . F 2.665.57
*
Ser 66 . . B . . T . 2.19 -1.43 . F 2.663.30
*
Glu 67 . . . . T T . 2.10 -1.04 . F 3.402.21
*
10 Lys 68 . . . . T T . 1.80 -0.66 * F 3.061.89
*
Gly 69 . . . . T . . 1.09 -1.03 * F 2.52I
* .89
Ser 70 . . . . T T . 1.33 -0.84 * F 2.230.59
.
Ser 71 . . . . T T . 0.78 -0.44 * F 1.590.47
.
Ser 72 . . . . T T . 0.89 0.20 * F 0.650.35
.
15 Cys 73 . . . . T T . 0.63 -0.23 * F 1.250.52
.
Asn 74 . . . . T . . 0.39 -0.19 * . 0.900.60
.
Val 75 . . B . . . . 0.02 -0.07 * . 0.500.45
*
Arg 76 . . B . . T . 0.01 0.11 * . 0.100.45
.
Pro 77 . . B . . T . 0.31 0.03 * . 0.440.40
.
20 Ala 78 . . B . . T . 1.02 -0.37 * . 1.380.91
.
Cys 79 . . B . . T . 1.02 -1.01 * . 2.020.93
.
Thr 80 . . B . . . . 1.63 -1.01 * F 2.311.00
*
Asp 81 . . . . T T . 0.82 -0.69 . F 3.401.55
*
Lys 82 . . . . T T . 0.79 -0.40 . F 2.762.50
.
25 Asp 83 . . . . T T . 1.07 -0.21 . F 2.422.72
.
Tyr 84 . . B . . T . 1.70 -0.21 . . 1.532.35
.
Phe 85 . . B B . . . I.70 0.29 . . 0.191.60
.
Tyr 86 . . B B . . . 1.11 0.77 . . -0.451.38
.
Thr 87 . . B B . . . 0.40 1.27 . . -0.600.89
.
30 His 88 . . B B . . . 0.40 1.09 . . -0.600.55
.
Thr 89 . . B B . . . 0.06 0.30 . . -0.300..59
.
Ala 90 . . B B . . . 0.76 0.04 * . 0.000.41
.
Cys 91 . . . B T . . 0.66 -0.04 * . 1.300.49
.
Asp 92 . . . . T T . 0.97 -0.11 * . 2.000.33
.
35 Ala 93 . . . . . T C 0.69 -0.60 * F 2.550.57
.
Asn 94 . . . . . T C 1.00 -0.61 * F 3.001.54
.
Gly 95 . . . . . T C 0.78 -0.79 * F 2.701.60
.
Glu 96 . A . . . . C 0.84 -0.10 * F 1.701.30
.
Thr 97 . A B . . . . 0.60 0.01 * F 0.450.80
.
40 Gln 98 . A B . . . . 1.23 0.37 * F 0.301.27
*
Leu 99 . A B . . . . 0.94 -0.06 * . 0.451.47
*
Met 100. A B . . . . 0.70 0.86 * . -0.451.07
*
Tyr 101. A B . . . . 0.74 0.87 * . -0.600.62
*
Lys 102. A . . T . . 0.84 0.47 * . -0.051.51
*
Trp 103. A . . T . . 0.89 0.21 * . 0.252.36
*
Ala 104. A . . . . C 0.81 -0.40 * F 0.803.01
.
Lys 105. A . . . . C 0.74 -0.47 * F 0.801.06
*
Pro 106. A . . T . . 0.69 0.10 * F 0.250.54
.
Lys 107. . . . T . . 0.64 -0.43 * F 1.050.71
.
Ile 108. . B . . . . 0.93 -0.93 * . 0.800.62
.
Cys 109. . B . . T . 0.71 -0.93 * . 1.000.67
.
Ser I10. . B . . T . 0.67 -0.67 * F 1.150.28
.
Glu 111. . B . . T . 0.53 -0.67 . F 1.150.68
*
Asp 112A . . . . T . -0.10-0.93 * F 1.301.26
*
Leu 1 A A . . . . . -0.07-1.00 * F 0.750.95
13 *
Glu 114A A . . . . . 0.64 -0.74 * F 0.750.41
*
Gly 115A A . . . . . 0.13 -0.74 * F 0.750.49
*
Ala 116. A B . . . . -0.08-0.06 * . 0.300.49
*
Val 117. A B . . . . -0.67-0.31 * . 0.300.43
*
Lys 118. A B . . . . -0.160.19 * . -0.300.44
*
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
41
Leu 119 A B . . . -0.500.14* * . -0.300.59
. .
Pro 120 . B . T . -1.010.07* * . 0.10 0.78
. .
Ala 121 . . T T . -0.380.07* * F 0.65 0.29
. .
Ser 122 . . T T . 0.17 0.07* * F 0.65 0.70
. .
Gly 123 . . T T . 0.09 -0.13* * F 1.25 0.66
. .
Val 124 . B . . . 0.23 -0.06. . F 0.79 0.89
. .
Lys 125 . B . . . 0.23 O.OI. . F 0.33 0.35
. .
Thr 126 . B . . . 0.61 0.06. . F 0.47 0.55
. .
His 127 . B . . . 0.24 0.06* . . 0.61 1.15
. .
Cys 128 . B . T . 0.59 -0.01. . . 1.40 0.31
. .
Pro 129 . B . T . 1.23 0.39* * F 0.81 0.34
. .
Pro 130 . . T T . 0.84 0.33. . F 1.07 0.39
. .
Cys 131 . . T T . 0.46 0.26. . F 0.93 0.72
. .
Asn 132 . . . T C -0.210.47. . F 0.29 0.40
. .
Pro 133 . . T T . 0.50 0.83. . F 0.35 0.23
. .
Gly 134 . . T T . 0.40 0.40. . . 0.20 0.85
. .
Phe 135 . B . T . 0.61 0.31. . . 0.10 0.76
. .
Phe 136 . B . . . 1.28 0.31. . F 0.30 0.79
. .
Lys 137 . . T . . 0.98 0.29. . F 1.10 1.28
. .
Thr 138 . . T . . 0.88 0.24. . F 1.35 1.99
. .
Asn 139 . . T . . 0.56 -0.06* . F 2.20 3.31
. .
Asn 140 . . T T . 1.26 -0.27* . F 2.50 0.89
. .
Ser 141 . . T T . 1.74 0.13* * F 1.80 1.06
. .
Thr 142 . . T T . 1.03 0.07. . F 1.55 1.02
. .
Cys 143 . . T T . 1.13 0.24. . F 1.15 0.34
. .
Gln 144 . B . . . 0.89 0.27. . F 0.30 0.39
. .
Pro 145 . B . . . 0.54 0.64. . F -0.250.43
. .
Cys 146 . B . T . 0.54 0.59. . . -0.200.79
. .
Pro 147 . B . T . 0.61 0.40. . . -0.200.61
. .
Tyr 148 . . T T . 0.98 0.76. . . 0.20 0.62
. .
Gly 149 . . T T . 0.98 0.71. . . 0.35 1.55
. .
Ser 150 . . T . . 0.84 0.54. . F 0.30 1.61
. .
Tyr 151 . . T 'f . 1.21 0.54. . F 0.50 1.02
. .
Ser 152 . . T T . 1.42 0.17. . F 1.11 1.38
. .
Asn 153 . . T T . 1.00 -0.26. . F 2.02 1.71
. .
Gly 154 . . T T . 1.03 -0.07* . F 2.18 0.59
. .
Ser 155 . . T T . 1.44 -0.34* . F 2.49 0.63
. .
Asp 156 . . T T . 1.02 -0.73* . F 3.10 0.77
. .
Cys 157 . B . T . 1.11 -0.56* * F 2.39 0.42
. .
Thr 158 . B . T . 0.52 -0.56* * F 2.39 0.48
. .
Arg 159 . B . . . 0.52 -0.44* . F 1.89 0.29
. .
Cys 160 . B . T . 0.51 -0.01. . . 1.94 0.54
. .
Pro 161 . . T 'f . 0.51 -0.10. . . 2.34 0.54
. .
Ala 162 . . T T . 0.97 -0.59. * F 3.10 0.47
. .
Gly 163 . . . T C 0.69 -0.16* * F 2.44 1.37
. .
Thr 164 . . . . C -0.28-0.23. * F 1.78 0.89
. .
Glu 165 . B . . . 0.04 -0.01. . F 1.27 0.66
. .
Pro 166 . B . . . -0.44-0.09. * F 0.96 0.66
. .
Ala 167 . B . . . 0.14 0.27. * . -0.100.39
. .
Val 168 . B . . . 0.24 -0.21. * . 0.50 0.39
. .
Gly 169 . B . . . 0.60 0.54. * . -0.400.40
. .
Phe 170 A B . . . 0.31 0.11. * . -0.300.79
. .
Glu 171 A B . . . 0.23 0.53. * . -0.451.12
. .
Tyr 172 A . T . . 0.82 0.80* * . -0.051.19
. .
Lys 173 A . T . . 1.37 0.77* * . -0.052.21
. .
Trp 174 A . 'f . . 0.90 0.47* * . -0.051.84
. .
Trp 175 A . T . . 1.39 1.16* * . -0.200.97
. .
Asn 176 . . . . C 1.08 0.83* * . -0.200.75
. .
Thr 177 . . . . C 1.32 1.31* . F 0.10 1.03
. .
Leu 178 . . . . C 0.68 0.80* * F 0.10 1.57
. .
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
42
Pro 179 . . . . T C 0.97 0.50* . F 0.12 0.97
.
Thr 180 . . . . T C 0.94 0.10* . F 0.54 1.16
.
Asn 181 . . . . T C 0.63 0.10* * F 0.51 2.03
.
Met 182 . B . . T . 0.09 -0.10. * F 0.88 1.90
.
Glu 183 . B B . . . 0.09 0.11. * F -0.300.98
.
Thr 184 . B B . . . -0.000.31. . F -0.270.50
.
Thr 185 . B B . . . -0.030.30* . F -0.240.68
.
Val 186 . B B . . . -0.920.11* * F -0.210.39
.
Leu 187 . B B . . . -0.320.80* * F -0.480.19
.
Ser 188 . B B . . . -1.020.71. * F -0.450.21
.
Gly 189 . . B . . C -0.711.01. * . -0.400.24
.
Ile 190 A B B . . . -0.640.37* * . -0.300.51
.
Asn 191 A B B . . . 0.26 0.44* * . -0.600.60
.
Phe 192 A B B . . . 0.72 0.06* * . -0.151.21
.
Glu 193 A B B . . . 0.42 0.06* * . -0.151.71
. -
Tyr 194 . B . . T . 0.46 -0.01* * . 0.85 1.05
.
Lys 195 . . . T T . 1.00 0.07* * F 0.80 1.76
.
Gly 196 . . . T T . 0.71 -0.29* * F 1.40 1.00
.
Met 197 . . . . T C 1.41 0.63. * F 0.15 0.67
.
Thr 198 . . . . . C 0.56 -0.13. * F 0.85 0.58
.
Gly 199 A . . . C 0.21 0.51. * . -0.400.44
.
Trp 200 A B . . . . -0.180.59* * . -0.420.45
.
Glu 201 A B . . . . 0.17 0.40* . . -0.240.31
.
Val 202 A B . . . . 0.73 -0.09* . . 0.84 0.52
.
Ala 203 A B . . . . 0.16 -0.01* . . 1.02 0.67
.
Gly 204 . . . T . . 0.26 -0.24* . . 1.80 0.27
.
Asp 205 . . B T . . 0.23 0.51* . . 0.52 0.57
.
His 206 . B B . . . -0.360.36* . . 0.24 0.82
.
Ile 207 . B B . . . -0.090.36. . . 0.06 0.83
.
Tyr 208 . B B . . . 0.16 0.43* . . -0.420.50
.
Thr 209 . B B . . . -0.090.86. . . -0.600.37
.
Ala 210 . B B . . . -0.390.86. . . -0.600.53
.
Ala 211 . B . . . . -0.360.56. . . -0.120.45
.
Gly 212 . . . . . C 0.53 -0.20. . . 1.26 0.52
.
Ala 213 . . . . . C 0.78 -0.29. . F 1.69 0.83
.
Ser 214 . . . . T C 0.39 -0.79. . F 2.62 1.38
.
Asp 215 . . . T T . 0.38 -0.50. . F 2.80 1.21
.
Asn 216 . . . . T C 0.08 -0.31. . F 2.32 1.18
.
Asp 217 . B . . T . -0.39-0.13. . F 1.69 0.62
.
Phe 218 . B B . . . -0.110.17. . . 0.26 0.30
.
Met 219 . B B . . . -0.620.66. . . -0.320.27
.
Ile 220 . B B . . . -1.480.94. . . -0.600.14
.
Leu 221 . B B . . . -2.331.59. . . -0.600.12
.
Thr 222 . B B . . . -2.541.44. . . -0.600.09
.
Leu 223 . B B . . . -2.191.26. . . -0.600.19
.
Val 224 . B B . . . -2.291.00* * . -0.600.23
.
Val 225 . B B . . . -1.291.10* * . -0.600.14
.
Pro 226 . B . . . . -0.690.61* * . -0.400.33
.
Gly 227 . . . T . . -0.590.36* . F 0.45 0.68
.
Phe 228 . B . . . . 0.22 0.14* . F 0.45 1.42
.
Arg 229 . . . . . C 0.78 -0.10* * F 1.50 1.59
.
Pro 230 . . . . T C 0.78 -0.14* . F 1.95 2.16
.
Pro 231 . . . T T . 0.39 0.07* * F 1.80 1.85
.
Gln 232 . . . T T . 0.14 -0.10* * F 2.50 0.93
.
Ser 233 . B . . 'I' . 0.84 0.40* * F 0.95 0.61
.
Val 234 . B . . . . 0.42 -0.03* * . 1.55 0.66
.
Met 235 . B . . . . 0.63 0.03. . . 1.00 0.55
.
Ala 236 . B . . . . 0.84 -0.37. * . 1.65 0.71
.
Asp 237 . B . . T . 0.89 -0.36. . F 2.20 1.54
.
Thr 238 . . . . T C 1.19 -1.00. . F 3.00 3.11
.
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
43
Glu 239 A . . . T . 1.19 -1.61. . F 2.50 5.33
.
Asn 240 A . . . T . 1.20 -1.47* * F 2.20 2.37
.
Lys 241 A . . . . . 1.90 -0.97* * F 1.70 1.66
.
Glu 242 A . . . . . I -1.46* * F 1 1.88
. .01 .40
Val 243 A . B . . . 1.01 -0.77* * . 0.60 0.82
.
Ala 244 . B B . . . 0.31 -0.69* * . 0.60 0.59
.
Arg 245 . B B . . . -0.540.10* * . -0.300.29
.
Ile 246 . B B . . . -1.290.74* * . -0.600.29
.
Thr 247 . B B . . . -1.290.89* * . -0.600.25
.
Phe 248 . B B . . . -0.740.39* * . -0.300.22
.
Val 249 . B B . . . -0.970.87* * . -0.600.46
.
Phe 250 . B B . . . -1.740.87* * . -0.600.26
.
Glu 251 . B B . . . -1.160.96* * . -0.600.16
.
Thr 252 . . B T . . -1.700.56* * . -0.200.29
.
IS Leu 253 . . B T . . -I.000.56* * . -0.200.25
.
Cys 254 . . B T . . -0.810.17* * . 0.10 0.23
.
Ser 255 . . . T T . -0.110.74* * . 0.20 0.09
.
Val 256 . . . T T . -0.920.26* * . 0.50 0.18
.
Asn 257 . B . 'f T . -0.860.26. * . 0.50 0.28
.
Cys 258 . B . . T . -0.740.44. * . -0.200.33
.
Glu 259 . B B . . . -0.680.84. * . -0.600.38
.
Leu 260 . B B . . . -1.230.81. * . -0.600.24
.
Tyr 261 . B B . . . -0.721.06. * . -0.600.33
.
Phe 262 . B B . . . -1.580.91. * . -0.600.19
.
Met 263 . B B . . . -0.911.56. * . -0.600.17
.
Val 264 . B B . . . -1.211.27. * . -0.600.17
.
Gly 265 . B B . . . -0.290.90. * . -0.320.27
.
Val 266 . B B . . . -0.360.11. * . 0.26 0.53
.
Asn 267 . . . . T C 0.34 -0.01. * F 2.04 1.03
.
Ser 268 . . . . T C 0.63 -0.26. * F 2.32 1.67
.
Arg 269 . . . T T . 1.28 -0.20. * F 2.80 3.24
.
Thr 270 . . . T T . 0.77 -0.41. * F 2.52 3.12
.
Asn 271 . . . . . C 1.62 -0.17* * F 1.84 1.73
.
Thr 272 . . . . . C 1.31 -0.56* * F 1.86 1.53
.
Pro 273 . B . . . . 1.32 -0.07* * F 1.08 1.53
.
Val 274 . B . . . . 1.26 0.36. * F 0.05 1.00
.
Glu 275 . B . . . . 1.22 -0.04* . F 0.80 1.38
.
Thr 276 . B . . . . 0.92 -0.10* . F 0.99 0.88
.
Trp 277 . B . . . . 1.28 -0.14. . F 1.48 1.60
.
Lys 278 . . . T . . 1.14 -0.79. . F 2.52 1.85
.
Gly 279 . . . T . . 2.04 -0.36. . F 2.56 1.27
.
Ser 280 . . . T T . 2.04 -0.84. . F 3.40 2.41
.
Lys 281 . . . . T C 2.06 -1.36. . F 2.86 2.08
.
Gly 282 . . . T T . 2.10 -0.97. . F 2.72 2.82
.
Lys 283 . . . T T . 1.74 -0.64. . F 2.38 3.30
.
Gln 284 . . B T . . 1.84 -0.54* . F 1.64 2.38
.
Ser 285 . B B . . . 1.26 0.21* * F 0.00 3.77
.
Tyr 286 . B B . . . 0.32 0.47* * . -0.451.32
.
Thr 287 . B B . . . 0.67 1.16* . . -0.600.53
.
Tyr 288 . B B . . . 0.62 0.76. . . -0.600.69
.
Ile 289 . B B . . . 0.62 0.37* . . -0.300.76
.
Ile 290 . B B . . . 0.61 0.01. . . -0.040.85
.
Glu 291 . B B . . . 0.54 0.01. . F 0.37 0.78
.
Glu 292 . B . . . . 0.54 -0.26. . F 1.58 1.61
.
Asn 293 . . B T . . 0.49 -0.46. . F 2.04 3.32
.
Thr 294 . . B T . ' 0.68 -0.76. . F 2.60 2.57
. .
Thr 295 . . B . . C 1.26 0.03. * F 1.24 1.29
.
Thr 296 . . B . . C 0.97 0.51. . F 0.68 1.15
.
Ser 297 . . B . . C 0.38 1.03* * F 0.27 0.84
.
Phe 298 . B B . . . -0.321.04* * . -0.340.59
.
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
44
Thr 299 . B B . . . -0.011.34* . . -0.600.35
.
Trp 300 . B B . . . 0.41 1.26* . . -0.600.46
.
Ala 301 . . B . . C 0.41 0.87* . . -0.251.03
.
Phe 302 . . B T . . 0.40 0.57* * . -0.051.03
.
Gln 303 . . B T . . 0.40 0.57* * . -0.051.42
.
Arg 304 . . B . . C 0.68 0.44. . F -0.101.21
.
Thr 305 . . B . . C 0.97 0.44. . F -0.101.91
.
Thr 306 . . B . . C 0.97 -0.34* . F 0.80 1.91
.
Phe 307 . . B . . C 1.37 -0.24* * . 0.50 0.98
.
His 308 . . B . . C 1.48 0.14* * . -0.100.91
.
Glu 309 . . B . . C 1.41 -0.34* * . 0.65 1.24
.
Ala 310 . . . T . C 1.48 -0.83* . F 1.84 2.86
.
Ser 311 . . . 1' . . 1.48 -0.86* . F 2.18 3.30
.
Arg 312 . . . T . . 2.18 -0.87* . F 2.52 2.75
.
Lys 313 . . . T . . 2.21 -0.47* . F 2.56 4.38
.
Tyr 314 . . . T T . 1.36 -0.97* . F 3.40 5.45
.
Thr 315 . . . T T . 1.36 -0.71* . F 3.06 2.07
.
Asn 316 . B . . T . 1.70 -0.21* . F 2.02 1.04
.
Asp 317 . B . . T . 0.70 -0.21* . F 1.68 1.33
.
Val 318 . B B . . . 0.41 -0.29* . F 0.79 0.65
.
Ala 319 . B B . . . 0.36 -0.01* . . 0.30 0.63
.
Lys 320 . B B . . . -0.22-0.03* . . 0.30 0.51
.
Ile 321 . B B . . . -0.220.66* . . -0.600.48
.
Tyr 322 . B B . . . -1.080.41. . . -0.600.76
.
Ser 323 . B B . . . -0.530.56. . . -0.600.28
.
Ile 324 . B B . . . 0.06 1.04. . . -0.600.58
.
Asn 325 . B B . . . -0.840.76. . . -0.600.60
.
Val 326 . B B . . . -0.560.64. . . -0.600.33
.
Thr 327 . B B . . . -0.310.87. * . -0.600.47
.
Asn 328 . B B . . . -0.360.59* * . -0.600.47
.
Val 329 . B . . T . -0.320.61* * . -0.200.62
.
Met 330 . B . . T . -0.910.61* . . -0.200.32
.
Asn 331 . B . . T . -0.360.63* . . -0.200.20
.
Gly 332 . B . . T . -0.290.61* . . -0.200.36
.
Val 333 . B . . . . -0.960.73* . . -0.400.57
.
Ala 334 . B . . T . 0.01 0.69* . . -0.200.19
.
Ser 335 . B . . T . 0.40 0.29* . . 0.10 0.38
.
Tyr 336 . B . . T . -0.270.29* * . 0.10 0.79
.
Cys 337 . B . . T . -0.510.21* . . 0.10 0.42
.
Arg 338 . B . . 1' . -0.470.21. . . 0.10 0.32
.
Pro 339 . B . . T . 0.12 0.51. . . -0.200.17
.
Cys 340 . B . . T . -0.17-0.24. . . 0.70 0.54
.
Ala 341 . B . . T . -0.22-0.31* . . 0.70 0.28
.
Leu 342 . B . . . . 0.44 0.07* . . -0.100.24
.
Glu 343 . B . . . . -0.52-0.36* . . 0.75 0.75
.
Ala 344 . B . . . . -0.66-0.29. . F 1.15 0.55
.
Ser 345 . B . . . . -0.29-0.36. . F 1.40 0.66
.
Asp 346 . . . T T . 0.00 -0.66. . F 2.55 0.51
.
Val 347 . . . T T . 0.14 -0.27. . F 2.50 0.68
.
Gly 348 . . . T T . -0.17-0.20* . F 2.25 0.27
.
Ser 349 . . . T T . 0.12 -0.10* . F 2.00 0.23
.
Ser 350 . . . T . . -0.240.29* . F 0.95 0.42
.
Cys 351 . B . . T . -0.460.21. . F 0.50 0.23
.
Thr 352 . B . . T . -0.190.21. . F 0.25 0.26
.
5$ Ser 353 . B . . T . -0.190.33. . F 0.25 0.20
.
Cys 354 . B . . T . -0.130.37. . . 0.10 0.37
.
Pro 355 . B . . T . -0.080.56. . . -0.200.40
.
Ala 356 . . . T T . -0.300.83. . . 0.20 0.47
.
Gly 357 . B . . T . 0.01 1.13* . . -0.200.61
.
Tyr 358 . B . . T . 0.42 0.56* . . -0.200.66
.
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
Tyr 359 . B B . . . 1.09 0.13* . . 0.19 1.28
.
Ile 360 . B B . . . I.00 -0.37* . . 1.13 2.16
.
Asp 361 . B B . . . 1.24 -0.41* . . 1.47 1.84
.
Arg 362 . . . T . . 1.28 -0.74* . F 2.86 1.16
.
5 Asp 363 . . . T T . 0.86 -1.01* . F 3.40 2.40
.
Ser 364 . . . 'C T . 1.07 -1.13* . F 2.91 0.77
.
Gly 365 . . . T T . 1.66 -0.63* . F 2.57 0.53
.
Thr 366 . . . T T . 0.99 -0.24. . F 1.93 0.43
.
Cys 367 . . . T T . 0.67 0.33. * . 0.84 0.17
.
10 His 368 . . . T T . 0.46 0.37. . . 0.50 0.27
.
Ser 369 . . . T T . 0.76 0.37. . . 0.50 0.29
.
Cys 370 . B . . T . 0.79 0.29. . . 0.10 0.86
.
Pro 371 . . . . T C 0.21 0.20. . F 0.45 0.91
.
Pro 372 . . . T T . 0.07 0.39* . F 0.65 0.48
.
15 Asn 373 . . . T T . 0.14 0.69* * F 0.35 0.74
.
Thr 374 . B . . T . -0.140.11* * F 0.25 0.95
.
Ile 375 A B . . . . 0.49 0.19* . . -0.300.62
.
Leu 376 A B . . . . 0.70 0.26* . . -0.300.53
.
Lys 377 A B . . . . 0.70 0.26* . . -0.300.63
.
20 Ala 378 A B . . . . 0.46Ø20* . . -O.151.39
.
His 379 A B . . . 0.42 0.27. . . -0.152.64
.
Gln 380 . B . . T . 0.46 0.01* * F 0.40 1.31
.
Pro 381 . . . T T . 1.27 0.66. * . 0.20 0.96
.
Tyr 382 . . . T T . 0.63 0.56. * . 0.35 1.22
.
25 Gly 383 . . . T T . 0.56 0.56. * . 0.20 0.71
.
Val 384 . B B . . . -0.270.73. * . -0.600.25
.
Gln 385 . B B . . . -0.480.94. * . -0.600.12
.
Ala 386 . B B . . . -0.930.61. * . -0.600.18
.
Cys 387 . B B . . . -1.030.76. * . -0.60O.13
.
30 Val 388 . B B . . . -0.900.54. * . -0.600.08
.
Pro 389 . B . . . . -0.390.57. * . -0.400.12
.
Cys 390 . B . . . . -0.700.50. . . -0.400.21
.
Gly 391 . B . . T . -0.070.41. . F 0.29 0.42
.
Pro 392 . . . T 'T . 0.60 -0.23. . F 1.93 0.54
.
35 Gly 393 . . . T T . 1.46 -0.26. . F 2.42 1.61
.
Thr 394 . . . T T . 1.71 -0.43. . F 2.76 2.62
.
Lys 395 . . . T T . 1.49 -0.86. . F 3.40 3.39
.
Asn 396 . . . T T . 1.80 -0.60. . F 3.06 2.40
.
Asn 397 . B . . T . 1.71 -0.53. . F 2.32 2.26
.
40 Lys 398 . B . . T . 1.24 -0.63. . F 1.98 1.52
.
Ile 399 . B . . . . 0.89 0.06. . . 0.24 0.78
.
His 400 . B . . T . 0.60 0.23. . . 0.10 0.26
.
Ser 401 . B . . T . 0.60 0.59. . . -0.200.20
.
Leu 402 . B . . T . 0.60 0.99* . . -0.200.47
.
45 Cys 403 . B . . T . -0.110.30* . . 0.10 0.57
.
Tyr 404 . . . T . . 0.47 0.37. . . 0.30 0.23
.
Asn 405 . . . T T . -0.200.47. . . 0.20 0.40
.
Asp 406 . . . T T . -0.200.57* . . 0.20 0.65
.
Cys 407 . B . . T . 0.72 0.39* . . 0.10 0.55
.
Thr 408 . B . . T . 1.39 -0.37. . . 0.70 0.67
.
Phe 409 . B . . . . 1.32 -0.37. . . 0.80 0.65
.
Ser 410 . . . T T . 1.11 0.11. . F 1.40 1.75
.
Arg 411 . . . T T . 0.80 -0.03. . F 2.30 1.87
.
Asn 412 . . . . T C 1.58 -0.03. . F 2.40 3.12
.
Thr 413 . . . . T C 1.58 -0.81. . F 3.00 4.55
.
Pro 414 . . . . T C 1.58 -0.71* . F 2.70 3.36
. ~
Thr 415 . . . T T . 1.88 0.07* . F 1.70 1.81
.
Arg 416 . B . . T . 1.52 0.07* . F 1.00 2.01
.
Thr 417 . B . . T . 1.52 0.34* * F 0.70 2.04
.
Phe 418 . B . . . . 1.13 0.31* * . 0.05 2.27
.
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
46
Asn 419 . B . . T . 1.04 0.61* * . -0.051.01
.
Tyr 420 . B . . T . 0.77 1.00* . . -0.200.93
.
Asn 421 . B . . T . -0.161.01* * . -0.051.09
.
Phe 422 . B . . T . -0.430.91* * . -0.200.56
.
Ser 423 A . . . . C 0.27 1.01* * . -0.400.36
.
Ala 424 A . . . . C -0.040.66* * . -0.400.36
.
Leu 425 A B . . . . -0.660.74* * . -0.600.60
.
Ala 426 A B . . . . -0.970.60. * . -0.600.33
.
Asn 427 A B . . . . -1.080.70* . . -0.600.47
.
Thr 428 . B B . . . -1.370.89* . . -0.600.47
.
Val 429 . B B . . . -1.120.70. . . -0.600.47
.
Thr 430 . B B . . . -0.660.63. . . -0.600.29
.
Leu 431 . B B . . . -0.280.66. . . -0.600.20
.
Ala 432 . B B . . . -0.580.60. . . -0.600.42
.
Gly 433 . . B . . C -0.970.34. * F 0.05 0.39
.
Gly 434 . . . . T C -0.420.64. . F 0.15 0.41
.
Pro 435 . . . . T C -0.410.44. * F 0.15 0.58
.
Ser 436 . . . . T C 0.44 0.33. * F 0.73 0.79
.
Phe 437 . B . . T . 0.69 -0.10. . F 1.56 1.59
.
Thr 438 . B . . . . 0.22 -0.10* . F 1.64 1.02
.
Scr 439 . B . . 'f . 0.61 0.16* . F 1.37 0.63
.
Lys 440 . . . T T . 0.58 -0.23* . F 2.80 1.45
.
Gly 441 . . . T T . 0.18 -0.26* . F 2.52 1.57
.
Leu 442 . . . . T C 0.84 0.04* . F 1.44 1.02
.
Lys 443 A B . . . . 1.12 0.16* . . 0.26 0.69
.
Tyr 444 A B . . . . 0.72 0.66* . . -0.320.95
.
Phe 445 A B . . . . 0.37 I.01* * . -0.601.00
.
His 446 A B . . . . -0.100.81* * . -0.600.72
.
His 447 A B . . . . 0.41 1.50* * . -0.600.38
.
Phe 448 A B . . . . -0.441.13* * . -0.600.59
.
Thr 449 A B . . . . -0.871.03. * . -0.600.36
.
Leu 450 A . . T . . -0.511.10. * . -0.200.14
.
Ser 451 . . B T . . -0.481.03. * . -0.200.16
.
Leu 452 . . B T . . -0.440.64. * . 0.14 0.18
.
Cys 453 . . B T . . -0.090.56. * . 0.48 0.37
.
Gly 454 . . B T . . 0.33 0.30* . F 1.27 0.28
.
Asn 455 . . . T T . 1.19 -0.09* . F 2.61 0.66
.
Gln 456 . . . T T . 0.89 -0.77* . F 3.40 2.45
.
Gly 457 . . . T T . 1.40 -0.73* . F 3.06 2.45
.
Arg 458 . . . T T . 1.21 -0.77* . F 2.72 2.04
.
Lys 459 . B B . . . 0.89 -0.53* . F 1.43 0.87
.
Met 460 . B B . . . 0.58 -0.36* . . 0.64 0.47
.
Ser 461 . B B . . . 0.58 -0.30* . . 0.30 0.35
.
Val 462 . B B . . . 0.92 -0.30* . . 0.30 0.29
.
Cys 463 . B . . T . -0.040.10* . . 0.10 0.47
.
Thr 464 . B . . T . -0.400.13* * . 0.10 0.26
.
Asp 465 . B . . T . 0.20 0.23* . F 0.25 0.51
.
Asn 466 . B . . T . -0.31-0.41* * F 1.00 1.59
.
Val 467 . B B . . . 0.66 -0.30. * F 0.45 0.91
.
Thr 468 . B B . . . 0.43 -0.79. * F 0.90 1.07
.
Asp 469 . B B . . . 0.53 -0.10. * F 0.45 0.46
.
Leu 470 . B B . . . 0.53 -0.07. * F 0.76 0.97
.
Arg 471 . B B . . . 0.19 -0.71. * F 1.52 1.16
.
Ile 472 . B . . T . 1.04 -0.77. * F 2.08 0.69
.
Pro 473 . B . . T . 1.06 -0.77. * F 2.54 1.44
.
Glu 474 . . . T T . 0.71 -1.07. * F 3 0.99
. .
I
0
Gly 475 . . . . T C 0.82 -0.64. * F 2.74 1.40
.
Glu 476 . . . T T . 0.41 -0.54* . F 2.48 0.78
.
Ser 477 . . . . T C 1.34 -0.59* . F 2.10 0.60
.
Gly 478 . . . T T . 1.26 -0.59* . F 2.27 1.22
.
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
47
Phe 479 . . . T T . 0.37 -0.63* . F 1.94 0.95
.
Ser 480 . . . . T C 0.40 0.06* . F 0.97 0.49
.
Lys 481 . . . T T . -0.190.16* . F 1.30 0.72
.
Ser 482 . . . T T . -0.130.23* . F 1.17 0.84
.
Ile 483 . B . . T . -0.640.20* . . 0.49 0.98
.
Thr 484 . B B . . . -0.610.46* . . -0.340.37
.
Ala 485 . B B . . . -0.311.03* . . -0.470.15
.
Tyr 486 . B B . . . -0.941.04* . . -0.600.36
.
Val 487 . B B . . . -1.500.86. . . -0.600.25
.
Cys 488 . B B . . . -1.501.01. . . -0.600.19
.
Gln 489 . B B . . . -2.081.20. . . -0.600.08
.
Ala 490 . B B . . . -1.701.13. . . -0.600.08
.
Val 491 . B B . . . -1.670.91. . . -0.600.23
.
Ile 492 . B B . . . -0.810.77. . . -0.600.20
.
Ile 493 . B B . . . -1.000.37. . . -0.300.35
.
Pro 494 . B . . T . -1.310.51. * . -0.200.35
.
Pro 495 . B . . T . -1.070.36. * F 0.42 0.71
.
Glu 496 . B . . T . -0.460.10. . F 0.74 1.01
.
Val 497 . B . . T . 0.48 0.17. . F 0.91 1.02
.
Thr 498 . B . . T . 0.78 -0.26. * F 1.68 1.32
.
Gly 499 . B . . T . 0.64 -0.19. . F 1.70 0.77
.
Tyr 500 . B . . T . 0.00 0.24. . F 1.08 1.03
.
Lys 501 . B . . T . -0.300.24. . F 0.76 0.53
.
Ala 502 . B . . . . 0.26 0.14. * F 0.39 0.71
.
Gly 503 . B . . . . 0.57 0.10. * F 0.22 0.61
.
Val 504 . B . . . . 0.70 -0.26. * F 0.65 0.53
.
Ser 505 . B . . . . 0.09 0.17. * F 0.05 0.81
.
Ser 506 . B . . . . -0.260.31. * F 0.05 0.61
.
Gln 507 . B . . . . -0.480.27. . F 0.20 1.10
.
Pro 508 . B . . . . -0.720.31. . F 0.05 0.67
.
Val 509 A B . . . . 0.13 0.43* * F -0.450.51
.
Ser 510 A B . . . . 0.54 0.04* * . -0.300.49
.
Leu 511 A B . . . . 0.03 -0.36* . . 0.30 0.62
.
Ala 512 A B . . . . -0.86-0.10* * . 0.30 0.69
.
Asp 513 A B B . . . -0.99-0.06* * . 0.30 0.36
.
Arg 514 A B B . . . -0.99-0.01* * . 0.30 0.43
.
Leu 515 . B B . . . -I.00-0.06* . . 0.30 0.32
.
Ile 516 . B B . . . -0.50-0.07* . . 0.30 0.28
.
Gly 517 . B B . . . 0.09 0.41* . . -0.600.20
.
Val 518 . B B . . . -0.510.41* . . -0.600.41
.
Thr 519 . B B . . . -0.930.34* * F -0.150.58
.
Thr 520 . B B . . . -0.930.14. * F -0.150.85
.
Asp 521 . B B . . . -0.040.40. * F -0.450.94
.
Met 522 . B B . . . -0.04-0.24. * F 0.60 1.09
.
Thr 523 . B B . . . -0.08-0.30. * . 0.30 0.75
.
Leu 524 . B B . . . -0.08-0.10* * F 0.45 0.31
.
Asp 525 . . B T . . -0.070.39* * F 0.25 0.46
.
Gly 526 . . B T . . -0.280.16* * F 0.34 0.42
.
Ile 527 . . B . . C -0.270.10* . F 0.23 0.79
.
Thr 528 . . B . . C 0.04 -0.09* * F 0.92 0.48
.
Ser 529 . . . . T C 0.04 -0.09* . F 1.41 0.84
.
Pro 530 . . . . T C -0.660.17* . F 0.90 0.99
.
Ala 531 . B . . T . -0.340.27* . F 0.61 0.59
.
Glu 532 . B . . T . -0.270.29. * . 0.37 0.60
.
Leu 533 A B . . . . 0.04 0.59. . . -0.420.32
.
Phe 534 A B . . . . 0.04 0.16. . . -0.210.55
.
His 535 A B . . . . -0.560.04. . . -0.300.43
.
Leu 536 A B . . . . -0.310.73. . . -0.600.43
.
Glu 537 A B . . . . -1.200.47. . . -0.600.49
.
Ser 538 . . . T . . -0.600.37. . . 0.30 0.25
.
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
48
Leu 539 . . . T . . 0.10 0.30 . . 0.30 0.47
. .
Gly 540 . . . . . C -0.72 -0.39 . . 0.70 0.45
. .
Ile 541 . . B . . C -0.80 0.26 . F 0.05 0.25
. .
Pro 542 . B B . . . -1.50 0.56 . F -0.450.21
. .
Asp 543 . B B . . . -1.90 0.66 . . -0.600.19
. .
Val 544 . B B . . . -1.33 1.01 * . -0.600.23
. *
Ile 545 . B B . . . -0.88 1.09 . . -0.600.23
. *
Phe 546 . B B . . . -0.29 0.66 * . -0.600.27
. *
Phe 547 . B B . . . -0.08 1.04 * . -0.600.50
. .
Tyr 548 . B . . . . -0.08 0.80 * . 0.09 1.14
. .
Arg 549 . . . T T . -0.08 0.11 . F 1.48 2.19
. .
Ser 550 . . . T T . 0.50 -0.03 . F 2.42'1.88
. .
Asn 551 . . . T T . 1.20 -0.33 . F 2.76 1.73
. *
Asp 552 . . . T T . 1.60 -0.69 . F 3.40 1.53
. *
Val 553 . . . T . . 1.18 -0.30 . F 2.56 1.53
. .
Thr 554 . B . . . . 0.77 -0.11 . F 1.67 0.51
. *
Gln 555 . B . . . . 0.77 -0.13 . F 1.33 0.41
. *
Ser 556 . B . . . . 0.42 0.26 * F 0.67 0.74
. *
Cys 557 . B . . T . 0.53 0.04 * F 0.81 0.51
. *
Ser 558 . . . T T . 1.09 -0.44 * F 2.09 0.57
. *
Ser 559 . . . T T . 1.09 -0.46 * F 2.37 0.57
. .
Gly 560 . . . T T . 0.78 -0.36 . F 2.80 1.54
. .
Arg 561 . . B T . . 0.19 -0.44 * F 2.12 1.66
. .
Ser 562 . . B T . . 0.97 -0.14 * F 1.69 0.87
. *
Thr 563 . B B . . . 0.41 -0.53 * F 1.46 1.72
. *
Thr 564 . B B . . . 0.82 -0.31 * F 0.73 0.65
. .
Ile 565 . B B . . . 0.50 -0.31 * F 0.45 0.95
. .
Arg 566 . B B . . . 0.09 -0.13 * . 0.30 0.35
. .
Val 567 . B B . . . 0.18 -0.23 * . 0.64 0.33
. .
Arg 568 . B B . . . 0.49 -0.29 * . 0.98 0.73
. .
Cys 569 . B B . . . 0.84 -0.57 * . 1.62 0.64
. .
Ser 570 . . . . T C 1.42 -0.57 * F 2.86 1.73
. *
Pro 571 . . . T T . 0.46 -0.73 * F 3.40 1.27
. *
Gln 572 . . . T T . 1.10 -0.09 * F 2.76 1.76
. *
Lys 573 . B . . T . 0.64 -0.23 * F 2.02 2.03
. .
Thr 574 . B . . . . 1.01 -0.19 . F 1.48 1.30
. .
Val 575 . B . . T . 0.50 -0.23 . F 1.34 1.01
. .
Pro 576 . B . . T . -0.10 0.06 . F 0.25 0.42
. .
Gly 577 . B . . T . -0.91 0.74 . F -0.050.24
. .
Ser 578 . B . . T . -1.17 0.94 . F -0.050.26
. .
Leu 579 . B . . . . -1.20 0.73 * F -0.250.26
. .
Leu 580 . B . . . . -0.66 0.73 * F -0.400.26
. .
Leu 581 . B . . T . -1.11 0.79 . F -0.050.28
. .
Pro 582 . B . . T . -1.07 0.97 . F -0.050.18
. .
Gly 583 . . . T T . -0.77 0.67 . F 0.35 0.30
. .
Thr 584 . B . . T . -0.30 -0.01 . F 1.16 0.61
. .
Cys 585 . . . T T . 0.20 -0.27 . F 1.87 0.39
. .
Ser 586 . . . T T . 0.34 -0.21 . F 2.18 0.57
. .
Asp 587 . . . T T . 0.56 -0.07 . F 2.49 0.21
. .
Gly 588 . . . T T . 0.56 -0.56 . F 3.10 0.66
. .
Thr 589 . . . T . . 0.20 -0.70 . F 2.59 0.48
. *
Cys 590 . . . T T . 0.87 -0.51 . F 2.48 0.16
. *
Asp 591 . . . T T . 0.47 -0.11 . F 1.87 0.25
. .
Gly 592 . . . T T . 0.43 0.24 * F 0.96 0.15
. .
Cys 593 . . . T T . 0.08 0.26 . . 0.50 0.38
. .
Asn 594 A B . . . . -0.42 0.47 . . -0.600.20
. .
Phe 595 A B . . . . -0.04 1.16 * . -0.600.17
. .
His 596 A B . . . . -0.04 1.64 * . -0.600.33
. .
Phe 597 A B . . . . 0.00 1.07 * . -0.600.35
. *
Leu 598 A . . T . . 0.08 1.06 * . -0.200.54
. .
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
49
Trp 599 A . . T . . -0.51 0.77. * . -0.200.40
.
Glu 600 A . . T . . -0.40 0.77. * . -0.200.47
.
Ser 601 A . . T . . -1.03 0.49. . . -0.200.58
.
Ala 602 A . . T . . -0.54 0.37. . . 0.100.29
.
Ala 603 A . . T . . -0.54 -O.II. . . 0.700.26
.
Ala 604 A . . T . . -0.92 0.57. . . -0.200.16
.
Cys 605 . . . . T C -1.22 0.76. . . 0.000.09
.
Pro 606 . B . . T . - I 0.64. . . -0.200.1
. .78 1
Leu 607 . B . . T . -1 0.79* . ' -0.200.08
. .78 .
10Cys 608 . B . . T . -1.19 0.79* . . -0.200.16
.
Ser 609 . B B . . . -0.84 0.21. . . -0.300.17
.
Val 610 . B B . . . -0.21 0.54. . . -0.600.32
.
Ala 611 . B B . . . -0.59 0.36. . . -0.300.82
.
Asp 612 . B . . . . -0.67 0.29. . . -0.100.62
.
15Tyr 613 . B B . . . -0.86 0.59. . . -0.600.58
.
His 614 . B B . . . -0.86 0.59. . . -0.600.43
.
Ala 615 . B B . . . -0.30 0.47. . . -0.600.34
.
Ile 616 . B B . . . -0.38 0.86. * . -0.600.29
.
Val 617 . B B . . . - I 0.67. . . -0.600.12
. .23
20Ser 618 . B B . . . -1.58 0.81. . . -0.600.09
.
Ser 619 . B B . . . -1.89 0.81. . . -0.600. I
. 2
Cys 620 . B B . . . -2.19 0.56* . . -0.600.16
.
Val 621 . B B . . . -1.30 0.60* . . -0.600.09
.
Ala 622 . B B . . . -0.40 0.61* . . -0.600.11
.
25Gly 623 . B B . . . -0.41 0.23* . . -0.300.41
.
Ile 624 . B B . . . -0.42 0.14* . . -0.300.80
.
Gln 625 . B B . . . 0.00 -0.01. . F 0.601.15
.
Lys 626 . B B . . . 0.00 0.24* . F 0.001.82
.
Thr 627 . B B . . . 0.30 0.46* * F -0.301.92
.
30Thr 628 . B B . . . 0.76 0.69* . F -0.301.17
.
Tyr 629 . B B . . . 1.64 0.29* . . -0.151.14
.
Val 630 A B B . . . 1.43 0.29* . . -0.151.37
.
Trp 631 A B B . . . 1.43 0.23* * . -0.151.47
.
Arg 632 A B B . . . 0.93 -0.26* . F 0.601.88
.
35Glu 633 A B B . . . 0.58 -0.33* . F 0.852.09
.
Pro 634 A . . T . . 0.52 -0.40* . F 1.501.06
.
Lys 635 A . . T . . 1.03 -0.93* . F 1.900.73
.
Leu 636 A . . T . . 0.98 -0.50* . F 1.850.42
. '
Cys 637 . . . T T . -0.02 -0.07* . F 2.500.27
.
40Ser 638 . . . T T . -0.32 0.19. * F 1.650.09
.
Gly 639 . . . T T . -0.92 0.57* * F 1.100.15
.
Gly 640 . . . T T . -1.18 0.57* . F 0.850.23
.
Ile 641 . . . . . C -0.37 0.43* . F 0.200.27
.
Ser 642 . . . . . C 0.30 0.04. * F 0.250.47
.
45Leu 643 . B . . . . 0.71 0.01. * F 0.050.82
.
Pro 644 . B . . . . 0.20 -0.41. * F 0.802.30
.
Glu 645 . B B . . . 0.23 -0.46. * F 0.601.27
.
Gln 646 . B B . . . 0.23 -0.36. * F 0.602.23
.
Arg 647 . B B . . . -0.13 -0.36. * F 0.601.01
.
50Val 648 . B B . . . 0.72 -0.21. * . 0.300.31
.
Thr 649 . B B . . . 0.62 -0.21. * . 0.300.36
.
Ile 650 . B B . . . -0.27 -0.13. * . 0.300.27
.
Cys 651 . B B . . . -0.27 0.56. * . -0.600.25
.
Lys 652 . B B . . . -1.08 -0.09* * . 0.300.29
.
55Thr 653 . B B . . . -0.51 0.21* * . -0.300.36
.
Ile 654 . B B . . . -1.01 0.44* * . -0.600.71
.
Asp 655 . B B . . . -0.08 0.56* * . -0.600.29
.
Phe 656 . B B . . . -0.27 0.56* * . -0.600.40
.
Trp 657 . B B . . . -0.66 0.71* * . -0.600.43
.
60Leu 658 . B B . . . -1.23 0.46* * . -0.600.25
.
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
Lys 659. . B B . . . -0.641.14* * . -0.600.20
Val 660. . . B T . . -1.230.74* * . -0.200.26
Gly 661. . . B T . . -0.880.33* * . 0.100.32
Ile 662. . . B T . . -0.900.07* * . 0.100.16
5 Ser 663. . . . . T C -0.760.56* * . 0.000.31
Ala 664. . . . T T . -1.110.49. * F 0.350.17
Gly 665. . . . T T . -0.840.54. . F 0.350.34
Thr 666. . B . . T . -1.390.36. . F 0.250.26
Cys 667. . B B . . . -1.310.66. . . -0.600.18
10 Thr 668. . B B . . . -1.820.84. . . -0.600.15
Ala 669. . B B . . . -1.541.10. . . -0.600.09
Ile 670. . B B . . . -2.061.10. . . -0.600.23
Leu 671. . B B . . . -2.561.17. . . -0.600.12
Leu 672. . B B . . . -2.201.37. . . -0.600.10
15 Thr 673. . B B . . . -2.561.36. . . -0.600.20
Val 674. . B B . . . -2.211.24. . . -0.600.13
Leu 675. . B B . . . -2.021.31. . . -0.600.25
Thr 676. . B B . . . -1.501.41* . . -0.600.15
Cys 677. . B B . . . -0.641.84* . . -0.600.21
20 Tyr 678. . B B . . . -0.291.20. . . -0.600.51
Phe 679. . . B T . . 0.57 0.51. . . -0.200.70
Trp 680. . . B T . . 1.38 0.43* . . 0.292.10
Lys 681. . . . . T C 1.73 0.26* . F 1.282.32
Lys 682. . . . T T . 1.59 -0.50* * F 2.425.37
25 Asn 683. . . . . T C 1.83 -0.60* * F 2.864.21
Gln 684. . . . T T . 2.29 -1.51* * F 3.403.65
Lys 685. . B . . . . 2.62 -0.76* * F 2.462.86
Leu 686. . B . . . . 2.33 -0.76* * F 2.323.55
Glu 687. . B . . . . 1.99 -0.40* * . 1.733.21
30 1'yr688. . B . . T . 2.03 -0.41* * . 1.792.15
Lys 689. . B . . T . 1.22 -0.41* * F 1.805.22
Tyr 690. . B . . T . 0.32 -0.41* * F 2.002.49
Ser 691. . B . . T . 0.53 0.23* * F 1.201.18
Lys 692. A B . . . . 0.53 0.09* * F 0.450.58
35 Leu 693. A B . . . . 0.19 0.49* * .. -0.200.60
Val 694. A B . . . . -0.170.23* * . -0.100.45
Met 695. A B . . . . -0.730.33* * . -0.300.33
Asn 696. A B . . . . -0.391.01. * . -0.600.33
Ala 697. A B . . . . -0.430.33* * . -0.300.88
40 Thr 698. A B . . . . -0.29-0.31. * . 0.651.48
Leu 699. A B . . . . 0.57 -0.36* . F 0.850.49
Lys 700. A B . . . . 0.36 -0.76. * F 1.350.82
Asp 701. . . . T T . 0.14 -0.57. * F 2.350.47
Cys 702. . B . . T . 0.14 -0.63. . . 2.000.87
45 Asp 703. . B . . T . -0.13-0.81. . . 1.800.44
Leu 704. . B . . 'C . 0.68 -0.31. . . 1.300.27
Pro 705. . B . . . . 0.33 -0.31. . . 0.900.83
Ala 706. . . . T . . -0.33-0.50. * . 1.100.67
Ala 707A . . . . . . -0.260.07. . . -0.100.43
50 Asp 708A . . . . T . -1.14-0.11. . . 0.700.28
Ser 709. . B . . T . -0.930.14. . . 0.100.20
Cys 710. . B . . T . -0.720.26. . . 0.100.19
Ala 711. . B . . 'f . -0.48-0.24. . . 0.700.20
Ile 712. A B . . . . 0.11 0.19. . . -0.300.15
Met 713. A B . . . . 0.11 -0.20. . . 0.300.48
Glu 714. A B . . . . -0.44-0.77. . F 0.750.79
Gly 715. A . . . . C 0.22 -0.63* . F 0.950.83
Glu 716A A . . . . . 0.81 - * . F 0.901.46
I
.31
Asp 717A A . . . . . 1.70 -1.93* . F 0.901.41
Val 718A A . . . . . 1.49 -1.93* . F 0.902.38
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
51
Glu 719 A A . . . . . 0.60 -1.67* . F 0.90 1.13
Asp 720 A A . . . . . 0.24 -0.99* . F 0.75 0.48
Asp 721 A A . . . . . -0.07-0.20. * F 0.45 0.55
Leu 722 A A . . . . . -0.37-0.36* * . 0.30 0.46
Ile 723 A A . . . . . 0.53 0.03. * . -0.300.37
Phe 724 . A B . . . . 0.53 0.03. . . -0.300.44
Thr 725 . A B . . . . 0.50 0.43. . F -0.450.87
Ser 726 . . . . . T C 0.20 0.24. . F 0.60 1.68
Lys 727 . . . . '1' T . 0.20 -0.06. . F 1.40 2.60
Asn 728 . . . . . T C 0.74 -0.16* * F 1.48 1.49
His 729 . . . . . T C 1.56 -0.21* * F 1.76 1.10
Ser 730 . . . . . . C 1.57 -0.60. * . 1.99 1.07
Leu 731 . . . . T . . 1.87 -0.21. * . 2.02 0.90
Gly 732 . . . . T T . 1.79 -0.21. . F 2.80 1.06
Arg 733 . . . . T T . 0.98 -0.21* . F 2.52 1.07
Ser 734 . . . . T T . 0.80 0.09* . F 1.88 1.07
Asn 735 . . . . T T . 0.89 -0.17* * F 2.44 1.68
His 736 . . . . . . C 1.81 -0.17* * F 2.00 1.33
Leu 737 . . . . . . C 1.81 -0.17* * F 1.96 1.94
Pro 738 . . . . . T C 0.89 -0.13* * F 2.40 1.19
Pro 739 . . . . T T . 0.38 0.16. * F 1.61 0.72
Arg 740 . . . . T T . -0.220.34. * F 1.37 0.72
Gly 741 . . B . . T . -0.190.27. * F 0.73 0.46
Leu 742 . A B . . . . -0.19-0.16. * . 0.54 0.50
Leu 743 . A B . . . . -0.290.10* . . -0.300.21
Met 744 . A B . . . . -0.080.59* . . -0.600.31
Asp 745 . A B . . . . -0.860.56* . . -0.600.64
Ixu 746 . A B . . . . -0.400.44. . . -0.600.42
Thr 747 . A B . . . . 0.02 -0.24. * . 0.30 0.83
Gln 748 . A B . . . . 0.44 -0.43. . F 0.45 0.63
Cys 749 . A B . . . . 0.66 0.00. . . -0.300.98
Arg 750 . A B . . . . 0.27 -0.26. . . 0.30 0.87
TABLE
II
Res Pos I II III IV V VI VII VIII IX X XI XII XIII XIV
Met 1 . . B . . . . 0.08 0.20. . . -0.100.71
Ser 2 . . B . . T . 0.47 0.26. . . 0.10 0.80
Thr 3 . . B . . T . 0.51 0.23. . . 0.50 1.01
Gly 4 . . . . . T C 0.90 0.23. * . 0.95 1.01
Thr 5 . . . . T T . 0.94 -0.39. . F 2.15 1.26
Asn 6 . . . . . T C 0.69 -0.34. . F 2.05 0.86
Gly 7 . . . . T T . 0.69 -0.19. . F 2.50 0.65
Asp 8 . . . . T T . 0.79 -0.23. . F 2.25 0.60
Gly 9 . . B . . T . 0.54 -0.29. . F 1.60 0.58
Val 10 . . B . . . . 0.86 -0.19. * F 1.15 0.59
Ser 11 . . B . . . . 0.51 -0.21. . F 0.90 0.57
Pro 12 . . B . . T . 0.00 0.21* . F 0.25 0.57
Ala 13 . . B . . T . -0.860.43* . F -0.050.57
Asn 14 . . B . . T . -1.320.43* . F -0.050.31
Gly I . . B . . T . -0.470.73. . . -0.200.17
S
Val 16 . . B B . . . -0.060.30. . . -0.300.28
Val 17 . . B B . . . -0.14-0.20. . . 0.60 0.34
Leu 18 . . B B . . . 0.20 -0.21. . . 0.90 0.46
Asp 19 . . B . . T . -0.010.11* * F 1.15 0.96
Arg 20 . . B . . '1' . 0.44 -0.10* * F 2.20 2.01
Ser 21 . . . . . T C 0.41 -0.74* * F 3.00 4.77
Tyr 22 . . . . . T C 0.41 -0.74* * F 2.70 2.00
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
52
Pro 23 . . B B . . . 0.37 -0.10* * F 1.35 0.76
Arg 24 . . B B . . . -0.23 0.54* * . 0.00 0.42
~
Ile 25 . A B B . . . -0.34 0.77* * . -0.300.27
Val 26 . A B B . . . 0.07 0.01. * . -0.300.30
Val 27 . A B B . . . -0.54 -0.41. * . 0.30 0.30
Met 28 . A B B . . . -0.33 0.23. * . -0.300.31
Glu 29 . A B B . . . -1.04 -0.46. * . 0.30 0.73
Arg 30 A A . B . . . -0.37 -0.49. * . 0.30 0.98
Val 31 A A . B . . . 0.18 -0.70. * . 0.75 1.53
Glu 32 A A . B . . . 0.44 -0.83* * . 0.75 1.27
Met 33 A A . . . . . 1.04 -0.33* * . 0.30 0.66
Pro 34 A . . . . . . 0.83 0.07* * F 0.20 1.53
Thr 35 A . . . . . . 0.13 -0.14* * F 0.80 1.37
Ala 36 A A . . . . . 0.18 0.36. . F 0.00 1.40
Gln 37 A A . . . . . -0.63 0.43. . F -0.450.75
Pro 38 A A . . . . . -0.62 0.69. . F -0.450.43
Ala 39 A A . . . . . -1.27 0.70. . . -0.600.43
Leu 40 A A . . . . . -0.96 0.84* . . -0.600.18
Leu 41 A A . . . . . -0.32 0.84* . . -0.600.20
Ala 42 A A . . . . . -0.32 0.41* . . -0.600.40
Val 43 . A B . . . . -0.92 0.31* . . -0.300.85
Gln 44 . A B . . . . -0.68 0.31* . . -0.060.85
Lys 45 . A B . . . . -0.08 0.06* . F 0.33 0.83
Gln 46 . A . . T . . 0.52 -0.01. . F 1.72 1.74
lxu 47 . A . . T . . 1.11 -0.23. . F 1.96 1.55
Gly 48 . . . . . T C 1.37 -0.23. . F 2.40 1.34
Pro 49 . . . . . T C 0.70 0.39. . F 1.41 0.77
Pro 50 . . . . T T . 0.77 0.56* * F 1.07 0.50
Gln 51 . . . . T '1' . -0.09 -0.13* * . 1.58 0.99
Met 52 . . B B . . . 0.13 0.09* * . -0.060.47
Cys 53 . . B B . . . -0.19 0.16* * . -0.300.31
Arg 54 . . B B . . . -0.29 0.30* * . -0.300.10
Val 55 . . B B . . . -0.74 0.39* . . -0.300.14
Ala 56 . . B B . . . -1.33 0.34* * . -0.300.14
Cys 57 . . B B . . . -1.59 0.27* * . -0.300.07
Thr 5 . . B B . . . -1 .81 0.9. * . -0.600.07
8 l
Cys 59 . . B B . . . -1.92 0.96* * . -0.600.05
Ala 60 . . B B . . . -0.96 0.86* . . -0.600.15
Val 61 . . B B . . . -1.22 0.29* * . -0.300.20
Ile 62 . . B B . . . -0.56 0.44* . . -0.600.28
Asn 63 . . B B . . . -0.20 0.27* . . -0.300.48
Arg 64 . . B B . . . -0.39 -0.23* . . 0.45 1.30
Val 65 . . B B . . . 0.20 -0.23* . F 0.60 1.38
Gln 66 . . B B . . . 0.39 -0.51* . F 0.90 1.38
Lys 67 . . B B . . . 0.97 -0.34* . F 0.45 0.38
Val 68 . . B B . . . 0.76 0.14* . . -0.300.73
Asn 69 . . B B . . . 0.33 -0.07* * . 0.30 0.65
Cys 70 . . B B . . . 0.89 0.01* * F -0.150.47
Thr 71 . . B . . T . 0.89 0.40. * F 0.25 0.85
Pro 72 . . . . T T . 0.26 0.16. * F 0.65 0.85
Thr 73 . . . . T T . 0.26 0.26. * F 0.80 1.61
Ser 74 . . . . T T . -0.41 0.33. . F 0.65 0.83
Asn 75 . . B . . . . -0.09 0.41. . F -0.250.29
Ala 76 . . B . . . . 0.22 0.41. . . -0.400.20
Val 77 . . B . . . . -0.23 -0.07. . . 0.50 0.24
Cys 78 . . B . . T . -0.73 0.11. . . 0.10 0.08
Gly 79 . . B . . T . -0.64 0.40* * . 0.10 0.07
Asp 80 . . B . . T . -0.53 0.33* * . 0.10 0.14
Cys 81 . . B . . T . -0.64 -0.31* * . 0.70 0.51
Leu 82 . . B . . . . -0.03 -0.10* * . 0.50 0.44
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
53
Pro 83 . . B . . T . 0.740.23 * . 0.100.42
*
Arg 84 . . B . . T . 1.130.23 * . 0.251.52
*
Phe 85 . . B . . T . 0.82-0.34 * . 0.853.69
*
Tyr 86 . . B . . 'f . 1.60-0.54 * . 1.383.44
*
Arg 87 . . B B . . . 1.52-0.97 * F 1.363.44
*
Lys 88 . . B B . . . 1.39-0.29 * F 1.292.79
*
Thr 89 . . B B . . . 0.93-0.64 * F 1.821.76
*
Arg 90 . . . B T . . 0.82-0.97 * F 2.300.89
*
Ile 91 . . . B T . . 1.07-0.29 . F 1.770.37
.
Gly 92 . . . . T . . 0.960.11 . F 1.140.44
.
Gly 93 . A . . T . . 0.91-0.37 * F 1.310.38
.
Leu 94 . A . . . . C 1.220.03 * F 0.280.93
*
Gln 95 . A . . T . . 0.44-0.66 * F 1.301.62
.
Asp 96 . A . . T . . 0.44-0.51 . F 1.150.88
.
Gln 97 . A B . . . . 0.58-0.26 . F 0.450.75
.
Glu 98 . A B . . . . 0.26-0.51 . F 0.750.67
.
Cys 99 . A B . . . . 0.76-0.34 . . 0.300.21
.
Ile 100. . B . . . . 0.800.14 . . -0.100.18
.
Pro 101. . . . T . . 0.80-0.26 . . 0.900.21
.
Cys 102. . . . T T . 0.490.14 . . 0.500.67
*
Thr 103. . . . T T . 0.280.06 . F 1.101.37
*
Lys 104. . . . T T . 0.63-0.20 . F 2.001.37
.
Gln 105. . . . . T C 1.22-0.14 . F 2.103.69
.
Thr 106. . . . . T C 1.43-0.33 . F 2.403.43
.
Pro 107. . . . . T C 1.24-0.81 * F 3.002.97
.
Thr 108. . . . T T . 1.56-0.17 * F 2.601.27
.
Ser 109. . B . . T . 0.84-0.17 * F 1.901.53
.
Glu 110. A B . . . . 0.26-0.09 * F 1.050.53
*
Val 111. A B . . . . -0.13-0.01 * . 0.600.37
*
Gln 112. A B . . . . 0.080.29 * . -0.300.24
*
Cys 113A A . . . . . -0.420.30 * . -0.300.24
*
Ala 114A A . . . . . -0.420.99 * . -0.600.27
*
Phe 1 A A . . . . . -1.230.73 * . -0.600.21
15 .
Gln 116A A . . . . . -1.231.01 * . -0.600.32
.
Leu 117A A . . . . . -1.231.09 * . -0.600.23
.
Ser 118. A B . . . . -1.160.59 * . -0.600.47
.
Leu 119. A B . . . . -0.570.30 * . -0.300.27
.
Val 120A A . . . . . -0.46-0.10 * . 0.300.55
.
Glu 121A A . . . . . -0.67-0.29 * . 0.300.41
.
Ala 122A A . . . . . -0.17-0.24 . . 0.300.78
.
Asp 123. A . . T . . -0.72-0.44 . . 0.851.51
.
Ala 124. A . . . . C -0.12-0.44 . F 0.650.65
.
Pro 125. A . . . . C 0.52-0.01 * F 0.650.99
.
Thr 126. . . . . . C 0.52-0.09 . F 0.850.92
.
Val 127. . . . . . C 1.110.31 . F 0.401.57
.
Pro 128. . . . . . C 0.52-0.19 . F 1.001.76
.
Pro 129A . . . . . . 0.80-0.11 . F 0.801.23
.
Gln 130A . . . . . . 0.20-0.11 . F 0.802.40
.
Glu 131A . . . . . . -0.34-0.07 . F 0.801.28
.
Ala 132A . . B . . . -0.080.14 . F -0.150.61
.
Thr 133A . . B . . . -0.680.21 . . -0.300.36
.
Leu 134A . . B . . . -1.320.50 . . -0.600.17
.
Val 135A . . B . . . -1.621.14 . . -0.600.13
.
Ala 136A . . B . . . -1.921.03 . . -0.600.12
.
Leu 137A . . B . . . -2.140.93 . . -0.600.19
*
Val 138A . . B . . . -2.640.93 . . -0.600.21
*
Ser 139A . . B . . . -2.690.97 . . -0.600.17
.
Scr 140. . B B . . . -2.691.11 . . -0.600.15
*
Leu 141. . B B . . . -2.801.07 . . -0.600.15
*
Leu 142. . B B . . . -2.301.21 . . -0.600.10
*
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
54
Val 143 . . B B . . . -2.261.31. . . -0.600.11
Val 144 . A B B . . . -2.541.61. * . -0.600.11
Phe 145 . A B B . . . -2.941.43. . . -0.600.13
Thr 146 . A B B . . . -2.941.53. . . -0.600.15
Leu 147 A A . B . . . -2.481.57. . . -0.600.17
Ala 148 A A . B . . . -2.431.36. . . -0.60O.19
Phe 149 A A . B . . . -2.281.26. . . -0.600.11
Leu 150 A A . B . . . -2.281.56. . . -0.600.12
Gly 151 A A . B . . . -2.781.66. . . -0.600.10
Leu 152 A A . B . . . -2.211.84. . . -0.600.10
Phe 153 A A . B . . . -2.291.81. . . -0.600.18
Phe 154 A A . B . . . -1.541.70* . . -0.600.10
Leu 155 A A . B . . . -0.731.27* . . -0.600.24
Tyr 156 A A . B .. . . -1.090.99* . . -0.600.48
Cys 157 . A . B T . . -0.980.99* . . -0.200.48
Lys 158 . A . B T . . -0.280.99* . . -0.200.50
Gln 159 . A . B T . . 0.530.70* . . -0.200.51
Phe 160 . A . B T . . 1.31-0.06* . . 0.85 1.88
Phe 161 . A . B T . . 0.89-0.13* . . 1.16 1.28
Asn 162 . . . . T T . 1.560.44* * . 0.82 0.39
Arg 163 . . . . 'I' T . 1.620.44* * . 1.13 0.79
His 164 . . . . 'f T . 1.28-0.34* * . 2.49 1.79
Cys 165 . . . . T 'f . 1.63-0.70* * . 3.10 1.10
Gln 166 . . . . T T . 1.52-0.67* . F 2.79 0.56
Arg 167 . . . . T T . 0.710.01* * F 1.58 0.34
Gly 168 . . . . T T . 0.600.20* * F 1.27 0.52
Gly l69 . . . . T T . -0.070.03* . F 0.96 0.52
Leu 170 . A . . . . C 0.600.41. * . -0.400.23
Leu 171 . A B . . . . 0.010.41. * . -0.600.40
Gln 172 . A B . . . . -0.100.49. * . -0.600.41
Phe 173 A A . . . . . 0.290.06. * . -0.300.83
Glu 174 A A . . . . . 0.32-0.63. * . 0.75 2.01
Ala 175 A A . . . . . 0.54-0.83* * F 0.90 1.67
Asp 176 A A . . . . . 1.40-0.73* * F 0.90 1.95
Lys 177 A A . . . . . 1.40-1.51. * F 0.90 2.26
Thr 178 A A . . . . . 2.10-1.51* * F 0.90 3
.87
Ala 179 A A . . . . . 1.80-2.01* . F 0.90 4.01
Lys 180 A A . . . . . 1.58-1.63* . F 0.90 2.69
Glu 181 A A . . . . . 0.88-0.94* . F 0.90 1.54
Glu 182 A A . . . . . 0.62-0.64. . F 0.90 1.32
Ser 183 A . . . . . . 0.08-0.71. * F 1.10 1.02
Leu 184 . . B . . . . 0.46-0.07. * . 0.50 0.44
Phe 185 . . B . . . . 0.200.36. . . 0.20 0.39
Pro 186 . . . . . . C -0.100.79. . . 0.40 0.45
Val 187 . . . . . . C -0.060.79. . F 0.85 0.73
Pro 188 . . . . . T C 0.240.10. . F 1.80 1.69
Pro 189 . . . . . T C 0.74-0.69. . F 3.00 1.89
Ser 190 . . . . . T C 1.14-0.63. . F 2.70 3.67
Lys 191 . . . . . T C 0.77-0.89. . F 2.40 3.18
Glu 192 A A . . . . . 1.62-0.81. . F 1.50 2.08
Thr 193 A A . . . . . 1.53-1.24. . F 1.20 2.69
Ser 194 A A . . . . . 1.74-1.24. * F 0.90 I
.80
Ala 195 A A . . . . . 1.19-0.84. * F 0.90 1.80
Glu 196 A A . . . . . 0.84-0.20. * F 0.45 0.93
Ser 197 A . . . . . . 0.56-0.30. * F 0.65 0.93
Gln 198 A . . . . . . 0.280.23. * F 0.05 0.96
Val 199 . . B . . . . 0.370.23. * . -0.100.56
Ser 200 . . B . . . . 0.610.66. * . -0.400.65
Trp 201 . . . . . . C 0.310.70. * . -0.200.37
Ala 202 . . . . . T C -0.200.69. . . 0.00 0.67
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
Pro 203 . . . . T C -0.790.73 . F 0.15 0.41
. *
Gly 204 . . . T T . 0.07 0.84 . F 0.35 0.40
. *
Ser 205 . . . . T C -0.440.33 . F 0.45 0.68
. *
Leu 206 . B . . . . -0.860.51 . . -0.400.36
. *
5 Ala 207 . B . . . . -0.570.87 . . -0.400.32
. *
Gln 208 . B . . . . -1.170.83 . . -0.400.32
. .
L.eu209 . B . . . . -0.821.13 . . -0.400.32
. .
Phe 210 . B . . . . -0.820.44 . . -0.400.52
. .
Ser 211 . B . . . . -0.870.33 . . -0.100.40
. .
10 L.cu212 . B . . . . -0.490.57 . . -0.400.36
. .
Asp 213 . . . T . . -1.380.31 . F 0.45 0.65
. .
Ser 214 . . . . . C -0.780.21 . F 0.25 0.34
. .
Val 215 . . . . . C -0.080.26 * F 0.25 0.64
. .
Pro 216 . . . . . C 0.22 -0.03 . F 0.85 0.66
. .
15 Ile 217 . B . . . . 1.03 0.37 . F 0.05 0.86
. .
Pro 218 . B . . . . 1.03 0.39 . F 0.46 2.00
. .
Gln 219 . B . . . . 0.99 0.14 * F 0.72 2.24
. .
Gln 220 . B . . . . 1.63 0.14 . F 0.98 3.16
. .
Gln 221 . . . . . C 1.84 -0.11 . F 2.04 3.16
. .
20 Gln 222 . . . . . C 2.13 -0.54 . F 2.60 3.16
. .
Gly 223 . . . . T C 1.96 -0.33 . F 2.24 1.80
. .
Pro 224 . . . . T C 1.57 -0.30 . F 1.98 1.33
. .
Glu 225 . B . . T . 1.18 -0.27 . . 1.22 0.98
. .
Met 226 . B . . T . 0.79 -0.24 . . 1.1l 1.27
. *
25
TableIII
Res Pos 11 III N V VI VII VIII IX XI XII XIII X1V
I X
30 Met 1 . . B . . . . 0.20 -0.17 . . 0.93 1.00
.
Ala 2 . . B . . . . 0.56 -0.17 . . 1.06 0.78
.
Glu 3 . . . . . T C 0.64 -0.10 . . 1.74 0.83
.
Pro 4 . . . . . T C 1.00 -0.14 . . 2.17 1.12
.
Gly 5 . . . . T T . 1.36 -0.26 . F 2.80 1.51
.
35 His 6 . . . . . T C 1.14 -0.26 . . 2.17 1.18
.
Ser 7 . A . . . . C 1.43 0.43 . . 0.44 0.63
.
His 8 . A . . . . C 0.84 0.39 * . 0.46 0.86
.
His 9 . A . . . . C 1.17 0.46 * . -0.120.64
.
Leu 10 . A B . . . . 0.66 -0.04 * . 0.30 0.93
.
40 Ser 11 . A B B . . . 0.80 0.21 * . -0.300.51
.
Ala 12 . A B B . . . 0.76 -0.29 * . 0.30 0.73
.
Arg 13 . A B B . . . 0.90 -0.36 * . 0.30 0.87
.
Val 14 . . B B . . . 0.62 -1.04 * F 1.16 1.28
.
Arg 15 . . B B . . . 1.43 -0.94 * F 1.42 1.83
*
45 Gly 16 . . . . T . . 1.84 -1.44 * F 2.28 1.61
*
Arg 17 . . B . . . . 2.54 -1.44 * F 2.14 4.26
*
Thr 18 . . . . . . C 1.54 -2.09 * F 2.60 4.26
*
Glu 19 . . B . . . . 2.19 -1.40 * F 2.14 3
* .02
Arg 20 . . B B . . . 2.19 -1.40 * F 1.68 2.38
*
50 Arg 21 . . B B . . . 1.72 -1.40 * F 1.42 3.23
*
Ile 22 . . B B . . . 1.32 -1.20 * F 1.16 1.54
*
Pro 23 . . B B . . . 1.74 -0.29 . F 0.45 0.83
*
Arg 24 . . . B T . . 0.93 -0.29 . F 0.85 0.83
*
L.eu25 . . B B . . . 0.01 0.40 . . -0.300.97
*
55 Trp 26 . . B B . . . -0.910.40 * . -0.300.52
*
Arg 27 . . B B . . . -0.310.66 . . -0.600.22
*
L.eu28 . . B B . . . -0.691.57 . . -0.600.28
*
L.eu29 . . B B . . . -1.141.39 . . -0.600.27
*
L.eu30 . . B B . . . -0.640.90 * . -0.600.14
*
Trp 31 . . . B . . C -0.941.39 * . -0.400.24
*
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
56
Ala 32 . . . B . . C -1.761.20 * . -0.400.29
*
Gly 33 . . . B . . C -0.941.30 . . -0.400.30
.
Thr 34 . . . B . . C -0.991.01 . . -0.400.50
.
Ala 35 . . B B . . . -0.490.74 . . -0.600.37
.
Phe 36 . . B B . . . -0.200.73 . . -0.600.54
.
Gln 37 . . B B . . . 0.04 0.70 . . -0.600.64
.
Val 38 . . B B . . . 0.08 0.64 . . -0.600.63
.
Thr 39 . . B B . . . 0.04 0.63 . F -0.301.05
.
Gln 40 . . . B T . . 0.42 0.27 . F 0.250.60
.
10Gly 41 . . . . T . . 1.12 0.30 . F 0.601.25
.
Thr 42 . . . . . . C 0.31 -0.34 . F 1.001.50
*
Gly 43 . . . . . T C 1.13 -0.14 . F 1.050.72
.
Pro 44 . . . . T C 0.86 -0.04 . F 1.050.98
.
Glu 45 . . B . . T . 0.19 0.03 . F 0.250.69
.
15Leu 46 . . B . . T . 0.58 0.11 . . 0.100.37
.
His 47 . . B . . . . 0.89 -0.31 . . 0.500.48
.
Ala 48 . . . . . . C 0.93 -0.74 . . 1.000.48
.
Cys 49 A . . . . T . 1.14 -0.36 . . 0.700.78
.
Lys 5 0 . . . . T . 0.90 -1.04 . F I 1.00
A . .15
20Glu 51 A . . . . T . 1.68 -0.79 . F 1.301.55
.
Ser 52 . . . . T T . 1.47 -0.79 . F 1.703.93
*
Glu 53 . A . . T . . 2.06 -0.60 . F 1.303.08
.
Tyr 54 . A . . 'f . . 2.48 -0.60 . . 1.153.08
.
His 55 . A . . T . . 2.12 0.16 * . 0.253.60
.
25'1'yr56 . A . . T . . 1.53 0.26 * . 0.253.00
.
Glu 57 . A B . . . . 1.17 0.76 . . -0.451.93
.
Tyr 58 . A B . . . . 1.17 0.57 . . -0.600.76
.
Thr 59 . A B . . . . 1.11 0.07 . . -0.300.81
.
Ala 60 . A B . . . . 0.83 -0.30 . . 0.640.63
.
30Cys 61 . A B . . . . 0.73 0.19 . . 0.380.58
.
Asp 62 . A . . T . . 0.43 -0.14 . F 1.870.40
.
Ser 63 . . . . T T . 0.79 -0.24 . F 2.610.53
*
Thr 64 . . . . T T . 0.81 -0.74 * F 3.401.92
*
Gly 65 . . . . T T . 1.51 -0.40 * F 2.761.21
.
35Ser 66 . . . . T T . 1.32 -0.40 * F 2.421.77
.
Arg 67 . . . B T . . 0.73 -0.14 * F 1.530.91
*
'1'rp68 . . B B . . . 0.18 -0.13 * . 0.640.93
.
Arg 69 . . B B . . . 0.28 0.09 * . -0.300.51
.
Val 70 . . B B . . . 0.59 0.13 * . -0.300.41
.
40Ala 71 . . B B . . . 0.58 0.63 * . -0.600.53
.
Val 72 . . B B . . . 0.26 0.20 * . -0.300.39
.
Pro 73 . . . . T . . 0.20 0.63 * . 0.000.81
*
His 74 . . . . T . . -0.720.41 * . 0.000.79
*
Thr 75 . . . . . T C -0.530.60 . F 0.150.88
.
45Pro 76 . . . . T T . -0.260.53 . F 0.350.30
.
Gly 77 . . . . T T . 0.30 0.59 . F 0.350.32
.
Leu 78 . . B . . T . -0.300.47 . -0.200.30
.
Cys 79 . . B . . . . -0.480.67 . . -0.400.16
.
Thr 80 . . B . . . . -0.170.67 . . -0.400.25
.
50Ser 81 . . B . . . . -0.170.24 . F 0.050.51
*
Leu 82 . . B . . T . -0.68-0.01 . F 1.301.46
*
Pro 83 . . B . . T . 0.18 0.06 * F 0.850.75
*
Asp 84 . . . . . T C 0.50 -0.43 * F 2.101.12
*
Pro 85 . . . . 'f T . 0.50 -0.39 . F 2.601.34
*
55Val 86 . . . . T . . 0.80 -0.59 . F 3.001.25
*
Lys 87 . . B . . . . 0.94 -1.01 . F 2.301.30
*
Gly 88 . . B . . . . 0.86 -0.44 . F 1.550.45
*
Thr 89 . . B . . . . 0.16 -0.49 . F 1.250.81
*
Glu 90 . . B . . . . 0.07 -0.34 . F 0.950.35
.
60Cys 91 . . B . . T . 0.26 0.04 * . 0.100.48
.
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
57
Ser 92 . . B . . T . 0.21 0.19 * . 0.10 0.18
.
Phe 93 . . B . . T . -0.030.10 * . 0.10 0.16
.
Ser 94 . . . . T T . -0.070.60 * . 0.20 0.31
.
Cys 95 . . . . 'f . . -0.070.46 * . 0.00 0.23
.
Asn 96 . . . . T T . -0.100.07 . . 0.50 0.46
*
Ala 97 . . . . . T C -0.610.07 . . 0.30 0.30
*
Gly 98 . . . . . T C 0.09 0.37 . F 0.45 0.46
*
Glu 99 A . . . . '1' . -0.21-0.20 * . 0.70 0.47
.
Phe 100 . A B . . . . 0.50 0.01 . . -0.300.46
.
Leu 101 . A B . . . . 0.50 -0.49 . . 0.30 0.94
.
Asp 102 A A . . . . . 1.09 -0.91 . . 0.94 0.90
.
Met 103 . A . . T . . 1.13 -0.51 . . 1.83 1.81
.
Lys 104 . A . . T . . 0.47 -0.91 . F 2.32 2.94
*
Asp 105 . . . . T T . 1.21 -1.03 . F 2.91 0.94
*
Gln 106 . . . . T T . 1.81 -1.03 * F 3.40 1.90
.
Ser 107 . . . . '1' T . 1 -1 . F 3 1
.14 .21 .06 .47
.
Cys 108 . . . . T T . 1.16 -0.64 * F 2.57 0.47
.
Lys 109 . . B . . T . I.11 -0.14 * F 1.53 0.28
.
Pro 110 . . B . . T . 0.77 -0.54 * F 1.69 0.36
.
Cys 111 . . B . . T . 0.88 -0.50 * . 1.10 0.66
.
Ala 112 . . B . . T . 0.93 -1.07 * F 1.75 0.64
.
Glu 113 . . B . . . . 1.30 -0.31 * F 1.45 0.65
.
Gly 114 . . B . . T . 0.44 -0.36 * F 2.00 1.63
.
Arg 115 . . B . . T . 0.31 -0.24 * F 1.80 1.33
.
Tyr 116 . . B . . T . 0.67 -0.31 * . 1.30 0.76
.
Ser 117 . . B . . T . 0.91 0.17 * . 0.65 1.11
.
Leu 118 . . B B . . . 0.02 0.17 * . -0.100.56
*
Gly 119 . . . B T . . 0.48 0.86 * F -0.050.25
*
Thr 120 . . B B . . . -0.330.10 * F -0.150.37
*
Gly 121 . . B B . . . -0.090.50 * F -0.450.39
*
Ile 122 . A B B . . . 0.21 -0.19 * . 0.30 0.65
*
Arg 123 . A B B . . . 0.73 -0.61 * . 0.60 0.78
.
Phe 124 . A B B . . . 1.08 -0.19 * . 0.30 0.83
.
Asp 125 . A . B . . C 1.39 -0.61 * . 0.95 1.97
.
Glu 126 . A . . T . . 0.92 -1.30 * F 1.30 1.75
.
Trp 127 . A . . T . . 1.60 -0.61 * F 1.30 1.66
.
Asp 128 . A . . . . C 1.46 -0.97 * F 1.10 1.54
.
Glu 129 . A . . . . C 1.81 -0.47 . F 0.80 1.21
*
Leu 130 . . . . . T C 1.11 -0.04 . . 1.05 1.14
*
Pro 131 . . . . . T C 0.52 -0.17 . . 0.90 0.59
*
His 132 . . . . T T . 0.51 0.33 . . 0.50 0.34
*
Gly 133 . . . . . T C -0.300.71 . . 0.00 0.56
*
Phe 134 . A . . . . C -0.600.71 . . -0.400.30
*
Ala 135 . A . . . . C -0.380.67 * . -0.400.29
*
Ser 136 . A . . . . C -0.170.67 * . -0.400.30
.
Leu 137 . A . . . . C -0.730.64 * . -0.400.56
.
Ser 138 . . . . . 'f C -0.390.47 * . 0.00 0.55
.
Ala 139 . . . . . T C -0.50-0.03 * . 0.90 0.71
.
Asn 140 . . B . . T . 0.09 0.27 * . 0.10 0.71
.
Met 141 . . B . . T . 0.39 -0.41 * . 0.70 0.88
.
Glu 142 . . B . . . . 0.90 -0.80 * . 0.95 1.45
.
Leu 143 A . . . . T . 0.61 -0.91 * . 1.15 1.21
.
Asp 144 A . . . . T . 0.61 -0.81 * F 1.30 1.24
.
Asp 145 A . . . . T . 0.61 -0.93 * F 1.15 0.72
.
Ser 146 A . . . . T . 0.91 -0.93 . F 1.58 1.51
.
Ala 147 A . . . . . . 0.60 -1.23 . F 1.66 1.22
.
Ala 148 A . . . . . . 1.07 -0.74 . F 1.94 1.05
.
Glu 149 . . . . T . . 1.07 -0.31 . F 2.17 0.78
.
Ser 150 . . . . T T . 0.40 -0.30 . F 2.80 1.23
*
Thr 151 . . . . T T . 0.39 -0.23 . F 2.37 0.66
.
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
58
Gly 152 . . . . T T . 0.68 -0.24. . F 2.09 0.55
Asn 153 . . . . T T . 0.97 0.14. . F 1.21 0.55
Cys 154 . . . . T T . 1.01 0.14. . F 0.93 0.51
Thr 155 . . . . T T . 1.02 -0.34. . F 1.40 1.02
Ser 156 . . . . T T . 0.48 0.14. . F 0.65 0.67
Ser 157 . . . . T T . 0.61 0.39* * F 0.88 0.93
Lys 158 . . . . T . . 0.72 0.24* * F 0.91 0.99
Trp 159 . . B . . . . 1.04 -0.24* * F 1.49 1.45
Val 160 . . B . . T . 1.36 -0.20* * F 1.92 1.07
Pro 161 . . B . . T . 1.41 -0.59* * F 2.30 0.90
Arg 162 . . . . T T . 0.82 0.17* * F 1.72 1.33
Gly 163 . . B . . T . 0.19 -0.06* * F 1.69 1.26
Asp 164 . . . . T . . -0.22-0.20* * F 1.51 0.82
Tyr 165 . . B . . . . 0.63 0.16* . . 0.13 0.36
lle 166 . . B . . . . 0.53 0.56. * . -0.400.59
Ala 167 . . B . . . . 0.42 0.61. * . -0.400.51
Phe 168 . . B . . . . 0.77 0.61. . . -0.090.54
Asn 169 . . B . . T . 0.10 -0.14. . . 1.47 1.35
Thr 170 . . . . . T C 0.03 -0.26. . F 1.98 0.71
Asp 171 . . . . T T . 0.33 -0.27. * F 2.64 1.19
Glu 172 . . . . T T . 0.61 -0.56. . F 3.10 0.75
Cys 173 . . . B T . . 0.50 -0.47. . . 1.94 0.75
Thr 174 . . B B . . . -0.10-0.27. . . 1.23 0.37
Ala 175 . . B B . . . -0.030.34. . . 0.32 0.21
Thr 176 . . B B . . . -0.621.10. . . -0.290.62
Leu 177 . . B B . . . -1.481 * * . -0.600.43
.03
Met 178 . . B B . . . -0.811.19. * . -0.600.32
Tyr 179 . . B B . . . -1.311.09. * . -0.600.35
Ala 180 . . B B . . . -0.681.29. * . -0.600.35
Val 181 . . B B . . . -0.370.60. * . -0.600.71
Asn 182 . . B B . . . 0.14 0.39. * . -0.300.79
Leu 183 . . B B . . . 0.40 0.01. * F 0.21 1.05
Lys 184 . . B B . . . 0.33 -0.06. * F 1.02 1.40
Gln 185 . . . . T T . 0.07 -0.21. * F 2.03 1.25
Ser 186 . . . . . T C 0.92 0.03. * F 1.44 1.13
Gly 187 . . . . . T C 0.22 -0.26. * F 2.10 0.91
Thr 188 . . B . . T . 1.03 0.53. * F 0.79 0.45
Val 189 . . B B . . . 0.74 0.13. * F 0.48 0.59
Asn 190 . . B B . . . 0.50 0.50. * . -0.180.93
Phe 191 . . B B . . . 0.56 0.83. * . -0.241.01
Glu 192 . . B B . . . 0.69 1.10. * . -0.452.13
Tyr 193 . . B B . . . 1.00 0.89. * . -0.452.04
Tyr 194 . . . . T . . 1.56 0.49. * . 0.15 3.94
Tyr 195 . . . . . T C 1.26 0.09. * . 0.45 3.05
Pro 196 . . . . T T . 1.07 0.47. . F 0.50 2.61
Asp 197 . . . . T T . 0.18 0.40. . F 0.50 1.17
Ser 198 . . . . . T C -0.280.33. . F 0.45 0.52
Ser 199 . . B B . . . -0.030.36. * F -0.150.29
Ile 200 . . B B . . . -0.49-0.07. * . 0.30 0.30
Ile 201 . . B B . . . -0.980.71. * . -0.600.20
Phe 202 . . B B . . . -1.831.11* * . -0.600.13
Glu 203 . . B B . . . -1.531.37. * . -0.600.13
Phe 204 . . B B . . . -1.231.09. * . -0.600.33
Phe 205 . . B B . . . -0.340.80* * . -0.600.62
Val 206 . . . B T . . 0.54 0.01* . . 0.10 0.59
Gln 207 . . . . T T . 0.58 0.41. * F 0.50 1.19
Asn 208 . . . . T T . 0.58 0.20. . F 0.65 0.73
Asp 209 . . . . T T . 1.07 -0.19. . F 1.40 1.71
Gln 210 . . . . 1' T . 1.77 -0.40. . F 1.74 1.53
Cys 211 . . . . T . . 2.03 -0.40. * F 1.88 1.53
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
59
Gln 212 . B . . T . 2.03-0.30 * F 1.870.93
. .
Pro 213 . B . . T . 2.03-0.30 * F 2.210.89
. .
Asn 214 . . . T T . 1.73-0.70 . F 3.402.78
. .
Ala 215 . . . T T . 1.84-0.89 * F 3.062.15
. .
Asp 216 . . . T . . 2.22-1.29 . F 2.752.73
. .
Asp 2l7 . . . T T . 1.62-0.80 . F 2.841.78
. *
Ser 218 . . . . T C 1.88-0.59 . F 2.531.75
. *
Arg 219 . . . T T . 1.57-1.09 . F 2.622.09
. *
Trp 220 . B . . T . 1.84-0.60 . . 2.301.81
. *
Met 221 A B . . . . 1.84-0.11 . . 1.371.95
. *
Lys 222 A B . . . . 1.89-0.50 * F 1.291.72
. *
Thr 223 A . . . . C 1.84-0.50 . F 1.263.27
. *
Thr 224 A . . . . C 1.44-0.99 . F 1.333.27
. *
Glu 225 A . . . . C 1.73-0.69 . F I.101.72
. *
Lys 226 A . . . . C 1.63-0.69 * F 1.102.06
. *
Gly 227 A . . T . . 1.56-0.39 . F 1.001.24
. *
Trp 228 A . . . . C 1.57-0.37 . . 0.500.97
. .
Glu 229 A . . . . C 1.020.01 . . -0.100.65
. .
Phe 230 A B . . . . 1.020.66 . . -0.600.49
. .
His 231 A B . . . . 0.170.23 . . -0.300.81
. .
Ser 232 A B . . . . 0.510.00 . . -0.300.38
. .
Val 233 A B . . . . 0.910.40 . . -0.260.71
. *
Glu 234 A . . . . C 0.57-0.39 . . 1.331.03
. .
Leu 235 A . . . . C 1.27-0.46 . . 1.520.76
. .
Asn 236 . . . T T . 1.30-0.44 . F 2.761.64
. .
Arg 237 . . . T T . 0.74-0.69 . F 3.401.52
. *
Gly 238 . . . 'f T . 0.79-0.04 . F 2.761.37
. *
Asn 239 . . . T T . 0.54-0.04 . F 2.270.70
. *
Asn 240 . . B . . C 1.070.31 * F 0.730.56
. *
Val 241 . B B . . . 1.181.23 * . -0.260.60
. .
Leu 242 . B B . . . 0.760.80 * . -0.600.73
. .
Tyr 243 . B B . . . 0.790.89 * . -0.600.65
. .
Trp 244 . B B . . . 0.200.97 * . -0.451.27
. .
Arg 245 . B B . . . -0.500.83 . . -0.451.56
. .
Thr 246 . B B . . . 0.060.93 . F -0.450.86
. .
Thr 247 . B B . . . 0.010.56 * . -0.451.10
. .
Ala 248 . B B . . . -0.030.29 . . -0.300.42
. .
Phe 249 . B B . . . -0.061.20 * . -0.600.30
. .
Ser 250 . B B . . . -0.121.20 * . -0.600.30
. .
Val 251 . . B T . . -0.670.71 * . -0.200.60
. *
Trp 252 . B B . . . -0.570.86 . . -0.360.51
. *
'fhr 253 . B B . . . 0.070.50 . F 0.030.59
. *
Lys 254 . . B . . C 0.560.11 * F 0.921.60
. ' *
Val 255 . . . . T C -0.00-0.10 . F 2.162.35
. *
Pro 256 . . . . T C 0.04-0.37 . F 2.401.21
. *
Lys 257 . B . . T . -0.52-0.17 * F 1.810.50
. *
Pro 258 . B . . T . -0.100.47 * F 0.670.50
. .
Val 259 . B B . . . -0.14-0.17 . . 0.780.63
. *
Leu 260 . B B . . . -0.18-0.20 . . 0.540.51
. .
Val 261 . B B . . . -0.560.49 . . -0.600.23
. .
Arg 262 . B B . . . -1.490.56 . . -0.600.31
. .
Asn 263 . B B . . . -1.590.60 . . -0.600.27
. *
Ile 264 . B B . . . -1.080.40 . . -0.600.52
. .
Ala 265 . B B . . . -1.120.19 . . -0.300.26
. *
Ile 266 . B B . . . -0.860.83 . . -0.600.12
. *
Thr 267 . B B . . . -1.210.93 . . -0.600.17
. .
Gly 268 . B B . . . - I .00 * . -0.600.27
. I .
.52
Val 269 . B B . . . -0.930.99 . . -0.600.56
. .
Ala 270 . B B . . . -0.340.69 . . -0.600.52
. .
'fyr 271 . B B . . . -0.120.20 . . -0.300.90
. *
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
Thr 272 . B . . T . -0.51 0.34 . F 0.25 0.65
. .
Ser 273 . B . T T . -0.38 0.49 . F 0.35 0.56
. .
Glu 274 . . . T T . -0.19 0.41 . F 0.35 0.55
. .
Cys 275 . B . . T . 0.44 0.23 . . 0.10 0.20
. .
5 Phe 276 . B . . . . 0.48 -0.26 . . 0.50 0.31
. .
Pro 277 . . . T . . 0.44 -0.21 . . 0.90 0.27
. .
Cys 278 . . . T . . 0.43 0.21 . . 0.30 0.50
. .
Lys 279 . . . . T C 0.19 0.13 . F 0.45 0.84
. .
Pro 280 . . . T T . 0.27 0.10 * F 0.65 0.85
. .
10 Gly 281 . . . T T . 0.97 0.17 * F 0.80 1.60
. *
1'hr282 . B . . T . 1.22 -0.40 . F 1.34 1.34
. *
Tyr 283 . B . . . . 1.89 -0.40 . F 1.48 1.73
. *
Ala 284 . B . . . .. 1.50 -0.43 . F 1.82 3.03
. *
Asp 285 . B . . T . 1.41 -0.43 . F 2.36 2.08
. *
15 I_ys286 . . . T 1' . 1.46 -0.53 . F 3.40 1.78
. *
Gln 287 . . . T T . 1.07 -0.90 . F 3.06 2.36
. *
Gly 288 . . . T T . 0.64 -0.61 . F 2.72 1.22
. *
Ser 289 . . . T T . 1.28 -0.04 . F 1.93 0.33
. *
Ser 290 . B . T T . 0.47 -0.04 . F 1.59 0.38
. *
20 Phe 291 . B . . T . -0.24 0.24 . . 0.10 0.32
. *
Cys 292 . B . . T . -0.46 0.39 . . 0.10 0.13
. *
Lys 293 . B . . . . -0.70 0.43 . . -0.400.15
. *
Leu 294 . B . . . . -0.40 0.54 . . -0.400.17
. .
Cys 295 . B . . . . -0.40 0.16 . . -0.100.51
. .
25 Pro 296 . B . . . . 0.06 -0.03 . . 0.50 0.34
. .
Ala 297 . . . T . . 0.42 0.73 . . 0.00 0.65
. .
Asn 298 . . . T T . 0.38 0.43 . F 0.84 1.62
. .
Ser 299 . . . T T . 1.23 0.26 . F 1.48 1.69
. *
Tyr 300 . . . T T . 1.56 -0.17 . F 2.42 3.34
. .
30 Ser 301 . . . . T C 1.77 -0.24 . F 2.56 2.05
. *
Asn 302 . . . T T . 2.04 -0.64 . F 3.40 2.65
. *
Lys 303 . . . T T . 1.74 -0.54 . F 3.06 2.44
. .
Gly 304 . . . T T . 1.38 -0.91 * F 2.72 2.44
. .
Glu 305 . . . T T . 1.59 -0.73 . F 2.23 0.81
. *
35 Thr 306 . . . T T . 1.89 -0.63 . F 1.89 0.55
. *
Ser 307 . B . . T . 1.22 -0.23 . F 0.85 0.97
. *
Cys 308 . B . . T . I.18 -0.09 * . 1.04 0.30
. .
His 309 . B . . T . 1.31 -0.09 . . 1.38 0.35
. .
Gln 310 . . . T . . 1.31 -0.14 . . 1.92 0.40
. .
40 Cys 311 . . . T . . 1.67 -0.53 . . 2.71 1.25
. .
Asp 312 . . . T T . 1.72 -I.10 . F 3.40 1.84
. .
Pro 3l3 . . . T T . 2.09 -0.84 . F 3.06 1.66
. .
Asp 314 . . . T T . 2.12 -0.86 . F 3.06 4.15
. .
Lys 315 . . . T T . 2.17 -1.43 . F 3.06 4.31
. *
45 Tyr 316 . B . . T . 2.49 -1.43 . F 2.66 5.57
. *
Ser 317 . B . . T . 2.19 -1.43 . F 2.66 3.30
. *
Glu 318 . . . T T . 2 .10 -1.04 . F 3 2.21
. * .40
Lys 319 . . . T T . 1.80 -0.66 . F 3.06 1.89
. *
Gly 320 . . . T . . 1.09 -1.03 * F 2.52 1.89
. *
50 Ser 321 . . . T T . 1.33 -0.84 * F 2.23 0.59
. .
Ser 322 . . . T T . 0.78 -0.44 * F 1.59 0.47
. .
Ser 323 . . . T T . 0.89 0.20 * F 0.65 0.35
. .
Cys 324 . . . T T . 0.63 -0.23 * F 1.25 0.52
. .
Asn 325 . . . T . . 0.39 -0.19 * . 0.90 0.60
. .
55 Val 326 . B . . . . 0.02 -0.07 * . 0.50 0.45
. .
Arg 327 . B . . T . 0.01 0.11 * . 0.10 0.45
. .
Pro 328 . B . . T . 0.31 0.03 * . 0.44 0.40
. .
Ala 329 . B . . T . 1.02 -0.37 * . 1.38 0.91
. .
Cys 330 . B . . T . 1.02 -1.01 * . 2.02 0.93
. .
60 Thr 331 . B . . . . 1.63 -1.01 * F 2.31 1.00
. *
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
61
Asp 332 . . . . T T . 0.82 -0.69* . F 3.40 1.55
Lys 333 . . . . T T . 0.79 -0.40. . F 2.76 2.50
Asp 334 . . . . T T . 1.07 -0.21. . F 2.42 2.72
Tyr 335 . . B . . T . 1.70 -0.21. . . 1.53 2.35
Phe 336 . . B B . . . 1.70 0.29. . . 0.19 1.60
Tyr 337 . . B B . . . I.11 0.77. . . -0.451.38
Thr 338 . . B B . . . 0.40 1.27. . . -0.600.89
His 339 . . B B . . . 0.40 1.09. . . -0.600.55
Thr 340 . . B B . . . 0.06 0.30. . . -0.300.59
Ala 341 . . B B . . . 0.76 0.04. * . 0.00 0.41
Cys 342 . . . B T . . 0.66 -0.04. * . 1.30 0.49
Asp 343 . . . . T T . 0.97 -0.11. * . 2.00 0.33
Ala 344 . . . . . T C 0.69 -0.60. * F 2.55 0.57
Asn 345 . . . . . T C 1.00 -0.61. * F 3.00 1.54
Gly 346 . . . . . T C 0.78 -0.79. * F 2.70 1.60
Glu 347 . A . . . . C 0.84 -0.10. * F 1.70 1.30
Thr 348 . A B . . . . 0.60 0.01. * F 0.45 0.80
Gln 349 . A B . . . . 1.23 0.37* *. F 0.30 1.27
Leu 350 . A B . . . . 0.94 -0.06* * . 0.45 1.47
Met 351 . A B . . . . 0.70 0.86* * . -0.451.07
Tyr 352 . A B . . . . 0.74 0.87* * . -0.600.62
Lys 353 . A . . T . . 0.84 0.47* * . -0.051.51
Trp 354 . A . . T . . 0.89 0.21* * . 0.25 2.36
Ala 355 . A . . . . C 0.81 -0.40. * F 0.80 3.01
Lys 356 . A . . . . C 0.74 -0.47* * F 0.80 1.06
Pro 357 . A . . T . . 0.69 0.10. * F 0.25 0.54
Lys 358 . . . . T . . 0.64 -0.43. * F 1.05 0.71
Ile 359 . . B . . . . 0.93 -0.93. * . 0.80 0.62
Cys 360 . . B . . T . 0.71 -0.93. * . 1.00 0.67
Ser 361 . . B . . T . 0.67 -0.67. * F 1.15 0.28
Glu 362 . . B . . T . 0.53 -0.67* . F 1.15 0.68
Asp 363 A . . . . T . -0.10-0.93* * F 1.30 1.26
Leu 364 A A . . . . . -0.07-1.00* * F 0.75 0.95
Glu 365 A A . . . . . 0.64 -0.74* * F 0.75 0.41
Gly 366 A A . . . . . 0.13 -0.74* * F 0.75 0.49
Ala 367 . A B . . . . -0.08-0.06* * . 0.30 0.49
Val 368 . A B . . . . -0.67-0.31* * . 0.30 0.43
Lys 369 . A B . . . . -0.160.19* * . -0.300.44
Leu 370 . A B . . . . -0.500.14* * . -0.300.59
Pro 371 . . B . . T . -1.010.07* * . 0.10 0.78
Ala 372 . . . . T T . -0.380.07* * F 0.65 0.29
Ser 373 . . . . T T . 0.17 0.07* * F 0.65 0.70
Gly 374 . . . . T T . 0.09 -0.13* * F 1.25 0.66
Val 375 . . B . . . . 0.23 -0.06. . F 0.79 0.89
Lys 376 . . B . . . . 0.23 0.01. . F 0.33 0.35
Thr 377 . . B . . . . 0.61 0.06. . F 0.47 0.55
His 378 . . B . . . . 0.24 0.06* . . 0.61 1.15
Cys 379 . . B . . T . 0.59 -0.01. . . 1.40 0.31
Pro 380 . . B . . T . 1.23 0.39* * F 0.81 0.34
Pro 381 . . . . T T . 0.84 0.33. . F 1.07 0.39
Cys 382 . . . . T T . 0.46 0.26. . F 0.93 0.72
Asn 383 . . . . . T C -0.210.47. . F 0.29 0.40
Pro 384 . . . . T T . 0.50 0.83. . F 0.35 0.23
Gly 385 . . . . T T . 0.40 0.40. . . 0.20 0.85
Phe 386 . . B . . T . 0.61 0.31. . . 0.10 0.76
Phe 387 . . B . . . . 1.28 0.31. . F 0.30 0.79
Lys 388 . . . . T . . 0.98 0.29. . F 1.10 1.28
Thr 389 . . . . T . . 0.88 0.24. . F 1.35 1.99
Asn 390 . . . . T . . 0.56 -0.06* . F 2.20 3.31
Asn 391 . . . . T T . 1.26 -0.27* . F 2.50 0.89
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
62
Ser 392 . . . T 'C . 1.74 0.13* . F 1.80 1.06
.
Thr 393 . . . T T . 1.03 0.07. . F 1.55 1.02
.
Cys 394 . . . 'f T . 1.13 0.24. . F 1.15 0.34
.
Gln 395 . B . . . . 0.89 0.27. . F 0.30 0.39
.
Pro 396 . B . . . . 0.54 0.64. . F -0.250.43
.
Cys 397 . B . . T . 0.54 0.59. . . -0.200.79
.
Pro 398 . B . . 'f . 0.61 0.40. . . -0.200.61
.
Tyr 399 . . . T T . 0.98 0.76. . . 0.20 0.62
.
Gly 400 . . . T T . 0.98 0.71. . . 0.35 1.55
.
Ser 401 . . . T . . 0.84 0.54. . F 0.30 1.61
.
Tyr 402 . . . T T . 1.21 0.54. . F 0.50 1.02
.
Ser 403 . . . T T . 1.42 0.17. . F 1.11 1.38
.
Asn 404 . . . T T . 1.00 -0.26. . F 2.02 1.71
.
Gly 405 . . . T T . 1.03 -0.07* . F 2.18 0.59
.
Ser 406 . . . T '1' . 1.44 -0.34* . F 2.49 0.63
.
Asp 407 . . . T 'f . 1.02 -0.73* . F 3.10 0.77
.
Cys 408 . B . . T . 1.11 -0.56* * F 2.39 0.42
.
Thr 409 . B . . T . 0.52 -0.56* * F 2.39 0.48
.
Arg 410 . B . . . . 0.52 -0.44* . F 1.89 0.29
.
Cys 411 . B . . 'f . 0.51 -0.01. . . 1.94 0.54
.
Pro 412 . . . T T . 0.51 -0.10. . . 2.34 0.54
.
Ala 413 . . . T T . 0.97 -0.59. * F 3.10 0.47
.
Gly 414 . . . . T C 0.69 -0.16* * F 2.44 1.37
.
Thr 415 . . . . . C -0.28-0.23. * F 1.78 0.89
.
Glu 416 . B . . . . 0.04 -0.01. . F 1.27 0.66
.
Pro 417 . B . . . . -0.44-0.09. . F 0.96 0.66
.
Ala 418 . B . . . . 0.14 0.27. * . -0.100.39
.
Val 419 . B . . . . 0.24 -0.21. * . 0.50 0.39
.
Gly 420 . B . . . . 0.60 0.54. * . -0.400.40
.
Phe 421 A B . . . . 0.31 0.11. * . -0.300.79
.
Glu 422 A B . . . . 0.23 0.53. * . -0.451.12
.
Tyr 423 A . . T . . 0.82 0.80* * . -0.051.19
.
Lys 424 A . . T . . 1.37 0.77* * . -0.052.21
.
Trp 425 A . . T . . 0.90 0.47* * . -0.051.84
.
Trp 426 A . . 'f . . 1.39 1.16* * . -0.200.97
.
Asn 427 . . . . . C 1.08 0.83* * . -0.200.75
.
'Chr428 . . . . . C 1.32 1.31* . F 0.10 1.03
.
Leu 429 . . . . . C 0.68 0.80* * F 0.10 1.57
.
Pro 430 . . . . T C 0.97 0.50* . F 0.12 0.97
.
Thr 431 . . . . T C 0.94 O.10* . F 0.54 1.16
.
Asn 432 . . . . T C 0.63 0.10* * F 0.51 2.03
.
Met 433 . B . . T . 0.09 -0.10. * F 0.88 1.90
.
Glu 434 . B B . . . 0.09 0.11. * F -0.300.98
.
Thr 43S . B B . . . -0.000.31. . F -0.270.50
.
Thr 436 . B B . . . -0.030.30* . F -0.240.68
.
Val 437 . B B . . . -0.920.11* * F -0.210.39
.
Leu 438 . B B . . . -0.320.80* * F -0.480.19
.
Ser 439 . B B . . . -1.020.71. * F -0.450.21
.
Gly 440 . . B . . C -0.711.01. * . -0.400.24
.
Ile 441 A B B . . . -0.640.37* * . -0.300.51
.
Asn 442 A B B . . . 0.26 0.44* * . -0.600.60
.
Phe 443 A B B . . . 0.72 0.06* * . -0.151.21
.
Glu 444 A B B . . . 0.42 0.06* * . -0.151.71
.
Tyr 445 . B . . T . 0.46 -0.01* * . 0.85 1.05
.
Lys 446 . . . T T . 1.00 0.07* * F 0.80 1.76
.
Gly 447 . . . T T . 0.71 -0.29* * F 1.40 1.00
.
Met 448 . . . . T C 1.41 0.63. * F 0.15 0.67
.
Thr 449 . . . . . C 0.56 -0.13. * F 0.85 0.58
.
Gly 450 A . . . . C 0.21 0.51. * . -0.400.44
.
'1'rp451 A B . . . . -0.180.59* * . -0.420.45
.
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
63
Glu 452 . A B . . . . 0.17 0.40* . . -0.240.31
Val 453 . A B . . . . 0.73 -0.09* . . 0.84 0.52
Ala 454 . A B . . . . 0.16 -O.OI* . . 1.02 0.67
Gly 455 . . . . T . . 0.26 -0.24* . . 1.80 0.27
Asp 456 . . . B T . . 0.23 0.51* . . 0.52 0.57
His 457 . . B B . . . -0.360.36* . . 0.24 0.82
Ile 458 . . B B . . . -0.090.36. . . 0.06 0.83
Tyr 459 . . B B . . 0.16 0.43* . . -0.420.50
Thr 460 . . B B . . . -0.090.86. . . -0.600.37
Ala 461 . . B B . . . -0.390.86. . . -0.600.53
Ala 462 . . B . . . . -0.360.56. . . -0.120.45
Gly 463 . . . . . . C 0.53 -0.20. . . 1.26 0.52
Ala 464 . . . . . . C 0.78 -0.29. . F 1.69 0.83
Ser 465 . . . . . T C 0.39 -0.79. . F 2.62 1.38
Asp 466 . . . . T 'C . 0.38 -0.50. . F 2.80 1.21
Asn 467 . . . . . T C 0.08 -0.31. . F 2.32 1.18
Asp 468 . . B . . T . -0.39-0.13. . F 1.69 0.62
Phe 469 . . B B . . . -0.110.17. . . 0.26 0.30
Met 470 . . B B . . . -0.620.66. . . -0.320.27
Ile 471 . . B B . . . -1.480.94. . . -0.600.14
Leu 472 . . B B . . . -2.331.59. . . -0.600.12
Thr 473 . . B B . . . -2.541.44. . . -0.600.09
Leu 474 . . B B . . . -2.191.26. . . -0.600.19
Val 475 . . B B . . . -2.291.00* * . -0.600.23
Val 476 . . B B . . . -1.291.10* . . -0.600.14
Pro 477 . . B . . . . -0.690.61* * . -0.400.3
3
Gly 478 . . . . T . . -0.590.36* . F 0.45 0.68
Phe 479 . . B . . . . 0.22 0.14* . F 0.45 1.42
Arg 480 . . . . . . C 0.78 -0.10* * F 1.50 1.59
Pro 481 . . . . . 'f C 0.78 -0.14* . F 1.95 2.16
Pro 482 . . . . T 'f . 0.39 0.07* * F 1.80 1.85
Gln 483 . . . . T T . 0.14 -0.10* * F 2.50 0.93
Ser 484 . . B . . T . 0.84 0.40* * F 0.95 0.61
Val 485 . . B . . . . 0.42 -0.03* * . 1.55 0.66
Met 486 . . B . . . . 0.63 0.03. . . 1.00 0.55
Ala 487 . . B . . . . 0.84 -0.37. * . 1.65 0.71
Asp 488 . . B . . 'f . 0.89 -0.36. . F 2.20 1.54
Thr 489 . . . . . T C 1.19 -1.00. . F 3.00 3.11
Glu 490 A . . . . T . 1.19 -1.61. . F 2.50 5.33
Asn 491 A . . . . T . 1.20 -1.47* * F 2.20 2.37
Lys 492 A . . . . . . 1.90 -0.97* * F 1.70 1.66
Glu 493 A . . . . . . 1.01 -1.46* * F 1.40 1.88
Val 494 A . . B . . . 1.01 -0.77* * . 0.60 0.82
Ala 495 . . B B . . . 0.31 -0.69* * . 0.60 0.59
Arg 496 . . B B . . . -0.540.10* * . -0.300.29
Ile 497 . . B B . . . -1.290.74* * . -0.600.29
Thr 498 . . B B . . . -1.290.89* * . -0.600.25
Phe 499 . . B B . . . -0.740.39* * . -0.300.22
Val 500 . . B B . . . -0.970.87* * . -0.600.46
Phe 501 . . B B . . . -1.740.87* * . -0.600.26
Glu 502 . . B B . . . -1.160.96* * . -0.600.16
Thr 503 . . . B T . . -1.700.56* . . -0.200.29
Leu 504 . . . B T . . -1.000.56* * . -0.200.25
Cys 505 . . . B T . . -0.810.17* * . 0.10 0.23
Ser 506 . . . . T T . -0.110.74* * . 0.20 0.09
Val 507 . . . . T T . -0.920.26* * . 0.50 0.18
Asn 508 . . B . T T . -0.860.26. * . 0.50 0.28
Cys 509 . . B . . T . -0.740.44. * . -0.200.33
Glu 510 . . B B . . . -0.680.84. * . -0.600.38
Leu 511 . . B B . . . -1.230.81. * . -0.600.24
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
64
Tyr 512 . B B . . . -0.721.06 * . -0.600.33
. .
Phe 513 . B B . . . -1.580.91 * . -0.600.19
. .
Met 514 . B B . . . -0.911.56 * . -0.600.17
. .
Val 515 . B B . . . -1.211.27 * . -0.600.17
. .
Gly 516 . B B . . . -0.290.90 * . -0.320.27
. .
Val 517 . B B . . . -0.360.11 * . 0.26 0.53
. .
Asn 518 . . . . T C 0.34 -0.01 * F 2.04 1.03
. .
Ser 519 . . . . T C 0.63 -0.26 * F 2.32 1.67
. .
Arg 520 . . . T T . 1.28 -0.20 * F 2.80 3.24
. .
Thr 521 . . . T T . 0.77 -0.41 * F 2.52 3.12
. .
Asn 522 . . . . . C 1.62 -0.17 * F 1.84 1.73
. *
Thr 523 . . . . . C 1.31 -0.56 * F 1.86 1.53
. *
Pro 524 . B . . . . 1.32 -0.07 * F 1.08 1.53
. *
Val 525 . B . . . . 1.26 0.36 * F 0.05 1.00
. .
Glu 526 . B . . . . 1.22 -0.04 . F 0.80 1.38
. *
Thr 527 . B . . . . 0.92 -0.10 . F 0.99 0.88
. *
Trp 528 . B . . . . 1.28 -0.14 . F 1.48 1.60
. .
Lys 529 . . . T . . 1.14 -0.79 . F 2.52 1.85
. .
Gly 530 . . . T . . 2.04 -0.36 . F 2.56 1.27
. .
Ser 5 31 . . . T T . 2.04 -0.84 . F 3 2.41
. . .40
Lys 532 . . . . T C 2.06 -1.36 . F 2.86 2.08
. .
Gly 533 . . . T T . 2.10 -0.97 . F 2.72 2.82
. .
Lys 534 . . . T T . 1.74 -0.64 . F 2.38 3.30
. .
Gln 535 . . B T . . 1.84 -0.54 . F 1.64 2.38
. *
Ser 536 . B B . . . 1.26 0.21 * F 0.00 3.77
. *
Tyr 537 . B B . . . 0.32 0.47 * . -0.451.32
. *
Thr 538 . B B . . . 0.67 1.16 . . -0.600.53
. *
Tyr 539 . B B . . . 0.62 0.76 . . -0.600.69
. .
Ile 540 . B B . . . 0.62 0.37 . . -0.300.76
. *
Ile 541 . B B . . . 0.61 0.01 . . -0.040.85
. .
Glu 542 . B B . . . 0.54 0.01 . F 0.37 0.78
. .
Glu 543 . B . . . . 0.54 -0.26 . F 1.58 1.61
. .
Asn 544 . . B T . . 0.49 -0.46 . F 2.04 3.32
. .
Thr 545 . . B T . . 0.68 -0.76 . F 2.60 2.57
. .
Thr 546 . . B . . C 1.26 0.03 * F 1.24 1.29
. .
Thr 547 . . B . . C 0.97 0.51 . F 0.68 1.15
. .
Ser 548 . . B . . C 0.38 1.03 * F 0.27 0.84
. *
Phe 549 . B B . . . -0.321.04 * . -0.340.59
. *
Thr 550 . B B . . . -0.011.34 . . -0.600.35
. *
Trp 551 . B B . . . 0.41 1.26 . . -0.600.46
. *
Ala 552 . . B . . C 0.41 0.87 . . -0.251.03
. *
Phe 553 . . B T . . 0.40 0.57 * . -0.051.03
. *
Gln 554 . . B T . . 0.40 0.57 * . -0.051.42
. *
Arg 555 . . B . . C 0.68 0.44 . F -0.101.21
. .
Thr 556 . . B . . C 0.97 0.44 . F -0.101.91
. .
Thr 557 . . B . . C 0.97 -0.34 . F 0.80 1.91
. *
Phe 558 . . B . . C 1.37 -0.24 * . 0.50 0.98
. *
His 559 . . B . . C 1.48 0.14 * . -0.100.91
. *
Glu 560 . . B . . C 1.41 -0.34 * . 0.65 1.24
. *
Ala 561 . . . T . C 1.48 -0.83 . F 1.84 2.86
. *
Ser 562 . . . T . . 1.48 -0.86 . F 2.18 3.30
. *
Arg 563 . . . T . . 2.18 -0.87 . F 2.52 2.75
. *
Lys 564 . . . T . . 2.21 -0.47 . F 2.56 4.38
. *
Tyr 565 . . . T T . 1.36 -0.97 . F 3.40 5.45
. *
Thr 566 . . . T T . 1.36 -0.71 . F 3.06 2.07
. *
Asn 567 . B . . T . 1.70 -0.21 . F 2.02 1.04
. *
Asp 568 . B . . T . 0.70 -0.21 . F 1.68 1.33
. *
Val 569 . B B . . . 0.41 -0.29 . F 0.79 0.65
. *
Ala 570 . B B . . . 0.36 -0.01 . . 0.30 0.63
. *
Lys 571 . B B . . . -0.22-0.03 . . 0.30 0.51
. *
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
Ile 572 . B B . . . -0.22 0.66* . . -0.600.48
.
Tyr 573 . B B . . . -1.08 0.41. . . -0.600.76
.
Ser 574 . B B . . . -0.53 0.56. . . -0.600.28
.
Ile 575 . B B . . . 0.06 1.04. . . -0.600.58
.
5 Asn 576 . B B . . . -0.84 0.76. . . -0.600.60
.
Val 577 . B B . . . -0.56 0.64. . . -0.600.33
.
Thr 578 . B B . . . -0.31 0.87. * . -0.600.47
.
Asn 579 . B B . . . -0.36 0.59* * . -0.600.47
.
Val 580 . B . . T . -0.32 0.61* . . -0.200.62
.
10Met 581 . B . . T . -0.91 0.61* . . -0.200.32
.
Asn 582 . B . . T . -0.36 0.63* . . -0.200.20
.
Gly 583 . B . . T . -0.29 0.61* . . -0.200.36
.
Val 584 . B . . . . -0.96 0.73* . . -0.400.57
.
Ala 585 . B . . 'f . 0.01 0.69* . . -0.200.19
.
15Ser 586 . B . . T . 0.40 0.29* . . 0.10 0.38
.
Tyr 587 . B . . T . -0.27 0.29* * . 0.10 0.79
.
Cys 588 . B . . T . -0.51 0.21* . . 0.10 0.42
.
Arg 589 . B . . T . -0.47 0.21. . . 0.10 0.32
.
Pro 590 . B . . T . 0.12 0.51. . . -0.200.17
.
20Cys 591 . B . . T . -0.17 -0.24. . . 0.70 0.54
.
Ala 592 . B . . T . -0.22 -0.31* . . 0.70 0.28
.
Leu 593 . B . . . . 0.44 0.07* . . -0.100.24
.
Glu 594 . B . . . . -0.52 -0.36* . . 0.75 0.75
.
Ala 595 . B . . . . -0.66 -0.29. . F 1.15 0.55
.
25Ser 596 . B . . . . -0.29 -0.36. . F 1.40 0.66
.
Asp 597 . . . T T . 0.00 -0.66. . F 2.55 0.51
.
Val 598 . . . T T . 0.14 -0.27. . F 2.50 0.68
.
Gly 599 . . . T T . -0.17 -0.20* . F 2.25 0.27
.
Ser 600 . . . T T . 0.12 -0.10* . F 2.00 0.23
.
30Ser 601 . . . T . . -0.24 0.29* . F 0.95 0.42
.
Cys 602 . B . . T . -0.46 0.21. . F 0.50 0.23
.
Thr 603 . B . . T . -0.19 0.21. . F 0.25 0.26
.
Ser 604 . B . . T . -0.19 0.33. . F 0.25 0.20
.
Cys 605 . B . . T . -0.13 0.37. . . 0.10 0.37
.
35Pro 606 . B . . T . -0.08 0.56. . . -0.200.40
.
Ala 607 . . . T T . -0.30 0.83. . . 0.20 0.47
.
Gly 608 . B . . T . 0.01 1..13* . . -0.200.61
.
Tyr 609 . B . . T . 0.42 0.56* . . -0.200.66
.
Tyr 610 . B B . . . 1.09 0.13* . . 0.19 1.28
.
40Ilc 611 . B B . . . I.00 -0.37* . . 1.13 2.16
.
Asp 612 . B B . . . 1.24 -0.41* . . 1.47 1.84
.
Arg 613 . . . T . . 1.28 -0.74* . F 2.86 1.16
.
Asp 614 . . . T T . 0.86 -1.01* . F 3.40 2.40
.
Ser 615 . . . T T . 1.07 -1.13* . F 2.91 0.77
.
45Gly 616 . . . T T . 1.66 -0.63* . F 2.57 0.53
.
Thr 617 . . . T T . 0.99 -0.24. . F 1.93 0.43
.
Cys 618 . . . T T . 0.67 0.33. . . 0.84 0.17
.
His 619 . . . T T . 0.46 0.37. . . 0.50 0.27
.
Ser 620 . . . T T . 0.76 0.37. . . 0.50 0.29
.
50Cys 621 . B . . T . 0.79 0.29. . . 0.10 0.86
.
Pro 622 . . . . 'C C 0.21 0.20. . F 0.45 0.91
.
Pro 623 . . . T T . 0.07 0.39* . F 0.65 0.48
.
Asn 624 . . . T T . 0.14 0.69* . F 0.35 0.74
.
Thr 625 . B . . 'f . -0.14 0.11* * F 0.25 0.95
.
55Ile 626 A B . . . . 0.49 0.19* . . -0.300.62
.
Leu 627 A B . . . . 0.70 0.26* . . -0.300.53
.
Lys 628 A B . . . . 0.70 0.26* . . -0.300.63
.
Ala 629 A B . . . . 0.46 0.20* . . -0.151.39
.
His 630 A B . . . . 0.42 0.27. . . -0.152.64
.
60Gln 631 . B . . T . 0.46 0.01* . F 0.40 1.31
.
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
66
Pro 632 . . . T T . 1.27 0.66. * . 0.20 0.96
.
Tyr 633 . . . T T . 0.63 0.56. * . 0.35 1.22
.
Gly 634 . . . T T . 0.56 0.56. * . 0.20 0.71
.
Val 635 . B B . . . -0.270.73. . . -0.600.25
.
Gln 636 . B B . . . -0.480.94. * . -0.600.12
.
Ala 637 . B B . . . -0.930.61. * . -0.60O.18
.
Cys 638 . B B . . . -1.030.76. * . -0.600.13
.
Val 639 . B B . . . -0.900.54. . . -0.600.08
.
Pro 640 . B . . . . -0.390.57. * . -0.400.12
.
10Cys 641 . B . . . . -0.700.50. . . -0.400.21
.
Gly 642 . B . . T . -0.070.41. . F 0.29 0.42
.
Pro 643 . . . T T . 0.60 -0.23. . F 1.93 0.54
.
Gly 644 . . . T T . 1.46 -0.26. . F 2.42 1.61
.
Thr 645 . . . 'f T . 1.71 -0.43. . F 2.76 2.62
.
15Lys 646 . . . T ~1' . 1.49 -0.86. . F 3.40 3.39
.
Asn 647 . . . T T . 1.80 -0.60. . F 3.06 2.40
.
Asn 648 . B . . T . 1.71 -0.53. . F 2.32 2.26
.
Lys 649 . B . . T . 1.24 -0.63. . F 1.98 1.52
.
Ile 650 . B . . . . 0.89 0.06. . . 0.24 0.78
.
20His 651 . B . . T . 0.60 0.23. . . 0.10 0.26
.
Ser 652 . B . . T . 0.60 0.59. . . -0.200.20
.
Leu 653 . B . . T . 0.60 0.99* . . -0.200.47
.
Cys 654 . B . . T . -0.110.30* . . 0.10 0.57
.
Tyr 655 . . . T . . 0.47 0.37. . . 0.30 0.23
.
25Asn 656 . . . T T . -0.200.47. . . 0.20 0.40
.
Asp 657 . . . T T . -0.200.57* . . 0.20 0.65
.
Cys 658 . B . . T . 0.72 0.39* . . 0.10 0.55
.
Thr 659 . B . . T . 1.39 -0.37. . . 0.70 0.67
.
Phe 660 . B . . . . 1.32 -0.37. . . 0.80 0.65
.
30Ser 661 . . . T T . 1.11 0.11. . F 1.40 1.75
.
Arg 662 . . . T T . 0.80 -0.03. . F 2.30 1.87
.
Asn 663 . . . . T C 1.58 -0.03. . F 2.40 3.12
.
Thr 664 . . . . T C 1.58 -0.81. . F 3.00 4.55
.
Pro 665 . . . . T C 1.58 -0.71* . F 2.70 3.36
.
35Thr 666 . . . T T . 1.88 0.07* . F 1.70 1.81
.
Arg 667 . B . . T . 1.52 0.07* . F 1.00 2.01
.
'rhr 668 . B . . T . 1.52 0.34* * F 0.70 2.04
.
Phc 669 . B . . . . 1.13 0.31* * . 0.05 2.27
.
Asn 670 . B . . T . 1.04 0.61* * . -0.05I.01
.
40Tyr 671 . B . . T . 0.77 1.00* . . -0.200.93
.
Asn 672 . B . . T . -0.161.01* * . -0.051.09
.
Phc 673 . B . . T . -0.430.91* * . -0.200.56
.
Ser 674 A . . . . C 0.27 1.01* * . -0.400.36
.
Ala 675 A . . . . C -0.040.66* * . -0.400.36
.
45Leu 676 A B . . . . -0.660.74* * . -0.600.60
. '
Ala 677 A B . . . . -0.970.60. * . -0.600.33
.
Asn 678 A B . . . . -1.080.70* . . -0.600.47
.
Thr 679 . B B . . . -1.370.89* . . -0.600.47
.
Val 680 . B B . . . -1.120.70. . . -0.600.47
.
50Thr 681 . B B . . . -0.660.63. . . -0.600.29
.
Leu 682 . B B . . . -0.280.66. . . -0.600.20
.
Ala 683 . B B . . . -0.580.60. . . -0.600.42
.
Gly 684 . . B . . C -0.970.34. . F 0.05 0.39
.
Gly 685 . . . . T C -0.420.64. . F 0.15 0.41
.
55Pro 686 . . . . T C -0.410.44. * F 0.15 0.58
.
Ser 687 . . . . T C 0.44 0.33. * F 0.73 0.79
.
Phe 688 . B . . T . 0.69 -0.10. . F 1.56 1.59
.
Thr 689 . B . . . . 0.22 -0.10* . F 1.64 1.02
.
Ser 690 . B . . T . 0.61 0.16* . F 1.37 0.63
.
60Lys 691 . . . T T . 0.58 -0.23* . F 2.80 1.45
.
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
67
Gly 692 . . . T T . 0.18 -0.26* . F 2.52 1.57
.
Leu 693 . . . . T C 0.84 0.04* . F 1.44 1.02
.
Lys 694 A B . . . . 1.12 0.16* . . 0.26 0.69
.
Tyr 695 A B . . . . 0.72 0.66* . . -0.320.95
.
Phe 696 A B . . . . 0.37 1.01* * . -0.601.00
.
His 697 A B . . . . -0.100.81* * . -0.600.72
.
His 698 A B . . . . 0.41 1.50* * . -0.600.38
.
Phe 699 A B . . . . -0.441.13* * . -0.600.59
.
Thr 700 A B . . . . -0.871.03. * . -0.600.36
.
Leu 701 A . . T . . -0.511.10. * . -0.200.14
.
Ser 702 . . B T . . -0.481.03. * . -0.200.16
.
Leu 703 . . B T . . -0.440.64. * . 0.14 0.18
.
Cys 704 . . B T . . -0.090.56. * . 0.48 0.37
.
Gly 705 . . B T . . 0.33 0.30* . F 1.27 0.28
.
Asn 706 . . . T 'f . 1.19 -0.09* . F 2.61 0.66
.
Gln 707 . . . T T . 0.89 -0.77* . F 3.40 2.45
.
Gly 708 . . . T T . 1.40 -0.73* . F 3.06 2.45
.
Arg 709 . . . T T . 1.21 -0.77* . F 2.72 2.04
.
Lys 710 . B B . . . 0.89 -0.53* . F 1.43 0.87
.
Met 711 . B B . . . 0.58 -0.36* . . 0.64 0.47
.
Ser 712 . B B . . . 0.58 -0.30* . . 0.30 0.35
.
Val 713 . B B . . . 0.92 -0.30* . . 0.30 0.29
.
Cys 714 . B . . T . -0.040.10* . . 0.10 0.47
.
Thr 715 . B . . 1' . -0.400.13* * . 0.10 0.26
.
Asp 716 . B . . T . 0.20 0.23* . F 0.25 0.51
.
Asn 717 . B . . T . -0.31-0.41* * F 1.00 1.59
.
Val 718 . B B . . . 0.66 -0.30. * F 0.45 0.91
.
Thr 719 . B B . . . 0.43 -0.79. * F 0.90 1.07
.
Asp 720 . B B . . . 0.53 -0.10. * F 0.45 0.46
.
Leu 721 . B B . . . 0.53 -0.07. * F 0.76 0.97
.
Arg 722 . B B . . . 0.19 -0.71. * F 1.52 1.16
.
Ile 723 . B . . T . 1.04 -0.77. * F 2.08 0.69
.
Pro 724 . B . . T . 1.06 -0.77. * F 2.54 1.44
.
Glu 725 . . . T T . 0.71 -1 . * F 3.10 0.99
. .07
Gly 726 . . . . T C 0.82 -0.64. * F 2.74 1.40
.
Glu 727 . . . T 'f . 0.41 -0.54* . F 2.48 0.78
.
Ser 728 . . . . 'f C 1.34 -0.59* . F 2.10 0.60
.
Gly 729 . . . T T . 1.26 -0.59* . F 2.27 1.22
.
Phe 730 . . . T T . 0.37 -0.63* . F 1.94 0.95
.
Ser 731 . . . . T C 0.40 0.06* . F 0.97 0.49
.
Lys 732 . . . T T . -0.190.16* . F 1.30 0.72
.
Ser 733 . . . T T . -0.130.23* . F 1.17 0.84
.
Ile 734 . B . . T . -0.640.20* . . 0.49 0.98
.
Thr 735 . B B . . . -0.610.46* . . -0.340.37
.
Ala 736 . B B . . . -0.311.03* . . -0.470.15
.
Tyr 737 . B B . . . -0.941.04* . . -0.600.36
.
Val 738 . B B . . . -1.500.86. . . -0.600.25
.
Cys 739 . B B . . . -1.501.01. . . -0.600.19
.
Gln 740 . B B . . . -2.081.20. . . -0.600.08
.
Ala 741 . B B . . . -1.701.13. . . -0.600.08
.
Val 742 . B B . . . -1.670.91. . . -0.600.23
.
Ile 743 . B B . . . -0.810.77. . . -0.600.20
.
Ile 744 . B B . . . -1.000.37. . . -0.300.35
.
Pro 745 . B . . T . -1.310.51. * . -0.200.35
.
Pro 746 . B . . T . -1.070. . * F 0.42 0.71
. 3
6
Glu 747 . B . . T . -0.460.10. . F 0.74 1.01
.
Val 748 . B . . T . 0.48 0.17. . F 0.91 1.02
.
Thr 749 . B . . T . 0.78 -0.26. * F 1.68 1.32
.
Gly 750 . B . . T . 0.64 -0.19. . F 1.70 0.77
.
Tyr 751 . B . . T . 0.00 0.24. . F 1.08 1.03
.
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
68
Lys 752 . B . . T . -0.300.24. . F 0.76 0.53
.
Ala 753 . B . . . . 0.26 0.14. * F 0.39 0.71
.
Gly 754 . B . . . . 0.57 0.10. * F 0.22 0.61
.
Val 755 . B . . . . 0.70 -0.26. * F 0.65 0.53
.
Ser 756 . B . . . . 0.09 0.17. * F 0.05 0.81
.
Ser 757 . B . . . . -0.260.31. * F 0.05 0.61
.
Gln 758 . B . . . . -0.480.27. . F 0.20 I.10
.
Pro 759 . B . . . . -0.720.31. . F 0.05 0.67
.
Val 760 A B . . . . 0.13 0.43* * F -0.450.51
.
Ser 761 A B . . . . 0.54 0.04* * . -0.300.49
.
Leu 762 A B . . . . 0.03 -0.36* . . 0.30 0.62
.
Ala 763 A B . . . . -0.86-0.10* * . 0.30 0.69
.
Asp 764 A B B . . . -0.99-0.06* * . 0.30 0.36
.
Arg 765 A B B . . . -0.99-0.01* * . 0.30 0.43
.
Leu 766 . B B . . . -1.00-0.06* . . 0.30 0.32
.
Ile 767 . B B . . . -0.50-0.07* . . 0.30 0.28
.
Gly 768 . B B . . . 0.09 0.41* . . -0.600.20
.
Val 769 . B B . . . -0.510.41* . . -0.600.41
.
Thr 770 . B B . . . -0.930.34* * F -0.150.58
.
Thr 771 . B B . . . -0.930.14. * F -0.150.85
.
Asp 772 . B B . . . -0.040.40. * F -0.450.94
.
Met 773 . B B . . . -0.04-0.24. * F 0.60 1.09
.
Thr 774 . B B . . . -0.08-0.30. * . 0.30 0.75
.
Leu 775 . B B . . . -0.08-0.10* * F 0.45 0.31
.
Asp 776 . . B T . . -0.070.39* * F 0.25 0.46
.
Gly 777 . . B T . . -0.280.16* * F 0.34 0.42
.
Ile 778 . . B . . C -0.270.10* . F 0.23 0.79
.
Thr 779 . . B . . C 0.04 -0.09* * F 0.92 0.48
.
Ser 780 . . . . T C 0.04 -0.09* . F 1.41 0.84
.
Pro 781 . . . . T C -0.660.17* . F 0.90 0.99 ,
.
Ala 782 . B . . T . -0.340.27* . F 0.61 0.59
.
Glu 783 . B . . T . -0.270.29. * . 0.37 0.60
.
Leu 784 A B . . . . 0.04 0.59. . . -0.420.32
.
Phe 785 A B . . . . 0.04 0.16. . . -0.210.55
.
His 786 A B . . . . -0.560.04. . . -0.300.43
.
Leu 787 A B . . . . -0.310.73. . . -0.600.43
.
Glu 788 A B . . . . -1.200.47. . . -0.600.49
.
Ser 789 . . . T . . -0.600.37. . . 0.30 0.25
.
Leu 790 . . . T . . 0.10 0.30. . . 0.30 0.47
.
Gly 791 . . . . . C -0.72-0.39. . . 0.70 0.45
.
Ile 792 . . B . . C -0.800.26. . F 0.05 0.25
.
Pro 793 . B B . . . -1.500.56. . F -0.450.21
.
Asp 794 . B B . . . -1.900.66. . . -0.600.19
.
Val 795 . B B . . . -1.331.01* * . -0.600.23
.
Ile 796 . B B . . . -0.881.09* . . -0.600.23
.
Phe 797 . B B . . . -0.290.66* * . -0.600.27
.
Phe 798 . B B . . . -0.081.04. * . -0.600.50
.
Tyr 799 . B . . . . -0.080.80. . . 0.09 1.14
.
Arg 800 . . . T T . -0.080.11. . F 1.48 2.19
.
Ser 801 . . . T T . 0.50 -0.03. . F 2.42 1.88
.
Asn 802 . . . T T . 1.20 -0.33* . F 2.76 1.73
.
Asp 803 . . . T T . 1.60 -0.69* . F 3.40 1.53
.
Val 804 . . . T . . 1.18 -0.30. . F 2.56 1.53
.
Thr 805 . B . . . . 0.77 -0.11* . F 1.67 0.51
.
Gln 806 . B . . . . 0.77 -0.13* . F 1.33 0.41
.
Ser 807 . B . . . . 0.42 0.26* * F 0.67 0.74
.
Cys 808 . B . . T . 0.53 0.04* * F 0.81 0.51
.
Ser 809 . . . T T . 1.09 -0.44* * F 2.09 0.57
.
Ser 810 . . . T T . 1.09 -0.46. * F 2.37 0.57
.
Gly 811 . . . '1' T . 0.78 -0.36. . F 2.80 1.54
.
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
69
Arg 812 . . B T . . 0.19 -0.44 . F 2.12 1.66
. .
Ser 813 . . B T . . 0.97 -0.14 * F 1.69 0.87
. *
Thr 814 . B B . . . 0.41 -0.53 * F 1.46 1.72
. *
Thr 815 . B B . . . 0.82 -0.31 * F 0.73 0.65
. .
Ile 816 . B B . . . 0.50 -0.31 * F 0.45 0.95
. .
Arg 817 . B B . . . 0.09 -0.13 * . 0.30 0.35
. .
Val 818 . B B . . . 0.18 -0.23 * . 0.64 0.33
. .
Arg 819 . B B . . . 0.49 -0.29 * . 0.98 0.73
. .
Cys 820 . B B . . . 0.84 -0.57 * . 1.62 0.64
. .
Scr 821 . . . . T C 1.42 -0.57 * F 2.86 1.73
. *
Pro 822 . . . T T . 0.46 -0.73 * F 3.40 1.27
. *
Gln 823 . . . 'f T . 1.10 -0.09 * F 2.76 1.76
. *
Lys 824 . B . . T . 0.64 -0.23 * F 2.02 2.03
. .
Thr 825 . B . . . . 1.01 -0.19 . F 1.48 1.30
. .
Val 826 . B . . T . 0.50 -0.23 . F 1.34 1.01
. .
Pro 827 . B . . T . -0.10 0.06 . F 0.25 0.42
. .
Gly 828 . B . . T . -0.91 0.74 . F -0.050.24
. .
Ser 829 . B . . T . -1.17 0.94 . F -0.050.26
. .
Leu 830 . B . . . . -1.20 0.73 * F -0.250.26
. .
Leu 831 . B . . . . -0.66 0.73 * F -0.400.26
. .
Leu 832 . B . . T . -1.11 0.79 . F -0.050.28
. .
Pro 833 . B . . 'f . -1.07 0.97 . F -0.050.18
. .
Gly 834 . . . T T . -0.77 0.67 . F 0.35 0.30
. .
Thr 835 . B . . T . -0.30 -0.01 . F 1.16 0.61
. .
Cys 836 . . . T T . 0.20 -0.27 . F 1.87 0.39
. .
Ser 837 . . . T T . 0.34 -0.21 . F 2.18 0.57
. .
Asp 838 . . . T 'f . 0.56 -0.07 . F 2.49 0.21
. .
Gly 839 . . . T T . 0.56 -0.56 . F 3.10 0.66
. .
Thr 840 . . . T . . 0.20 -0.70 . F 2.59 0.48
. *
Cys 841 . . . T T . 0.87 -0.51 . F 2.48 0.16
. *
Asp 842 . . . T T . 0.47 -0.11 . F 1.87 0.25
. .
Gly 843 . . . T T . 0.43 0.24 * F 0.96 0.15
. .
Cys 844 . . . T T . 0.08 0.26 . . 0.50 0.38
. .
Asn 845 A B . . . . -0.42 0.47 . . -0.600.20
. .
Phe 846 A B . . . . -0.04 1.16 * . -0.600.17
. .
His 847 A B . . . . -0.04 1.64 * . -0.600.33
. .
Phe 848 A B . . . . 0.00 1.07 * . -0.600.35
. *
Leu 849 A . . T . . 0.08 1.06 * . -0.200.54
. .
Trp 850 A . . T . . -0.51 0.77 * . -0.200.40
. .
Glu 851 A . . T . . -0.40 0.77 * . -0.200.47
. .
Ser 852 A . . T . . -1.03 0.49 . . -0.200.58
. .
Ala 853 A . . T . . -0.54 0.37 . . 0.10 0.29
. .
Ala 854 A . . T . . -0.54 -0.11 . . 0.70 0.26
. .
Ala 855 A . . T . . -0.92 0.57 . . -0.200.16
. .
Cys 856 . . . . T C -1.22 0.76 . . 0.00 0.09
. .
Pro 857 . B . . T . -1.78 0.64 . . -0.200.1
. . 1
Leu 858 . B . . T . - I 0.79 . . -0.200.08
. .78 *
Cys 859 . B . . T . -1.19 0.79 . . -0.200.16
. *
Ser 860 . B B . . . -0.84 0.21 . . -0.300.17
. .
Val 861 . B B . . . -0.21 0.54 . . -0.600.32
. .
Ala 862 . B B . . . -0.59 0.36 . . -0.300.82
. .
Asp 863 . B . . . . -0.67 0.29 . . -0.100.62
. .
Tyr 864 . B B . . . -0.86 0.59 . . -0.600.58
. .
His 865 . B B . . . -0.86 0.59 . . -0.600.43
. .
Ala 866 . B B . . . -0.30 0.47 . . -0.600.34
. .
Ile 867 . B B . . . -0.38 0.86 . . -0.600.29
. .
Val 868 . B B . . . -1.23 0.67 . . -0.600.12
. .
Ser 869 . B B . . . -1.58 0.81 . . -0.600.09
. .
Ser 870 . B B . . . -1.89 0.81 . . -0.600.
. . I
2
Cys 871 . B B . . . -2.19 0.56 . . -0.600.16
. *
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
Val 872 . B B . . . -1.30 0.60* . . -0.600.09
.
Ala 873 . B B . . . -0.40 0.61* . . -0.600.11
.
Gly 874 . B B . . . -0.41 0.23* . . -0.300.41
.
Ile 875 . B B . . . -0.42 0.14* . . -0.300.80
.
5 Gln 876 . B B . . . 0.00 -0.01. . F 0.60 1.15
.
Lys 877 . B B . . . 0.00 0.24* . F 0.00 1.82
.
Thr 878 . B B . . . 0.30 0.46* * F -0.301.92
.
Thr 879 . B B . . . 0.76 0.69* . F -0.301.17
.
Tyr 880 . B B . . . 1.64 0.29* . . -0.151.14
.
10 Val 881 A B B . . . 1.43 0.29* . . -0.151.37
.
Trp 882 A B B . . . 1.43 0.23* * . -0.151.47
.
Arg 883 A B B . . . 0.93 -0.26* . F 0.60 1.88
.
Glu 884 A B B . . . 0.58 -0.33* . F 0.85 2.09
.
Pro 885 A . . 'I' . . 0.52 -0.40* . F 1.50 1.06
.
15 Lys 886 A . . T . . 1.03 -0.93* . F 1.90 0.73
.
Leu 887 A . . T . . 0.98 -0.50* . F 1.85 0.42
.
Cys 888 . . . T T . -0.02 -0.07* . F 2.50 0.27
.
Ser 889 . . . 'f T . -0.32 0.19. * F 1.65 0.09
.
Gly 890 . . . T T . -0.92 0.57* * F 1.10 0.15
.
20 Gly 891 . . . T T . -1.18 0.57* . F 0.85 0.23
.
Ile 892 . . . . . C -0.37 0.43* . F 0.20 0.27
.
Ser 893 . . . . . C 0.30 0.04. . F 0.25 0.47
.
Leu 894 . B . . . . 0.71 0.01. * F 0.05 0.82
.
Pro 895 . B . . . . 0.20 -0.41. * F 0.80 2.30
.
25 Glu 896 . B B . . . 0.23 -0.46. * F 0.60 1.27
.
Gln 897 . B B . . . 0.23 -0.36. * F 0.60 2.23
.
Arg 898 . B B . . . -0.13 -0.36. * F 0.60 1.01
.
Val 899 . B B . . . 0.72 -0.21. * . 0.30 0.31
.
Thr 900 . B B . . . 0.62 -0.21. * . 0.30 0.36
.
30 Ile 901 . B B . . . -0.27 -0.13. * . 0.30 0.27
.
Cys 902 . B B . . . -0.27 0.56. * . -0.600.25
.
Lys 903 . B B . . . -1.08 -0.09* * . 0.30 0.29
.
Thr 904 . B B . . . -0.51 0.21* * . -0.300.36
.
Ile 905 . B B . . . -1.01 0.44* * . -0.600.71
.
35 Asp 906 . B B . . . -0.08 0.56* * . -0.600.29
.
Phe 907 . B B . . . -0.27 0.56* * . -0.600.40
.
Trp 908 . B B . . . -0.66 0.71* * . -0.600.43
.
Leu 909 . B B . . . -1.23 0.46* * . -0.600.25
.
Lys 910 . B B . . . -0.64 1.14* * . -0.600.20
.
40 Val 911 . . B T . . -1.23 0.74* * . -0.200.26
.
Gly 912 . . B T . . -0.88 0.33* * . 0.10 0.32
.
Ile 913 . . B T . . -0.90 0.07* * . 0.10 0.16
.
Ser 914 . . . . T C -0.76 0.56* * . 0.00 0.31
.
Ala 915 . . . 'f T . -1.11 0.49. * F 0.35 0.17
.
45 Gly 916 . . . T T . -0.84 0.54. . F 0.35 0.34
.
Thr 917 . B . . T . -1.39 0.36. . F 0.25 0.26
.
Cys 918 . B B . . . -1.31 0.66. . . -0.600.18
.
Thr 919 . B B . . . -1.82 0.84. . . -0.600.15
.
Ala 920 . B B . . . - I 1.10. . . -0.600.09
. .54
50 Ile 921 . B B . . . -2.06 1.10. . . -0.600.23
.
Leu 922 . B B . . . -2.56 1.17. . . -0.600.12
.
Leu 923 . B B . . . -2.20 1.37. . . -0.600.10
.
Thr 924 . B B . . . -2.56 1.36. . . -0.600.20
.
Val 925 . B B . . . -2.21 1.24. . . -0.600.13
.
55 Ixu 926 . B B . . . -2.02 1.31. . . -0.600.25
.
Thr 927 . B B . . . -1.50 1.41* . . -0.600.
. I
5
Cys 928 . B B . . . -0.64 1.84* . . -0.600.21
.
Tyr 929 . B B . . . -0.29 1.20. . . -0.600.51
.
Phe 930 . . B T . . 0.57 0.51. . . -0.200.70
.
60 '1'rp931 . . B T . . 1.38 0.43* . . 0.29 2.10
.
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
71
Lys 932 . . . . T C 1.73 0.26* . F 1.28 2.32
.
Lys 933 . . . T 1' . 1.59 -0.50* * F 2.42 5.37
.
Asn 934 . . . . T C 1.83 -0.60* * F 2.86 4.21
.
Gln 935 . . . T T . 2.29 -1.51* * F 3.40 3.65
.
Lys 936 . . B . . . 2.62 -0.76* * F 2.46 2.86
.
Leu 937 . . B . . . 2.33 -0.76* * F 2.32 3.55
.
Glu 938 . . B . . . 1.99 -0.40* * . 1.73 3.21
.
Tyr 939 . . B . T . 2.03 -0.41* * . 1.79 2.15
.
Lys 940 . . B . T . 1.22 -0.41* * F 1.80 5.22
.
Tyr 941 . . B . T . 0.32 -0.41* * F 2.00 2.49
.
Ser 942 . . B . T . 0.53 0.23* * F 1.20 1.18
.
Lys 943 . A B . . . 0.53 0.09* * F 0.45 0.58
.
Leu 944 . A B . . . 0.19 0.49* . . -0.200.60
.
Val 945 . A B . . . -0.170.23* * . -0.100.45
.
Met 946 . A B . . . -0.730.33* * . -0.300.33
.
Asn 947 . A B . . . -0.391.01. * . -0.600.33
.
Ala 948 . A B . . . -0.430.33* * . -0.300.88
.
Thr 949 . A B . . . -0.29-0.31. * . 0.65 1.48
.
Leu 950 . A B . . . 0.57 -0.36* . F 0.85 0.49
.
Lys 951 . A B . . . 0.36 -0.76. * F 1.35 0.82
.
Asp 952 . . . T T . 0.14 -0.57. * F 2.35 0.47
.
Cys 953 . . B . T . 0.14 -0.63. . . 2.00 0.87
.
Asp 954 . . B . T . -0.13-0.81. . . 1.80 0.44
.
Leu 955 . . B . T . 0.68 -0.31. . . 1.30 0.27
.
Pro 956 . . B . . . 0.33 -0.31. . . 0.90 0.83
.
Ala 957 . . . T . . -0.33-0.50. * . 1.10 0.67
.
Ala 958 A . . . . . -0.260.07. . . -0.100.43
.
Asp 959 A . . . T . - -0.1. . . 0.70 0.28
. I 1
.14
Ser 960 . . B . T . -0.930.14. . . 0.10 0.20
. .
Cys 961 . . B . T . -0.720.26. . . 0.10 0.19
.
Ala 962 . . B . T . -0.48-0.24. . . 0.70 0.20
.
Ile 963 . A B . . . 0.11 0.19. . . -0.300.15
.
Met 964 . A B . . . 0.11 -0.20. . . 0.30 0.48
.
Glu 965 . A B . . . -0.44-0.77. . F 0.75 0.79
.
Gly 966 . A . . . C 0.22 -0.63* . F 0.95 0.83
.
Glu 967 A A . . . . 0.81 -1.31* . F 0.90 1
. .46
Asp 968 A A . . . . 1.70 -1.93* . F 0.90 1
. .41
Val 969 A A . . . . 1.49 -1.93* . F 0.90 2.38
.
Glu 970 A A . . . . 0.60 -1.67* . F 0.90 1.13
.
Asp 971 A A . . . . 0.24 -0.99* . F 0.75 0.48
.
Asp 972 A A . . . . -0.07-0.20. * F 0.45 0.55
.
Leu 973 A A . . . . -0.37-0.36* * . 0.30 0.46
.
Ile 974 A A . . . . 0.53 0.03. * . -0.300.37
.
Phe 975 . A B . . . 0.53 0.03. . . -0.300.44
.
Thr 976 . A B . . . 0.50 0.43. . F -0.450.87
.
Ser 977 . . . . T C 0.20 0.24. . F 0.60 1.68
.
Lys 978 . . . T T . 0.20 -0.06. . F 1.40 2.60
.
Asn 979 . . . . T C 0.74 -0.16* . F 1.48 1.49
.
His 980 . . . . T C 1.56 -0.21* * F 1.76 1.10
.
Ser 981 . . . . . C 1.57 -0.60. * . 1.99 1.07
.
Leu 982 . . . T . . 1.87 -0.21. . . 2.02 0.90
.
Gly 983 . . . T T . 1.79 -0.21. . F 2.80 1.06
.
Arg 984 . . . T T . 0.98 -0.21* . F 2.52 1.07
.
Ser 985 . . . T T . 0.80 0.09* . F 1.88 1.07
.
Asn 986 . . . T T . 0.89 -0.17* * F 2.44 1.68
.
His 987 . . . . . C 1.81 -0.17* * F 2.00 1.33
.
Leu 988 . . . . . C 1.81 -0.17* * F 1.96 1.94
.
Pro 989 . . . . T C 0.89 -0.13* * F 2.40 1.19
.
Pro 990 . . . T T . 0.38 0.16. * F 1.61 0.72
.
Arg 991 . . . T T . -0.220.34. * F 1.37 0.72
.
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
72
Gly 992 . B . . T . -0.19 0.27 . * F 0.73 0.46
.
Leu 993 A B . . . . -0.19 -0.16 . . 0.54 0.50
. *
Leu 994 A B . . . . -0.29 0.10 * . . -0.30 0.21
.
Met 995 A B . . . . -0.08 0.59 * . . -0.60 0.31
.
Asp 996 A B . . . . -0.86 0.56 * . . -0.60 0.64
.
Leu 997 A B . . . . -0.40 0.44 . . . -0.60 0.42
.
Thr 998 A B . . . . 0.02 -0.24 . . 0.30 0.83
. .
Gln 999 A B . . . . 0.44 -0.43 . F 0.45 0.63
. .
Cys 1000 A B : . . . 0.66 0.00 . . . -0.30 0.98
.
Arg 1001 A B . . . . 0.27 -0.26 . . 0.30 0.87
. .
Table
IV
Res Pos II III N V VI VII VIII IX X XI XII XIII XIV
I
Met 1 . A . . . . C 0.68 -0.13. * . 0.50 0.57
Asp 2 . A . . T . . 1.07 -0.56. * . 1.00 0.78
Cys 3 . A . . T . . 1.46 -0.59. . . 1.34 0.98
Gln 4 . A . . T . . 1.60 -1.01. * . 1.83 1.71
Glu 5 . A . . 'f . . 1.70 -0.87. * F 2.32 1.61
Asn 6 . . . . T T . 2.30 0.04. * F 2.16 3.15
Glu 7 . . . . T T . 2.30 -0.53. . F 3.40 3.04
Tyr 8 . . . . T T . 2.68 -0.53* . F 3.06 3.04
Trp 9 . . . . T T . 2.33 0.39* * . 1.67 1.99
Asp 10 . . . . T 'f . 2.44 0.41* * . 1.03 1.14
Gln I1 . . . . T T . 1.78 0.41* . F 0.84 1.42
Trp 12 . . . . T T . 0.92 0.23* . . 0.50 0.72
Gly 13 . . . . T T . 0.86 -0.04* . . 1.10 0.32
Arg 14 . . . B T . . 0.48 0.44* . . -0.200.27
Cys 15 . . . B T . . 0.48 0.61* * . -0.200.14
Val 16 . . . B T . . 0.59 0.10* * . 0.35 0.24
Thr 17 . . . B T . . 0.21 -0.33* * . 1.20 0.24
Cys 18 . . . . T T . 0.21 0.24. * . 1.25 0.24
Gln 19 . . . . T T . -0.110.10. * . 1.50 0.32
Arg 20 . . . . T T . 0.21 -0.11. . F 2.50 0.34
Cys 21 . . . . T T . 1.07 -0.17. . F 2.25 0.63
Gly 22 . . . . . T C 1.38 -0.34. . F 1.80 0.63
Pro 23 . . . . T T . 1.23 -0.74. . F 2.05 0.56
Gly 24 . . . . T T . 0.93 -0.06. . F 1.81 0.86
Gln 25 . . . . T T . 0.87 -0.24. * F 2.02 1.16
Glu 26 . . . . T . . 1.53 -0.67* . F 2.43 1.50
Leu 27 . . . . T . . 1.21 -1.10* . F 2.74 2.54
Ser 28 . . . . T T . 1.08 -0.96* . F 3.10 0.79
Lys 29 . . . . T 'f . 1.18 -0.93* . F 2.79 0.45
Asp 30 . . . . T T . 0.83 -0.17* . F 2.18 0.85
Cys 31 . . . . T T . 0.83 -0.43. * F 2.21 0.63
Gly 32 . . . . T . . 1.30 -0.81. . . 2.19 0.55
.
Tyr 33 . . . . T . . 1.26 -0.39. . F 2.07 0.32
Gly 34 . . . . . T C 1.21 0.04. . F 1.81 0.60
Glu 35 . . . . T T . 0.62 -0.53. . F 3.40 1.01
Gly 36 . . . . T T . 1.04 -0.46. . F 2.61 0.65
Gly 37 . . . . T T . 1.10 -0.46* . F 2.42 1.03
Asp 38 . . . . T . . 1.31 0.03* . F 1.13 0.62
Ala 39 . . . . . . C 1.36 0.53* . . 0.14 0.86
Tyr 40 . . . . T . . 0.54 0.49* . . 0.15 1.16
Trp 41 . . B . . . . 0.68 0.74* . . -0.400.57
His 42 . . . . . . C 0.72 1.17* * . -0.200.88
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
73
Ser 43 . . . . . C 0.42 1.06. . . -0.200.75
.
Leu 44 . . . . T C I.01 0.69. . F 0.15 0.96
.
Pro 45 . . . T T . 1.0I 0.17* * F 1.04 1.22
.
Ser 46 . . . T T . 1.34 0.43* . F 0.98 1.42
.
Ser 47 . . . T T . 1.08 0.04. . F 1.52 3.45
.
Gln 48 . . . T . . 1.08 -0.26. * F 2.16 2.99
.
Tyr 49 . . . T . . 1.60 -0.30. * F 2.40 2.99
.
Lys 50 . . . T . . 1.47 0.23. * F 1.56 2.35
.
Ser 51 . . . T T . 1.73 0.27. * F 1.52 1.34
.
Ser 52 . . . T T . 2.00 0.37. * F 1.28 1.17
.
Trp 5 . . . T T . 2.04 0.1. * . 0.74 0.79
3 1
.
Gly 54 . . . T T . 1.62 0.11. * . 0.87 1.18
.
His 55 . . . T . . 1.58 0.30. * . 0.74 0.47
.
His 56 . . . T . . 1.58 0.31. . . 0.96 0.78
.
Lys 57 . . . T . . 1.21 -0.21. * . 1.93 1.06
.
Cys 58 . . . T T . 0.61 -0.07. * . 2.20 0.42
.
Gln 59 . . . T T . 0.64 0.11* * . 1.38 0.21
.
Ser 60 . . . T T . 0.01 0.10* * . 1.16 0.15
.
Cys 61 . . . T T . -0.540.67* * . 0.64 0.15
.
Ile 62 . B B . . . -1.440.60* * . -0.380.09
.
Thr 63 . B B . . . -1.670.84. * . -0.600.05
.
Cys 64 . B B . . . -1.671.14. . . -0.600.07
.
Ala 65 . B B . . . -1.260.97* * . -0.600.15
.
Val 66 . B B . . . -1.440.29* * . -0.300.20
.
Ile 67 . B B . . . -0.560.44* * . -0.340.28
.
Asn 68 . . B T . . -0.200.27* . . 0.62 0.48
.
Arg 69 . . B T . . -0.39-0.23* . . 1.63 1.30
.
Val 70 . . B 'r . . 0.20 -0.23* . F 2.04 i
. .38
Gln 71 . . B T . . 0.39 -0.51* . F 2.60 1.38
.
Lys 72 . . B T . . 0.97 -0.34* . F 1.89 0.38
.
Val 73 . . B T . . 0.76 0.14* . . 0.88 0.73
.
Asn 74 . . B T . . 0.33 -0.07* * . 1.22 0.65
.
Cys 75 . . B T . . 0.89 0.01* * F 0.51 0.47
.
Thr 76 . . . . T C 0.89 0.40. * F 0.45 0.85
.
Pro 77 . . . T T . 0.26 0.16. * F 0.65 0.85
.
Thr 78 . . . T T . 0.26 0.26. * F 0.80 1.61
.
Ser 79 . . . T T . -0.410.33. . F 0.65 0.83
.
Asn 80 . . . T . . -0.090.41. . F 0.15 0.29
.
Ala 81 . . . T . . 0.22 0.41. . . 0.00 0.20
.
Val 82 . . . T . . -0.23-0.07. . . 0.90 0.24
.
Cys 83 . . . T T . -0.730.11. . . 0.50 0.08
.
Gly 84 . . . T T . -0.640.40* * . 0.50 0.07
.
Asp 85 . . . T T . -0.530.33* * . 0.50 0.14
.
Cys 86 . B . . T . -0.64-0.31* * . 0.70 0.51
.
Leu 87 . B . . . . -0.03-0.10* * . 0.81 0.44
.
Pro 88 . . . T T . 0.74 0.23* * . 1.12 0.42
.
Arg 89 . . . T T . 1.13 0.23* * . 1.58 1.52
.
Phe 90 . . . T T . 0.82 -0.34* * . 2.49 3.69
.
Tyr 91 . . . T T . 1.60 -0.54* * . 3.10 3.44
.
Arg 92 . . B T . . 1.52 -0.97* * F 2.54 3.44
.
Lys 93 . . B T . . 1.39 -0.29* * F 1.93 2.79
.
Thr 94 . . B T . . 0.93 -0.64* * F 1.92 1.76
.
Arg 95 . . B T . . 0.82 -0.97* * F 1.46 0.89
.
Ile 96 . . B T . . 1.07 -0.29. . F 0.85 0.37
.
Gly 97 . . . T . . 0.96 0.11. . F 0.45 0.44
.
Gly 98 A . . T . . 0.91 -0.37. * F 0.85 0.38
.
Leu 99 A . . . . C 1.22 0.03* * F 0.05 0.93
.
Gln 100 A . . T . . 0.44 -0.66. * F 1.30 1.62
.
Asp 101 A . . T . . 0.44 -0.51. . F 1.15 0.88
.
Gln 102 A . . T . . 0.58 -0.26. . F 0.85 0.75
.
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
74
Glu 103. A . . T . . 0.26-0.51. . F 1.15 0.67
Cys 104. A . . T . . 0.76-0.34. * . 0.70 0.21
Ile 105. . B . . . . 0.800.14. . . -0.100.18
Pro 106. . . . T . . 0.80-0.26. . . 0.90 0.21
Cys 107. . . . T T . 0.490.14* . . 0.50 0.67
Thr 108. . . . T T . 0.280.06* . F 1.10 1.37
Lys 109. . . . T T . 0.63-0.20. . F 2.00 1.37
Gln 110. . . . . 'f C 1.22-0.14. . F 2.10 3.69
Thr 111. . . . . T C 1.43-0.33. . F 2.40 3.43
Pro 112. . . . . T C 1.24-0.81. * F 3.00 2.97
Thr 113. . . . T T . 1.56-0.17. * F 2.60 1.27
Ser 114. . . . . T C 0.84-0.17. * F 2.10 1.53
Glu 115. A . . T . . 0.26-0.09* * F 1.45 0.53
Val 116. A B . . . . -0.13-0.01* * . 0.60 0.37
Gln 117. A B . . . . 0.080.29* * . -0.300.24
Cys 118. A B . . . . -0.420.30* * . -0.300.24
Ala 119A A . . . . . -0.420.99* * . -0.600.27
Phe 120A A . . . . . -1.230.73. * . -0.600.21
Gln 121A A . . . . . -1.231.01. * . -0.600.32
Leu 122. A . . . . C -1.231.09. * . -0.400.23
Ser 123. A . . . . C -1.160.59. * . -0.400.47
Leu 124. A . . . . C -0.570.30. * . -0.100.27
Val 125. A . . . . C -0.46-0.10. * . 0.50 0.55
Glu 126. A . . . . C -0.67-0.29. * . 0.50 0.41
Ala 127. A . . T . . -0.17-0.24. . . 0.70 0.78
Asp 128. A . . T . . -0.72-0.44. . . 0.85 1.51
Ala 129. A . . . . C -0.12-0.44. * F 0.65 0.65
Pro 130. A . . . . C 0.52-0.01. * F 0.85 0.99
Thr 131. . . . . . C 0.52-0.09. . F 1.25 0.92
Val 132. . . . . . C 1.110.31. . F 1.00 1.57
Pro 133. . . . . . C 0.52-0.19. . F 1.80 1.76
Pro 134. . . . . . C 0.80-0.11. . F 2.00 1.23
Gln 135. . . . . . C 0.20-0.11. . F 1.80 2.40
Glu 136A . B . . . . -0.34-0.07. . F 1.40 1.28
Ala 137. . B B . . . -0.080.14. . F 0.25 0.61
Thr 138. . B B . . . -0.680.21. . . -0.100.36
Leu 139. . B B . . . -1.320.50. . . -0.600.17
Val 140. . B B . . . -1 1.14. . . -0.600.13
.62
Ala 141. . B B . . . -1.921.03. . . -0.600.12
Leu 142. . B B . . . -2.140.93* . . -0.600.19
Val 143. . B B . . . -2.640.93* . . -0.600.21
Ser 144. . B B . . . -2.690.97. . ~ -0.600.17
.
Ser 145. . B B . . . -2.691.11* . . -0.600.15
Leu 146. . B B . . . -2.801.07* . . -0.600.15
Leu 147. . B B . . . -2.301.21* . . -0.600.10
Val 148. . B B . . . -2.261.31. . . -0.600.11
Val 149. A B B . . . -2.541.61. * . -0.600.11
Phe 150. A B B . . . -2.941.43. . . -0.600.13
Thr 151. A B B . . . -2.941.53. . . -0.600.15
Leu 152. A B B . . . -2.481.57. . . -0.600.17
Ala 153A A . B . . . -2.431.36. . . -0.600.19
Phe 154. A . B T . . -2.281.26. . . -0.200.11
Leu 155. A . B T . . -2.281.56. . . -0.200.12
Gly 156. A . B T . . -2.781.66. . . -0.200.10
Leu 157. A . B T . . -2.211.84. . . -0.200.10
Phe 158. A . B T . . -2.291.81. . . -0.200.18
Phe 159. A . B T . . -1.541.70* . . -0.200.10
Leu 160. A . B T . . -0.731.27* . . -0.200.24
Tyr 161. A . B T . . -1.090.99* . . -0.200.48
Cys 162. A . B T . . -0.980.99* . . -0.200.48
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
Lys 163 . A . B T . . -0.280.99* . . -0.200.50
Gln 164 . A . B T . . 0.53 0.70* . . -0.200.51
Phe 165 . A . B T . . 1.31 -0.06* . . 0.85 1.88
Phe 166 . A . B T . . 0.89 -0.13* . . 1.16 1.28
5 Asn 167 . . . . T T . 1.56 0.44* ~ . 0.82 0.39
*
Arg 168 . . . . 'C T . 1.62 0.44* * . 1.13 0.79
His 169 . . . . T T . 1.28 -0.34* * . 2.49 1.79
Cys 170 . . . . T T . 1.63 -0.70* * . 3.10 1.10
Gln 171 . . . . T T . 1.52 -0.67* . F 2.79 0.56
10 Arg 172 . . . . T T . 0.71 0.01* * F 1.58 0.34
Gly 173 . . . . T T . 0.60 0.20* * F 1.27 0.52
Gly 174 . . . . T T . -0.070.03* * F 0.96 0.52
Lcu 175 . A . . . . C 0.60 0.41* * . -0.400.23
Leu 176 . A . . . . C 0.01 0.41. * . -0.400.40
15 Gln 177 . A B . . . . -0.100.49. * . -0.600.41
Phe 178 . A B . . . . 0.29 0.06. * . -0.300.83
Glu 179 A A . . . . . 0.32 -0.63. * . 0.75 2.01
Ala 180 A A . . . . . 0.54 -0.83* * F 0.90 1.67
Asp 181 A A . . . . . 1.40 -0.73* * F 0.90 1.95
20 Lys 182 A A . . . . . 1.40 -1.5. * F 0.90 2.26
I
Thr I A A . . . . . 2.10 -1.51* * F 0.90 3
83 .87
Ala 184 A A . . . . . 1.80 -2.01* * F 1.20 4.01
Lys 185 A A . . . . . 1.58 -1.63* . F 1.50 2.69
Glu 186 A A . . . . . 0.88 -0.94* . F 1.80 1.54
25 Glu 187 . A . . T . . 0.62 -0.64. . F 2.50 1.32
Ser 188 . . . . T . . 0.08 -0.71. * F 3.00 1.02
Leu 189 . . . . T . . 0.46 -0.07. * . 2.10 0.44
Phe 190 . . . . . . C 0.20 0.36. . . 1.00 0.39
Pro 191 . . . . . . C -0.100.79. . . 0.70 0.45
30 Val 192 . . . . . . C -0.060.79. . F 0.85 0.73
Pro 193 . . . . . T C 0.24 0.10. . F 1.50 1.69
Pro 194 . . . . . T C 0.74 -0.69. . F 2.70 1.89
Scr 195 . . . . . T C 1.14 -0.63. . F 3.00 3.67
Lys 196 . . . . . T C 0.77 -0.89. . F 2.70 3.18
35 Glu 197 . A . . . . C 1.62 -0.81. . F 2.00 2.08
Thr 198 . A . . . . C 1.53 -1.24. . F 1.70 2.69
Ser 199 . A . . . . C 1.74 -1.24. * F 1.40 1.80
Ala 200 . A . . . . C 1.19 -0.84. * F 1.10 1.80
Glu 201 . A . . T . . 0.84 -0.20. * F 0.85 0.93
40 Ser 202 . . . . . . C 0.56 -0.30. * F 0.85 0.93
Gln 203 . . . . T . . 0.28 0.23. * F 0.45 0.96
Val 204 . . . . . . C 0.37 0.23. * . 0.10 0.56
Ser 205 . . . . T . . 0.61 0.66. * . 0.00 0.65
Trp 206 . . . . . . C 0.31 0.70. * . -0.200.37
45 Ala 207 . . . . . T C -0.200.69. . . 0.00 0.67
Pro 208 . . . . . T C -0.790.73* . F 0.15 0.41
Gly 209 . . . . T T . 0.07 0.84* . F 0.35 0.40
Ser 210 . . . . . T C -0.440.33* . F 0.45 0.68
Leu 211 . . . . . . C -0.860.51* . . -0.200.36
50 Ala 212 . . . . . . C -0.570.87* . . -0.200.32
Gln 213 . . B . . . . -1.170.83. . . -0.400.32
Leu 214 . . B . . . . -0.821.13. . . -0.400.32
Phe 215 . . B . . . . -0.820.44. . . -0.400.52
Ser 216 . . B . . . . -0.870.3 . . . -0.100.40
3
55 Leu 217 . . . T . . -0.490.57. . . 0.00 0.36
Asp 218 . . . . T . . -1.380.31. . F 0.45 0.65
Ser 219 . . . . . . C -0.780.21. . F 0.25 0.34
Val 220 . . . . . . C -0.080.26. * F 0.25 0.64
Pro 221 . . . . . . C 0.22 -0.03. . F 0.85 0.66
60 Ile 222 . . . . . . C 1.03 0.37. . F 0.25 0.86
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
76
Pro 223 . . . . . . C 1.03 0.39 . F 0.66 2.00
.
Gln 224 . . . . T . . 0.99 0.14 . F 1.12 2.24
*
Gln 225 . . . . . . C 1.63 0.14 . F 1.18 3.16
*
Gln 226 . . . . . . C 1.84 -0.11 . F 2.04 3.16
.
Gln 227 . . . . . . C 2.13 -0.54 . F 2.60 3.16
.
Gly 228 . . . . . T C 1.96 -0.33 . F 2.24 1.80
.
Pro 229 . . . . . T C 1.57 -0.30 . . 1.83 1.33
.
Glu 230 . . . . T T . 1.18 -0.27 . . 1.62 0.98
.
Met 231 . . . . . T C 0.79 -0.24 * . 1.31 1.27
.
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
77
In another aspect, the invention provides an isolated nucleic acid molecule
comprising a
polynucleotide which hybridizes under stringent hybridization conditions to a
portion of the
polynucleotide in a TR13 nucleic acid molecule of the invention described
above, for instance,
the cDNA clone (HWLHM70) contained in ATCC Deposit No. PTA-349, the nucleic
acid
sequence disclosed in Figures 1 A-C or the complementary strand thereof, and
fragments
thereof (e.g., as described herein).
By "stringent hybridization conditions" is intended overnight incubation at
42°C in a
solution comprising: 50% formamide, Sx SSC (750 mM NaCI, 75mM trisodium
citrate), 50
mM sodium phosphate (pH 7.6), Sx Denhardt's solution, 10% dextran sulfate, and
20 g/ml
denatured, sheared salmon sperm DNA, followed by washing the filters in O.lx
SSC at about
65°C.
In another aspect, the invention provides an isolated nucleic acid molecule
comprising a
polynucleotide which hybridizes under stringent hybridization conditions to
molecule of the
invention described above, for instance, the TR13 cDNA clone (HWLHN83)
contained in
ATCC Deposit No. PTA-507, the nucleic acid sequence disclosed in figures 7A-D
or the
complementary strand thereto, and fragments thereof (e.g., as described
herein).
By a polynucleotide which hybridizes to a "portion" of a polynucleotide is
intended a
polynucleotide (either DNA or RNA) hybridizing to at least about 15
nucleotides (nt), and more
preferably at least about 20 nt, still more preferably at least about 30 nt,
and even more
2o preferably about 30-70 nt of the reference polynucleotide. These are useful
as diagnostic
probes and primers as discussed above and in more detail below. In this
context "about"
includes the particularly recited size, larger or smaller by several (5, 4, 3,
2, or 1) nucleotides,
at either terminus or at both termini.
By a portion of a polynucleotide of "at least 20 nt in length," for example,
is intended
20 or more contiguous nucleotides from the nucleotide sequence of the
reference polynucleotide
(e.g., cDNA deposited as ATCC Deposit No. PTA-507, or the nucleotide sequence
as shown
in SEQ ID N0:39 or the complementary strand thereto, or a fragment thereof).
By a portion of a polynucleotide of "at least 20 nt in length," for example,
is intended
20 or more contiguous nucleotides from the nucleotide sequence of the
reference polynucleotide
(e.g., cDNA desposited as ATCC Deposit No: PTA-349, or the nucleotide sequence
as shown
in SEQ ID NO:1 or the complementary strand thereto, or a fragment thereof).
Of course, a polynucleotide which hybridizes only to a poly A sequence (such
as the 3'
terminal poly(A) tract of the TR13 cDNA shown in SEQ ID NO:1 or SEQ ID N0:39),
or to a
complementary stretch of T (or U) resides, would not be included in a
polynucleotide of the
invention used to hybridize to a portion of a nucleic acid of the invention,
since such a
polynucleotide would hybridize to any nucleic acid molecule containing a poly
(A) stretch or the
complement thereof (e.g., practically any double-stranded cDNA clone generated
using oligo
dT as a primer).
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
78
In another aspect, the invention provides an isolated nucleic acid molecule
comprising a
polynucleotide which hybridizes under stringent hybridization conditions to a
portion of the
polynucleotide in a TR14 nucleic acid molecule of the invention described
above, for instance,
a cDNA clone (HMSHK47) contained in ATCC Deposit No. PTA-348, the nucleic acid
sequence disclosed in preferably in Figures l0A-H or, alternatively, in
Figures 4A-D or the
complementary strand thereto, and fragments thereof (e.g., as described
herein).
By a portion of a polynucleotide of "at least 20 nt in length," for example,
is intended
20 or more contiguous nucleotides from the nucleotide sequence of the
reference polynucleotide
(e.g., cDNA deposited as ATCC Deposit No: PTA-348, or the nucleotide sequence
as shown
preferably in SEQ ID N0:60 or, alternatively, in SEQ ID N0:4 or the
complementary strand
thereto, or a fragment thereof).
Of course, a polynucleotide which hybridizes only to a poly A sequence (such
as the 3'
terminal poly(A) tract of the TR14 cDNA shown preferably in SEQ ID N0:60 or,
alternatively,
in SEQ ID N0:4), or to a complementary stretch of T (or U) resides, would not
be included in
a polynucleotide of the invention used to hybridize to a portion of a nucleic
acid of the
invention, since such a polynucleotide would hybridize to any nucleic acid
molecule containing
a poly (A) stretch or the complement thereof (e.g., practically any double-
stranded cDNA clone
generated using oligo dT as a primer).
In specific embodiments, the polynucleotides of the invention are less than
100000 kb,
50000 kb, 10000 kb, 1000 kb, 500 kb, 400 kb, 350 kb, 300 kb, 250 kb, 200 kb,
175 kb, 150
kb, 125 kb, 100 kb, 75 kb, 50 kb, 40 kb, 30 kb, 25 kb, 20 kb, 15 kb, 10 kb,
7.5 kb, or 5 kb
in length.
In further embodiments, nucleic acids of the invention comprise at least 15,
at least 30,
at least 50, at least 100, or at least 250, at least 500, or at least 1000
contiguous nucleotides of
TR13 coding sequence, but consist of less than or equal to 1000 kb, 500 kb,
250 kb, 200 kb,
150 kb, 100 kb, 75 kb, 50 kb, 30 kb, 25 kb, 20 kb, 15 kb, 10 kb, or 5 kb of
genomic DNA
that flanks the S' or 3' coding nucleotide set forth in Figures lA-C (SEQ ID
NO:1) or in
Figures 7A-D (SEQ ID N0:39). In further embodiments, nucleic acids of the
invention
comprise at least 15, at least 30, at least 50, at least 100, or at least 250,
at least 500, or at least
1000 contiguous nucleotides of TR13 coding sequence, but do not comprise all
or a portion of
any TR13 intron. In another embodiment, the nucleic acid comprising TR13
coding sequence
does not contain coding sequences of a genomic flanking gene (i.e., S' or 3'
to the TR13 gene
in the genome). In other embodiments, the polynucleotides of the invention do
not contain the
coding sequence of more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3,
2, or 1
genomic flanking gene(s).
In further, nucleic acids of the invention comprise at least 15, at least 30,
at least 50, at
least 100, or at least 250, at least 500, or at least 1000 contiguous
nucleotides of TR14 coding
sequence, but consist of less than or equal to 1000 kb, 500 kb, 250 kb, 200
kb, 150 kb, 100
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
79
kb, 75 kb, 50 kb, 30 kb, 25 kb, 20 kb, 15 kb, 10 kb, or 5 kb of genomic DNA
that flanks the
5' or 3' coding nucleotide set forth preferably in Figures l0A-H (SEQ ID
N0:60) or,
alternatively, in Figures 4A-D (SEQ ID N0:4). In further embodiments, nucleic
acids of the
invention comprise at least 15, at least 30, at least 50, at least 100, or at
least 250, at least 500,
or at least 1000 contiguous nucleotides of TR14 coding sequence, but do not
comprise all or a
portion of any TR14 intron. In another embodiment, the nucleic acid comprising
TR14 coding
sequence does not contain coding sequences of a genomic flanking gene (i.e.,
5' or 3' to the
TR14 gene in the genome). In other embodiments, the polynucleotides of the
invention do not
contain the coding sequence of more than 1000, 500, 250, 100, 50, 25, 20, 15,
10, 5, 4, 3, 2,
to or 1 genomic flanking gene(s).
As indicated, nucleic acid molecules of the present invention which encode a
TR13
polypeptide may include, but are not limited to the coding sequence for the
mature polypeptide,
by itself; the coding sequence for the mature polypeptide and additional
sequences, such as
those encoding a leader or secretory sequence, such as a pre-, or pro- or
prepro- protein
~5 sequence; the coding sequence of the mature polypeptide, with or without
the aforementioned
additional coding sequences, together with additional, non-coding sequences,
including for
example, but not limited to introns and non-coding 5' and 3' sequences, such
as the
transcribed, non-translated sequences that play a role in transcription, mRNA
processing -
including splicing and polyadenylation signals, for example - ribosome binding
and stability of
2o mRNA; additional coding sequence which codes for additional amino acids,
such as those
which provide additional functionalities. Thus, for instance, the polypeptide
may be fused to a
marker sequence, such as a peptide, which facilitates purification of the
fused polypeptide. In
certain preferred embodiments of this aspect of the invention, the marker
sequence is a hexa-
histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.),
among others, many
25 of which are commercially available. As described in Gentz et al., Proc.
Natl. Acad. Sci.
USA 86: 821-824 (1989), for instance, hexa-histidine provides for convenient
purification of
the fusion protein. The "HA" tag is another peptide useful for purification
which corresponds
to an epitope derived from the influenza hemagglutinin protein, which has been
described by
Wilson et al., Cell 37:767-778 (1984). As discussed below, other such fusion
proteins
3o include, but are not limited to, the TR13 receptor fused to Fc at the N- or
C-terminus.
As indicated, nucleic acid molecules of the present invention which encode a
TR14
polypeptide may include, but are not limited to the coding sequence for the
mature polypeptide,
by itself; the coding sequence for the mature polypeptide and additional
sequences, such as
those encoding a leader or secretory sequence, such as a pre-, or pro- or
prepro- protein
35 sequence; the coding sequence of the mature polypeptide, with or without
the aforementioned
additional coding sequences, together with additional, non-coding sequences,
including for
example, but not limited to introns and non-coding 5' and 3' sequences, such
as the
transcribed, non-translated sequences that play a role in transcription, mRNA
processing -
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
including splicing and polyadenylation signals, for example - ribosome binding
and stability of
mRNA; additional coding sequence which codes for additional amino acids, such
as those
which provide additional functionalities. Thus, for instance, the polypeptide
may be fused to a
marker sequence, such as a peptide, which facilitates purification of the
fused polypeptide. In
5 certain preferred embodiments of this aspect of the invention, the marker
sequence is a hexa-
histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.),
among others, many
of which are commercially available. As described in Gentz et al., Proc. Natl.
Acad. Sci.
USA 86: 821-824 (1989), for instance, hexa-histidine provides for convenient
purification of
the fusion protein. The "HA" tag is another peptide useful for purification
which corresponds
to to an epitope derived from the influenza hemagglutinin protein, which has
been described by
Wilson et al., Cell 37:767-778 (1984). As discussed below, other such fusion
proteins
include, but are not limited to, the TR14 receptor fused to Fc at the N- or C-
terminus.
The present invention further relates to variants of the nucleic acid
molecules of the
present invention, which encode portions, analogs, or derivatives of the TR13
receptor.
15 Variants may occur naturally, such as a natural allelic variant. By an
"allelic variant" is
intended one of several alternate forms of a gene occupying a given locus on a
chromosome of
an organism. Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985). Non-
naturally occurring variants may be produced using art-known mutagenesis
techniques.
Such variants include those produced by nucleotide substitutions, deletions or
additions
2o which may involve one or more nucleotides. The variants may be altered in
coding or non
coding regions or both. Alterations in the coding regions may produce
conservative or non
conservative amino acid substitutions, deletions,. or additions. Especially
preferred among
these are silent substitutions, additions, and deletions, which do not alter
the properties and
activities of the TR13 receptor or portions thereof. Also especially preferred
in this regard are
25 conservative substitutions.
The present invention further relates to variants of the nucleic acid
molecules of the
present invention, which encode portions, analogs, or derivatives of the TR14
receptor.
Variants may occur naturally, such as a natural allelic variant. By an
"allelic variant" is
intended one of several alternate forms of a gene occupying a given locus on a
chromosome of
3o an organism. Genes 1l, Lewin, B., ed., John Wiley & Sons, New York (1985).
Non-
naturally occurring variants may be produced using art-known mutagenesis
techniques.
Such variants include those produced by nucleotide substitutions, deletions or
additions
which may involve one or more nucleotides. The variants may be altered in
coding or non-
coding regions or both. Alterations in the coding regions may produce
conservative or non-
35 conservative amino acid substitutions, deletions, or additions. Especially
preferred among
these are silent substitutions, additions, and deletions, which do not alter
the properties and
activities of the TR14 receptor or portions thereof. Also especially preferred
in this regard are
conservative substitutions.
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
81
Further embodiments of the invention include isolated nucleic acid molecules
comprising or alternatively consisting of, a polynucleotide having a
nucleotide sequence at least
90% identical, and more preferably at least 95%, 96%, 97%, 98%, or 99%
identical to: (a) a
nucleotide sequence encoding the polypeptide having the amino acid sequence in
SEQ ID N0:2;
(b) a nucleotide sequence encoding the polypeptide having the amino acid
sequence in SEQ ID
N0:2, but lacking the amino terminal methionine (amino acid positions 2 - 750
of SEQ ID
N0:2); (c) a nucleotide sequence encoding the polypeptide having the amino
acid sequence
encoded by the cDNA clone contained in ATCC Deposit No. PTA-349 (HWLHM70); (d)
a
nucleotide sequence encoding the mature TR13 polypeptide having the amino acid
sequence
to encoded by the cDNA clone contained in ATCC Deposit No. PTA-349 (HWLHM70);
(e) a
nucleotide sequence encoding any combination of one, two, three or all four of
the TR13
cysteine rich domains disclosed in Figures lA-C (amino acids 105 to 170, amino
acids 251 to
265, amino acids 331 to 410, and/or amino acids 580 to 610 of SEQ ID N0:2);
(f) a nucleotide
sequence encoding the polypeptide having the amino acid sequence at positions
from about 105
~5 to about 170 of SEQ ID N0:2; (g) a nucleotide sequence encoding the
polypeptide having the
amino acid sequence at positions from about 251 to about 265 of SEQ ID N0:2;
(h) a
nucleotide sequence encoding the polypeptide having the amino acid sequence at
positions from
about 331 to about 410 of SEQ ID N0:2; (i) a nucleotide sequence encoding the
polypeptide
having the amino acid sequence at positions from about 580 to about 610 of SEQ
ID N0:2; and
20 (j) a nucleotide sequence complementary to any of the nucleotide sequences
in (a), (b), (c),
(d), (e), (f), (g), (h), or (i), above. In this context "about" includes the
particularly recited size,
larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either
terminus or at both termini.
Further embodiments of the invention include isolated nucleic acid molecules
comprising or alternatively consisting of, a polynucleotide having a
nucleotide sequence at least
25 90% identical, and more preferably at least 95%, 96%, 97%, 98%, or 99%
identical to: (a) a
nucleotide sequence encoding the polypeptide having the amino acid sequence in
SEQ ID
N0:40; (b) a nucleotide sequence encoding the polypeptide having the amino
acid sequence in
SEQ ID N0:40, but lacking the amino terminal methionine (amino acid positions
2 - 1001 of
SEQ ID N0:40); (c) a nucleotide sequence encoding the polypeptide having the
amino acid
3o sequence encoded by the cDNA clone contained in ATCC Deposit No. PTA-507
(HWLHN83);
(d) a nucleotide sequence encoding the mature TR13 polypeptide having the
amino acid
sequence encoded by the cDNA clone contained in ATCC Deposit No. PTA-507
(HWLHN83);
(e) a nucleotide sequence encoding the TR13 receptor mature extracellular
domain (amino acid
positions from about 42 to about 906 of SEQ ID N0:40); (f) a nucleotide
sequence encoding
35 the TR13 receptor transmembrane domain (amino acid positions from about 134
to about 150
of SEQ ID N0:40); (g) a nucleotide sequence encoding the TR13 receptor
intracellular domain
(amino acid positions 932 to about 1001 of SEQ ID N0:40); (h) a nucleotide
seuqence
encoding the TR13 receptor extracellular and intracellular domains with all or
a part of the
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
82
transmembrane domain deleted (amino acid positions from about 42 to about 96
and 932 to
about 1001 of SEQ ID N0:40); (i) a nucleotide sequence encoding the
polypeptide having the
amino acid sequence at positions from about 271 to about 421 of SEQ ID N0:40;
(j) a
nucleotide sequence encoding the polypeptide having the amino acid sequence at
positions from
about 271 to about 286 of SEQ ID N0:40; (k) a nucleotide sequence encoding the
polypeptide
having the amino acid sequence at positions from about 290 to about 300 of SEQ
ID N0:40; (1)
a nucleotide sequence encoding the polypeptide having the amino acid sequence
at positions
from about 301 to about 320 of SEQ ID N0:40; (m) a nucleotide sequence
encoding the
polypeptide having the amino acid sequence at positions from about 329 to
about 361 of SEQ
ID N0:40; (n) a nucleotide sequence encoding the polypeptide having the amino
acid sequence
at positions from about 404 to about 421 of SEQ ID N0:40; (o) a nucleotide
sequence encoding
the polypeptide having the amino acid sequence at positions from about 585 to
about 595 of
SEQ ID N0:40; (p) a nucleotide sequence encoding any one of the TR13 conserved
domains as
shown in Figures 7A-D; (q) a nucleotide sequence encoding the polypeptide
having the amino
acid sequence at positions from about 661 to about 674 of SEQ ID N0:40; (r) a
nucleotide
sequence encoding the polypeptide having the amino acid sequence at positions
from about 710
to about 744 of SEQ ID N0:40; (s) a nucleotide sequence encoding the
polypeptide having the
amino acid sequence at positions from about 980 to about 991 of SEQ ID N0:40;
(t) a
nucleotide sequence encoding the polypeptide having the amino acid sequence at
positions from
2o about 45 to about 60 of SEQ ID N0:40; (u) a nucleotide sequence encoding
the polypeptide
having the amino acid sequence at positions from about 121 to about 135 of SEQ
ID N0:40;
(v) a nucleotide sequence encoding the polypeptide having the amino acid
sequence at positions
from about 145 to about 160 of SEQ ID N0:40; and (w) a nucleotide sequence
complementary
to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h),
(i), (j), (k), (1), (m),
(n), (o), (p), (q), (r), (s), (t), (u) or (v) above. In this context "about"
includes the particularly
recited size, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at
either terminus or at
both termini.
By a polynucleotide having a nucleotide sequence at least, for example, 95%
"identical"
to a reference nucleotide sequence encoding a TR13 polypeptide is intended
that the nucleotide
3o sequence of the polynucleotide is identical to the reference sequence
except that the
polynucleotide sequence may include up to five mismatches per each 100
nucleotides of the
reference nucleotide sequence encoding the TR13 polypeptide. In other words,
to obtain a
polynucleotide having a nucleotide sequence at least 95% identical to a
reference nucleotide
sequence, up to 5% of the nucleotides in the reference sequence may be deleted
or substituted
with another nucleotide, or a number of nucleotides up to 5% of the total
nucleotides in the
reference sequence may be inserted into the reference sequence. These
mismatches of the
reference sequence may occur at the 5' or 3' terminal positions of the
reference nucleotide
sequence or anywhere between those terminal positions, interspersed either
individually among
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
83
nucleotides in the reference sequence or in one or more contiguous groups
within the reference
sequence. The reference (query) sequence may be the entire TR13 encoding
nucleotide
sequence shown in Figures lA-C (SEQ ID NO:1) or Figures 7A-D (SEQ ID N0:39) or
any
TR13 polynucleotide fragment (e.g., a polynucleotide encoding the amino acid
sequence of any
of the TR13 N- and/or C- terminal deletions described herein), variant,
derivative or analog, as
described herein.
As a practical matter, whether any particular polynucleotide sequence is at
least 90%,
95%, 96%, 97%, 98% or 99% identical to, for instance, the nucleotide sequence
shown in
SEQ ID NO:1 or SEQ ID N0:39 or to the nucleotide sequence of the deposited
cDNA clone
(HWLHM70 or HWLHN83) can be determined conventionally using known computer
programs such as the Bestfit program (Wisconsin Sequence Analysis Package,
Version 8 for
Unix, Genetics Computer Group, University Research Park, 575 Science Drive,
Madison, WI
53711). Bestfit uses the local homology algorithm of Smith and Waterman,
Advances in
Applied Mathematics 2: 482-489 (1981), to find the best segment of homology
between two
sequences. When using Bestfit or any other sequence alignment program to
determine whether
a particular sequence is, for instance, 95% identical to a reference sequence
according to the
present invention, the parameters are set, of course, such that the percentage
of identity is
calculated over the full-length of the reference nucleotide sequence and that
gaps in homology
of up to 5% of the total number of nucleotides in the reference sequence are
allowed.
2o Further embodiments of the invention include isolated nucleic acid
molecules
comprising a polynucleotide having a nucleotide sequence at least 90%
identical, and more
preferably at least 95%, 96%, 97%, 98%, or 99% identical to: (a) a nucleotide
sequence
encoding the polypeptide having the amino acid sequence in SEQ ID N0:61; (b) a
nucleotide
sequence encoding the polypeptide having the amino acid sequence in SEQ ID NO:
61, but
lacking the amino terminal methionine (amino acid positions 2 - 231 of SEQ ID
N0:61 ); (c) a
nucleotide sequence encoding the polypeptide having the amino acid sequence
encoded by the
cDNA clone contained in ATCC Deposit No. HMSHK47; (d) a nucleotide sequence
encoding
the polypeptide having the amino acid sequence in SEQ ID NO:S; (e) a
nucleotide sequence
encoding the polypeptide having the amino acid sequence in SEQ ID NO: 5, but
lacking the
3o amino terminal methionine (amino acid positions 2 - 226 of SEQ ID NO:S);
(f) a nucleotide
sequence encoding the TR14 receptor extracellular domain (preferably
preferably amino acid
positions from about 1 to about 138 of SEQ ID N0:61 or, alternatively, amino
acid positions
from about 1 to about 133 of SEQ ID NO:S); (g) a nucleotide sequence encoding
the TR14
cysteine rich domain (preferably amino acid positions from about 70 to about
90 of SEQ ID
N0:61 or, alternatively, amino acid positions from about 65 to about 85 of SEQ
ID NO:S); (h)
a nucleotide sequence encoding the TR14 receptor transmembrane domain
(preferably, amino
acid positions from about 139 to about 155 of SEQ ID N0:61 or, alternatively,
amino acid
positions from about 134 to about 150 of SEQ ID NO:S) ; (i) a nucleotide
sequence encoding
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
84
the TR14 receptor intracellular domain (preferably, amino acid positions from
about 156 to
about 231 of SEQ ID N0:61 or, alternatively, from about amino acid positions
151 to about
226 of SEQ ID NO:S); (j) a nucleotide sequence encoding the TR14 receptor
extracellular and
intracellular domains with all or part of the transmembrane domain deleted
(preferably amino
acid positions from about 1 to about 138 and 156 to about 231 of SEQ ID N0:61
or,
alternatively, amino acid positions from about 1 to about 133 and 151 to about
226 of SEQ ~
NO:S); and (k) a nucleotide sequence complementary to any of the nucleotide
sequences in
(a), (b), (c), (d), (e), (f), (g), (h), (i), (j) or (k) above. In this context
"about" includes the
particularly recited size, larger or smaller by several (5, 4, 3, 2, or 1)
nucleotides, at either
to terminus or at both termini.
By a polynucleotide having a nucleotide sequence at least, for example, 95%
"identical"
to a reference nucleotide sequence encoding a TR14 polypeptide is intended
that the nucleotide
sequence of the polynucleotide is identical to the reference sequence except
that the
polynucleotide sequence may include up to five mismatches per each 100
nucleotides of the
t5 reference nucleotide sequence encoding the TR14 polypeptide. In other
words, to obtain a
polynucleotide having a nucleotide sequence at least 95% identical to a
reference nucleotide
sequence, up to 5% of the nucleotides in the reference sequence may be deleted
or substituted
with another nucleotide, or a number of nucleotides up to 5% of the total
nucleotides in the
reference sequence may be inserted into the reference sequence. These
mismatches of the
2o reference sequence may occur at the 5' or 3' terminal positions of the
reference nucleotide
sequence or anywhere between those terminal positions, interspersed either
individually among
nucleotides in the reference sequence or in one or more contiguous groups
within the reference
sequence. The reference (query) sequence may be the entire TR14 encoding
nucleotide
sequence shown preferably in Figures l0A-H (SEQ ID N0:60) or, alternatively,
in Figures
25 4A-D (SEQ ID N0:4) or any TR14 polynucleotide fragment (e.g., a
polynucleotide encoding
the amino acid sequence of any of the TR14 N- and/or C- terminal deletions
described herein),
variant, derivative or analog, as described herein.
As a practical matter, whether any particular polynucleotide sequence is at
least 90%,
95%, 96%, 97%, 98% or 99% identical to, for instance, the nucleotide sequence
shown
30 preferably in SEQ ID N0:60 or, alternatively, in SEQ ID N0:4 or to the
nucleotide sequence of
the deposited cDNA clone can be determined conventionally using known computer
programs
such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8
for Unix,
Genetics Computer Group, University Research Park, 575 Science Drive, Madison,
WI
53711 ). Bestfit uses the local homology algorithm of Smith and Waterman,
Advances in
35 Applied Mathematics 2: 482-489 (1981), to find the best segment of homology
between two
sequences. When using Bestfit or any other sequence alignment program to
determine whether
a particular sequence is, for instance, 95% identical to a reference sequence
according to the
present invention, the parameters are set, of course, such that the percentage
of identity is
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
calculated over the full-length of the reference nucleotide sequence and that
gaps in homology
of up to 5% of the total number of nucleotides in the reference sequence are
allowed.
In a specific embodiment, the identity between a reference (query) sequence (a
sequence
of the present invention) and a subject sequence, also referred to as a global
sequence
5 alignment, is determined using the FASTDB computer program based on the
algorithm of
Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). Preferred parameters
used in a
FASTDB alignment of DNA sequences to calculate percent identity are:
Matrix=Unitary, k-
tuple=4, Mismatch Penalty=1, Joining Penalty=30, Randomization Group Length=0,
Cutoff
Score=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the length
of the
to subject nucleotide sequence, whichever is shorter. According to this
embodiment, if the
subject sequence is shorter than the query sequence because of 5' or 3'
deletions, not because
of internal deletions, a manual correction is made to the results to take into
consideration the
fact that the FASTDB program does not account for 5' and 3' truncations of the
subject
sequence when calculating percent identity. For subject sequences truncated at
the 5' or 3'
~5 ends, relative to the query sequence, the percent identity is corrected by
calculating the number
of bases of the query sequence that are 5' and 3' of the subject sequence,
which are not
matched/aligned, as a percent of the total bases of the query sequence. A
determination of
whether a nucleotide is matched/aligned is determined by results of the FASTDB
sequence
alignment. This percentage is then subtracted from the percent identity,
calculated by the above
2o FASTDB program using the specified parameters, to arrive at a final percent
identity score.
This corrected score is what is used for the purposes of this embodiment. Only
bases outside
the 5' and 3' bases of the subject sequence, as displayed by the FASTDB
alignment, which are
not matched/aligned with the query sequence, are calculated for the purposes
of manually
adjusting the percent identity score. For example, a 90 base subject sequence
is aligned to a
25 100 base query sequence to determine percent identity. The deletions occur
at the 5' end of the
subject sequence and therefore, the FASTDB alignment does not show a
matched/alignment of
the first 10 bases at S' end. The 10 unpaired bases represent 10% of the
sequence (number of
bases at the 5' and 3' ends not matched/total number of bases in the query
sequence) so 10% is
subtracted from the percent identity score calculated by the FASTDB program.
If the remaining
30 90 bases were perfectly matched the final percent identity would be 90%. In
another example,
a 90 base subject sequence is compared with a 100 base query sequence. This
time the
deletions are internal deletions so that there are no bases on the 5' or 3' of
the subject sequence
which are not matched/aligned with the query. In this case the percent
identity calculated by
FASTDB is not manually corrected. Once again, only bases 5' and 3' of the
subject sequence
35 which are not matched/aligned with the query sequence are manually
corrected for. No other
manual corrections are made for the purposes of this embodiment.
The present application is directed to nucleic acid molecules comprising a
polynucleotide sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to
the nucleic
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
86
acid sequence for example, shown in SEQ ID NO:1 or SEQ ID N0:39, or to the
nucleic acid
sequence of the cDNA deposited as ATCC deposit No. PTA-349 or PTA-507,
irrespective of
whether they encode a polypeptide having TR13 receptor activity. This is
because even where
a particular nucleic acid molecule does not encode a polypeptide having TR13
functional
activity, one of skill in the art would still know how to use the nucleic acid
molecule, for
instance, as a hybridization probe or a polymerase chain reaction (PCR)
primer. Uses of the
nucleic acid molecules of the present invention that do not encode a
polypeptide having TR13
receptor activity include, inter alias (1) isolating the TR13 receptor gene or
allelic variants
thereof in a cDNA library; (2) in situ hybridization (e.g., "FISH") to
metaphase chromosomal
1o spreads to provide precise chromosomal location of the TR13 receptor gene,
as described in
Verma et al., Human Chromosomes: A Manual of Basic Technigues, Pergamon Press,
New
York (1988); and (3) Northern Blot analysis for detecting TR13 receptor mRNA
expression in
specific tissues.
Preferred, however, are nucleic acid molecules having sequences at least 90%,
95%,
96%, 97%, 98% or 99% identical to for example, the nucleic acid sequence shown
in SEQ ~
NO:1 or SEQ ID N0:39, or to the nucleic acid sequence of the cDNA deposited as
PTA-349 or
PTA-507, which do, in fact, encode a polypeptide having TR13 receptor
functional activity. By
"a polypeptide having TR13 functional receptor activity" is intended
polypeptides exhibiting
activity similar, but not necessarily identical, to an activity of the TR13
receptor of the invention
(either the full-length protein or, preferably, the mature protein), as
measured, for example, in a
particular biological assay.
Of course, due to the degeneracy of the genetic code, one of ordinary skill in
the art will
immediately recognize that a large number of the nucleic acid molecules having
a sequence at
least 90%, 95%, 96%, 97%, 98%, or 99% identical to, for example, the nucleic
acid sequence
of the deposited cDNA or the nucleic acid sequence shown in SEQ ID NO: I or
SEQ ID N0:39
will encode a polypeptide "having TR13 receptor functional activity." In fact,
since degenerate
variants of these nucleotide sequences all encode the same polypeptide, this
will be clear to the
skilled artisan even without performing the above described comparison assay.
It will be
further recognized in the art that, for such nucleic acid molecules that are
not degenerate
3o variants, a reasonable number will also encode a polypeptide having TR13
receptor activity.
This is because the skilled artisan is fully aware of amino acid substitutions
that are either less
likely or not likely to significantly effect protein function (e.g., replacing
one aliphatic amino
acid with a second aliphatic amino acid).
For example, guidance concerning how to make phenotypically silent amino acid
substitutions is provided in J.U. Bowie et al., "Deciphering the Message in
Protein Sequences:
Tolerance to Amino Acid Substitutions," Science 247:1306-1310 (1990), wherein
the authors
indicate that proteins are surprisingly tolerant of amino acid substitutions.
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
87
The present application is directed to nucleic acid molecules comprising a
polynucleotide sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to
the nucleic
acid sequence for example, shown preferably in SEQ ID N0:60 or, alternatively,
in SEQ ID
N0:4, or to the nucleic acid sequence of the cDNA deposited as ATCC Deposit
No. PTA-348,
and even more preferably to the polypeptide coding regions of these sequences,
irrespective of
whether they encode a polypeptide having TRI4 receptor activity. This is
because even where
a particular nucleic acid molecule does not encode a polypeptide having TR14
functional
activity, one of skill in the art would still know how to use the nucleic acid
molecule, for
instance, as a hybridization probe or a polymerase chain reaction (PCR)
primer. Uses of the
1o nucleic acid molecules of the present invention that do not encode a
polypeptide having TR14
receptor activity include, inter alias (1) isolating the TR14 receptor gene or
allelic variants
thereof in a cDNA library; (2) in situ hybridization (e.g., "FISH") to
metaphase chromosomal
spreads to provide precise chromosomal location of the TRI4 receptor gene, as
described in
Verma et al., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press,
New
York (1988); and (3) Northern Blot analysis for detecting TR14 receptor mRNA
expression in
specific tissues.
Preferred, however, are nucleic acid molecules having sequences at least 90%,
95%,
96%, 97%, 98% or 99% identical to for example, the nucleic acid sequence shown
preferably
in SEQ ID N0:60 or, alternatively, in SEQ ID N0:4, or to the nucleic acid
sequence of the
deposited cDNA, and even more preferably to the polypeptide coding regions of
these
sequences, which do, in fact, encode a polypeptide having TRI4 receptor
functional activity.
By "a polypeptide having TR14 functional receptor activity" is intended
polypeptides exhibiting
activity similar, but not necessarily identical, to an activity of the TR14
receptor of the invention
(either the full-length protein or, preferably, the mature protein), as
measured, for example, in a
particular biological assay.
Of course, due to the degeneracy of the genetic code, one of ordinary skill in
the art will
immediately recognize that a large number of the nucleic acid molecules having
a sequence at
least 90%, 95%, 96%, 97%, 98%, or 99% identical to, for example, the nucleic
acid sequence
of the deposited cDNA or the nucleic acid sequence shown preferably in SEQ 1D
N0:60 or,
3o alternatively, in SEQ ID N0:4 will encode a polypeptide "having TR14
receptor functional
activity." In fact, since degenerate variants of these nucleotide sequences
all encode the same
polypeptide, this will be clear to the skilled artisan even without performing
the above
described comparison assay. It will be further recognized in the art that, for
such nucleic acid
molecules that are not degenerate variants, a reasonable number will also
encode a polypeptide
having TR14 receptor activity. This is because the skilled artisan is fully
aware of amino acid
substitutions that are either less likely or not likely to significantly
effect protein function (e.g.,
replacing one aliphatic amino acid with a second aliphatic amino acid).
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
88
Polynucleotide assays
This invention is also related to the use of TR13 polynucleotides to detect
complementary polynucleotides such as, for example, as a diagnostic reagent.
Detection of a
mutated form of TR13 polynucleotide associated with a dysfunction will provide
a diagnostic
tool that can add or define a diagnosis of a disease or susceptibility to a
disease which results
from under-expression over-expression or altered expression of TR13 or a
soluble form
thereof, such as, for example, tumors or autoimmune disease.
Individuals carrying mutations in the TR13 gene may be detected at the nucleic
acid
level by a variety of techniques. Nucleic acids for diagnosis may be obtained
from a patient's
cells, such as from blood, urine, saliva, tissue biopsy and autopsy material.
The genomic
DNA may be used directly for detection or may be amplified enzymatically by
using PCR prior
to analysis. (Saiki et al., Nature 324:163-166 (1986)). RNA or cDNA may also
be used in the
same ways, or through routine modification of these polynucleotides. As an
example, PCR
primers complementary to the nucleic acid encoding TR13 can be used to
identify and analyze
TR13 expression and mutations. For example, deletions and insertions can be
detected by a
change in size of the amplified product in comparison to the normal genotype.
Point mutations
can be identified using techniques known in the art, for example, by
hybridizing amplified
DNA to radiolabeled TR13 RNA or alternatively, radiolabeled TR13 antisense DNA
sequences.
Perfectly matched sequences can be distinguished from mismatched duplexes by,
for example,
RNase A digestion or by differences in melting temperatures.
This invention is also related to the use of TR14 polynucleotides to detect
complementary polynucleotides such as, for example, as a diagnostic reagent.
Detection of a
mutated form of TR14 polynucleotide associated with a dysfunction will provide
a diagnostic
tool that can add or define a diagnosis of a disease or susceptibility to a
disease which results
from under-expression over-expression or altered expression of TR14 or a
soluble form
thereof, such as, for example, tumors or autoimmune disease.
Individuals carrying mutations in the TR14 gene may be detected at the nucleic
acid
level by a variety of techniques. Nucleic acids for diagnosis may be obtained
from a patient's
cells, such as from blood, urine, saliva, tissue biopsy and autopsy material.
The genomic
3o DNA may be used directly for detection or may be amplified enzymatically by
using PCR prior
to analysis. (Saiki et al., Nature 324:163-166 (1986)). RNA or cDNA may also
be used in the
same ways, or through routine modification of these polynucleotides. As an
example, PCR
primers complementary to the nucleic acid encoding TR14 can be used to
identify and analyze
TR14 expression and mutations. For example, deletions and insertions can be
detected by a
change in size of the amplified product in comparison to the normal genotype.
Point mutations
can be identified by hybridizing amplified DNA to radiolabeled TR14 RNA or
alternatively,
radiolabeled TR14 antisense DNA sequences. Perfectly matched sequences can be
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
89
distinguished from mismatched duplexes by, for example, RNase A digestion or
by differences
in melting temperatures.
Sequence differences between a reference gene and genes having mutations also
may be
revealed by direct DNA sequencing. In addition, cloned DNA segments may be
employed as
probes to detect specific DNA segments. The sensitivity of such methods can be
greatly
enhanced by appropriate use of PCR or another amplification method. For
example, a
sequencing primer is used with double-stranded PCR product or a single-
stranded template
molecule generated by a modified PCR. The sequence determination is performed
by
conventional procedures with radiolabeled nucleotide or by automatic
sequencing procedures
to with fluorescent-tags.
Genetic testing based on DNA sequence differences may be achieved by detection
of
alteration in electrophoretic mobility of DNA fragments in gels, with or
without denaturing
agents. Small sequence deletions and insertions can be visualized by high
resolution gel
electrophoresis. DNA fragments of different sequences may be distinguished on
denaturing
~5 formamide gradient gels in which the mobilities of different DNA fragments
are retarded in the
gel at different positions according to their specific melting or partial
melting temperatures (see,
e.g., Myers et al., Science 230:1242 (1985)).
Sequence changes at specific locations also may be revealed by nuclease
protection
assays, such as RNase and S1 protection or the chemical cleavage method (e.g.,
Cotton et al.,
20 Proc. Natl. Acad. Sci. USA 85: 4397-4401 (1985)).
Thus, the detection of a specific DNA sequence may be achieved by methods such
as,
for example, hybridization, RNase protection, chemical cleavage, direct DNA
sequencing or
the use of restriction enzymes, (e.g., restriction fragment length
polymorphisms ("RFLP") and
Southern blotting of genomic DNA.
25 In addition to more conventional gel-electrophoresis and DNA sequencing,
mutations
also can be detected by in situ analysis.
Vectors and Host Cells
The present invention also relates to vectors which include the isolated DNA
molecules
30 of the present invention, host cells which are genetically engineered with
the recombinant
vectors and/or nucleic acids of the invention and the production of TR13
polypeptides or
fragments thereof by recombinant techniques.
The present invention also relates to vectors which include the isolated DNA
molecules
of the present invention, host cells which are genetically engineered with the
recombinant
35 vectors and/or nucleic acids of the invention and the production of TR14
polypeptides or
fragments thereof by recombinant techniques.
Host cells can be genetically engineered to incorporate nucleic acid molecules
and express
polypeptides of the present invention. The polynucleotides may be introduced
alone or with
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
other polynucleotides. Such other polynucleotides may be introduced
independently, co-
introduced or introduced joined to the polynucleotides of the invention.
In accordance with the present invention the vector may be, for example, a
clone vector, a
single or double-stranded phage vector, a single or double-stranded RNA or DNA
viral vector.
5 Such vectors may be introduced into cells as polynucleotides, preferably
DNA, by well known
techniques for introducing DNA and RNA into cells. Viral vectors may be
replication
competent or replication defective. In the latter case viral propagation
generally will occur only
in complementing host cells.
Preferred among vectors, in certain respects, are those for expression of
polynucleotides and polypeptides of the present invention. Generally, such
vectors comprise
cis-acting control regions effective for expression in a host operatively
linked to the
polynucleotide to be expressed. Appropriate trans-acting factors either are
supplied by the
host, supplied by a complementing vector or supplied by the vector itself upon
introduction into
the host.
15 The polynucleotides may be joined to a vector containing a selectable
marker for
propagation in a host. Generally, a clone vector is introduced in a
precipitate, such as a calcium
phosphate precipitate, or in a complex with a charged lipid. If the vector is
a virus, it may be
packaged in vitro using an appropriate packaging cell line and then transduced
into host cells.
The DNA insert should be operatively linked to an appropriate promoter, such
as the
2o phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40
early and late
promoters and promoters of retroviral LTRs, to name a few. Other suitable
promoters will be
known to the skilled artisan. The expression constructs will further contain
sites for
transcription initiation, termination and, in the transcribed region, a
ribosome binding site for
translation. The coding portion of the mature transcripts expressed by the
constructs will
25 preferably include a translation initiating at the beginning and a
termination codon (UAA, UGA
or UAG) appropriately positioned at the end of the polypeptide to be
translated.
As indicated, the expression vectors will preferably include at least one
selectable
marker. Such markers include dihydrofolate reductase or neomycin resistance
for eukaryotic
cell culture and tetracycline or ampicillin resistance genes for culturing in
E. coli and other
3o bacteria. Representative examples of appropriate hosts include, but are not
limited to, bacterial
cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal
cells, such as
yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells;
animal cells such as
CHO, COS and Bowes melanoma cells; and plant cells. Appropriate culture
mediums and
conditions for the above-described host cells are known in the art.
35 Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9,
available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors,
pNHBA,
pNHl6a, pNHlBA, pNH46A, and pSport available from Stratagene; and ptrc99a,
pKK223-3,
pKK233-3, pDR540, pRlTS available from Pharmacia. Among preferred eukaryotic
vectors
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
91
are pWLNEO, pSV2CAT, pOG44, pXTI and pSG available from Stratagene; and pSVK3,
pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be
readily
apparent to the skilled artisan.
The present invention also relates to host cells containing the above-
described vector
constructs described herein, and additionally encompasses host cells
containing nucleotide
sequences of the invention that are operably associated with one or more
heterologous control
regions (e.g., promoter and/or enhancer) using techniques known of in the art.
The host cell
can be a higher eukaryotic cell, such as a mammalian cell (e.g., a human
derived cell), or a
lower eukaryotic cell, such as a yeast cell, or the host cell can be a
prokaryotic cell, such as a
t0 bacterial cell. The host strain may be chosen which modulates the
expression of the inserted
gene sequences, or modifies and processes the gene product in the specific
fashion desired.
Expression from certain promoters can be elevated in the presence of certain
inducers; thus
expression of the genetically engineered polypeptide may be controlled.
Furthermore, different
host cells have characteristics and specific mechanisms for the translational
and post-
translational processing and modification (e.g., phosphorylation, cleavage) of
proteins.
Appropriate cell lines can be chosen to ensure the desired modifications and
processing of the
foreign protein expressed.
Introduction of the construct into the host cell can be effected by calcium
phosphate
transfection, DEAE-dextran mediated transfection, cationic lipid-mediated
transfection,
electroporation, transduction, infection or other methods. Such methods are
described in many
standard laboratory manuals, such as Davis et al., Basic Methods In Molecular
Biology (1986).
In addition to encompassing host cells containing the vector constructs
discussed
herein, the invention also encompasses primary, secondary, and immortalized
host cells of
vertebrate origin, particularly mammalian origin, that have been engineered to
delete or replace
endogenous genetic material (e.g., TR13 coding sequence), and/or to include
genetic material
(e.g., heterologous polynucleotide sequences) that is operably associated with
TR13
polynucleotides of the invention, and which activates, alters, and/or
amplifies endogenous
TR13 polynucleotides. For example, techniques known in the art may be used to
operably
associate heterologous control regions (e.g., promoter and/or enhancer) and
endogenous TR13
polynucleotide sequences via homologous recombination (see, e.g., US Patent
Number
5,641,670, issued June 24, 1997; International Publication Number WO 96/29411,
published
September 26, 1996; International Publication Number WO 94/12650, published
August 4,
1994; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and
Zijlstra et al.,
Nature 342:435-438 (1989), the disclosures of each of which are incorporated
by reference in
their entireties).
In addition to encompassing host cells containing the vector constructs
discussed
herein, the invention also encompasses primary, secondary, and immortalized
host cells of
vertebrate origin, particularly mammalian origin, that have been engineered to
delete or replace
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
92
endogenous genetic material (e.g., TR14 coding sequence), and/or to include
genetic material
(e.g., heterologous polynucleotide sequences) that is operably associated with
TR14
polynucleotides of the invention, and which activates, alters, and/or
amplifies endogenous
TR14 polynucleotides. For example, techniques known in the art may be used to
operably
associate heterologous control regions (e.g., promoter and/or enhancer) and
endogenous TR14
polynucleotide sequences via homologous recombination (see, e.g., US Patent
Number
5,641,670, issued June 24, 1997; International Publication Number WO 96/29411,
published
September 26, 1996; International Publication Number WO 94/12650, published
August 4,
1994; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and
Zijlstra et al.,
Nature 342:435-438 (1989), the disclosures of each of which are incorporated
by reference in
their entireties).
The TR13 polypeptides of the invention may be expressed in a modified form,
such as
a fusion protein (comprising the polypeptide joined via a peptide bond to a
heterologous protein
sequence (of a different protein)), and may include not only secretion signals
but also additional
heterologous functional regions. Alternatively, such a fusion protein can be
made by protein
synthetic techniques, e.g., by use of a peptide synthesizer. Thus, a region of
additional amino
acids, particularly charged amino acids, may be added to the N-terminus of the
polypeptide to
improve stability and persistence in the host cell, during purification or
during subsequent
handling and storage. Also, peptide moieties may be added to the polypeptide
to facilitate
purification. Such regions may be removed prior to final preparation of the
polypeptide. The
addition of peptide moieties to polypeptides to engender secretion or
excretion, to improve
stability and to facilitate purification, among others, are familiar and
routine techniques in the
art. For example, in one embodiment, polynucleotides encoding TR13
polypeptides of the
invention may be fused to the pelB pectate lyase signal sequence to increase
the efficiency to
expression and purification of such polypeptides in Gram-negative bacteria.
See, US Patent
Nos. 5,576,195 and 5,846,818, the contents of which are herein incorporated by
reference in
their entireties.
The TR14 polypeptides of the invention may also be expressed in a modified
form,
such as a fusion protein (comprising the polypeptide joined via a peptide bond
to a
heterologous protein sequence (of a different protein)), and may include not
only secretion
signals but also additional heterologous functional regions. Alternatively,
such a fusion protein
can be made by protein synthetic techniques, e.g., by use of a peptide
synthesizer. Thus, a
region of additional amino acids, particularly charged amino acids, may be
added to the N-
terminus of the polypeptide to improve stability and persistence in the host
cell, during
purification or during subsequent handling and storage. Also, peptide moieties
may be added
to the polypeptide to facilitate purification. Such regions may be removed
prior to final
preparation of the polypeptide. The addition of peptide moieties to
polypeptides to engender
secretion or excretion, to improve stability and to facilitate purification,
among others, are
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
93
familiar and routine techniques in the art. For example, in one embodiment,
polynucleotides
encoding TR14 polypeptides of the invention may be fused to the pelB pectate
lyase signal
sequence to increase the efficiency to expression and purification of such
polypeptides in
Gram-negative bacteria. See, US Patent Nos. 5,576,195 and 5,846,818, the
contents of
which are herein incorporated by reference in their entireties.
A preferred fusion protein comprises a heterologous region from immunoglobulin
that
is useful to solubilize proteins. For example, EP-A-O 464 533 (Canadian
counterpart
2045869) discloses fusion proteins comprising various portions of constant
region of
immunoglobin molecules together with another human protein or part thereof. In
many cases,
the Fc part in a fusion protein is thoroughly advantageous for use in therapy
and diagnosis and
thus results, for example, in improved pharmacokinetic properties (EP-A 0232
262). On the
other hand, for some uses, it would be desirable to be able to delete the Fc
part after the fusion
protein has been expressed, detected and purified in the advantageous manner
described. This
is the case when the Fc portion proves to be a hindrance to use in therapy and
diagnosis, for
example, when the fusion protein is to be used as an antigen for
immunizations. In drug
discovery, for example, human proteins, such as the hILS-receptor, have been
fused W th Fc
portions for the purpose of high-throughput screening assays to identify
antagonists of hIL-5.
See, D. Bennett et al., Journal of Molecular Recognition 8:52-58 ( 1995) and
K. Johanson et
al., The Journal of Biological Chemistry 270:16:9459-9471 (1995).
2o Polypeptides of the present invention include naturally purified products,
products of
chemical synthetic procedures, and products produced by recombinant techniques
from a
prokaryotic or eukaryotic host, including, for example, bacterial, yeast,
higher plant, insect and
mammalian cells. Depending upon the host employed in a recombinant production
procedure,
the polypeptides of the present invention may be glycosylated or non-
glycosylated. In
addition, polypeptides of the invention may also include an initial modified
methionine residue,
in some cases as a result of host-mediated processes.
In addition, TR13 polypeptides of the 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 TR13 polypeptide fragment 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
TR13 polypeptide
sequence. Non-classical amino acids include, but are not limited to, to the D-
isomers of the
common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-
aminobutyric acid,
Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino
isobutyric
acid, 3-amino propionic acid, ornithine, norleucine, norvaline,
hydroxyproline, sarcosine,
citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine,
phenylglycine,
cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acids such as
b-methyl amino
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
94
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).
In addition, TR14 polypeptides of the 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 TR14 polypeptide fragment 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
TR14 polypeptide
sequence. Non-classical amino acids include, but are not limited to, to the D-
isomers of the
common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-
aminobutyric acid,
Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino
isobutyric
acid, 3-amino propionic acid, ornithine, norleucine, norvaline,
hydroxyproline, sarcosine,
citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine,
phenylglycine,
cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acids such as
b-methyl amino
~5 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).
The invention additionally, encompasses TR13 polypeptides (proteins) which are
differentially modified during or after translation, e.g., by glycosylation,
acetylation,
phosphorylation, amidation, derivatization by known protecting/blocking
groups, proteolytic
2o 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.
25 The invention additionally, encompasses TR14 polypeptides (proteins) which
are
differentially modified during or after translation, e.g., by glycosylation,
acetylation,
phosphorylation, amidation, derivatization by known protecting/blocking
groups, proteolytic
cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any
of numerous
chemical modifications may be carried out by known techniques, including but
not limited to,
3o specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin,
papain, V8 protease,
NaBH4, acetylation, formylation, oxidation, reduction, metabolic synthesis in
the presence of
tunicamycin; etc.
Additional post-translational modifications encompassed by the invention
include, for
example, e.g., N-linked or O-linked carbohydrate chains, processing of N-
terminal or C
35 terminal ends), attachment of chemical moieties to the amino acid backbone,
chemical
modifications of N-linked or O-linked carbohydrate chains, and addition or
deletion of an N-
terminal methionine residue as a result of procaryotic host cell expression.
The polypeptides
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
may also be modified with a detectable label, such as an enzymatic,
fluorescent, isotopic or
affinity label to allow for detection and isolation of the protein.
Also provided by the invention are chemically modified derivatives of TR13
polypeptides (proteins) which may provide additional advantages such as
increased solubility,
5 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, for example, 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.
Also provided by the invention are chemically modified derivatives of TR14
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.
15 4,179,337). The chemical moieties for derivitization may be selected from
water soluble
polymers such as, for example, 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
20 moieties.
The polymer may be of any molecular weight, and may be branched or unbranched.
For polyethylene glycol, the preferred molecular weight is between about 1 kDa
and about 100
kDa (the term "about" indicating that in preparations of polyethylene glycol,
some molecules
will weigh more, some less, than the stated molecular weight) for ease in
handling and
25 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). The polymer may be of any molecular
weight, and may be
branched or unbranched. For polyethylene glycol, the preferred molecular
weight is between
3o 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
35 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,
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
96
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.
As noted above, the polyethylene glycol may have a branched structure.
Branched
polyethylene glycols are described, for example, in U.S. Patent No. 5,643,575;
Morpurgo et
al., Appl. Biochem. Biotechnol. 56:59-72 (1996); Vorobjev et al., Nucleosides
Nucleotides
18:2745-2750 (1999); and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999),
the
disclosures of each of which are incorporated herein by reference.
The polyethylene glycol molecules (or other chemical moieties) should be
attached to
to the polypeptide (proteins) with consideration of effects on functional or
antigenic domains of
the protein. There are a number of attachment methods available to those
skilled in the art,
e.g., EP 0 401 384, herein incorporated by reference (coupling PEG to G-CSF),
see also
Malik et al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation of GM-
CSF using
tresyl chloride). For example, polyethylene glycol may be covalently bound
through amino
acid residues via a reactive group, such as, a free amino or carboxyl group.
Reactive groups
are those to which an activated polyethylene glycol molecule may be bound. The
amino acid
residues having a free amino group may include lysine residues and the N-
terminal amino acid
residues; those having a free carboxyl group may include aspartic acid
residues glutamic acid
residues and the C-terminal amino acid residue. Sulfhydryl groups may also be
used as a
2o reactive group for attaching the polyethylene glycol molecules. Preferred
for therapeutic
purposes is attachment at an amino group, such as attachment at the N-terminus
or lysine
group.
As suggested above, polyethylene glycol may be attached to proteins via
linkage to any
of a number of amino acid residues. For example, polyethylene glycol can be
linked to a
proteins via covalent bonds to lysine, histidine, aspartic acid, glutamic
acid, or cysteine
residues. One or more reaction chemistries may be employed to attach
polyethylene glycol to
specific amino acid residues (e.g., lysine, histidine, aspartic acid, glutamic
acid, or cysteine) of
the protein or to more than one type of amino acid residue (e.g., lysine,
histidine, aspartic acid,
glutamic acid, cysteine and combinations thereof) of the protein.
3o One may specifically desire polypeptides (proteins) chemically modified at
the N-
terminus. Using polyethylene glycol as an illustration of the present
composition, one may
select from a variety of polyethylene glycol molecules (by molecular weight,
branching, etc.),
the proportion of polyethylene glycol molecules to protein (or peptide)
molecules in the reaction
mix, the type of pegylation reaction to be performed, and the method of
obtaining the selected
N-terminally pegylated protein. The method of obtaining the N-terminally
pegylated
preparation (i.e., separating this moiety from other monopegylated moieties if
necessary) may
be by purification of the N-terminally pegylated material from a population of
pegylated protein
molecules. Selective proteins chemically modified at the N-terminus
modification may be
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
97
accomplished by reductive alkylation which exploits differential reactivity of
different types of
primary amino groups (lysine versus the N-terminal) available for
derivatization in a particular
protein. Under the appropriate reaction conditions, substantially selective
derivatization of the
protein at the N-terminus with a carbonyl group containing polymer is
achieved.
As indicated above, pegylation of the proteins of the invention may be
accomplished by
any number of means. For example, polyethylene glycol may be attached to the
protein either
directly or by an intervening linker. Linkerless systems for attaching
polyethylene glycol to
proteins are described in Delgado et al., Crit. Rev. 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
1o No. 5,349,052; WO 95/06058; and WO 98/32466, the disclosures of each of
which are
incorporated herein by reference.
One system for attaching polyethylene glycol directly to amino acid residues
of proteins
without an intervening linker employs tresylated MPEG, which is produced by
the modification
of monmethoxy polyethylene glycol (MPEG) using tresylchloride (C1SOZCHZCF3).
Upon
reaction of protein with tresylated MPEG, polyethylene glycol is directly
attached to amine
groups of the protein. Thus, the invention includes protein-polyethylene
glycol conjugates
produced by reacting proteins of the invention with a polyethylene glycol
molecule having a
2,2,2-trifluoreothane sulphonyl group.
Polyethylene glycol can also be attached to proteins using a number of
different
intervening linkers. For example, U.S. Patent No. 5,612,460, the entire
disclosure of which
is incorporated herein by reference, discloses urethane linkers for connecting
polyethylene
glycol to proteins. Protein-polyethylene glycol conjugates wherein the
polyethylene glycol is
attached to the protein by a linker can also be produced by reaction of
proteins with compounds
such as MPEG-succinimidylsuccinate, MPEG activated with 1,1'-
carbonyldiimidazole,
MPEG-2,4,5-trichloropenylcarbonate, MPEG-p-nitrophenolcarbonate, and various
MPEG-
succinate derivatives. A number additional polyethylene glycol derivatives and
reaction
chemistries for attaching polyethylene glycol to proteins are described in WO
98/32466, the
entire disclosure of which is incorporated herein by reference. Pegylated
protein products
produced using the reaction chemistries set out herein are included within the
scope of the
invention.
The number of polyethylene glycol moieties attached to each protein of the
invention
(i.e., the degree of substitution) may also vary. For example, the pegylated
proteins of the
invention may be linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15,
17, 20, or more
polyethylene glycol molecules. Similarly, the average degree of substitution
within ranges
such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12, 11-13, 12-14, 13-
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).
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
98
As mentioned the TR13 and TR14 polypeptides (proteins) of the invention may be
modified by either natural processes, such as posttranslational processing, or
by chemical
modiEcation 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
TR13 or TR14 polypeptide. TR13 or TR14 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 TR13 or TR14 polypeptides may result from posttranslation
natural
processes or may be made by synthetic methods. Modifications include
acetylation, acylation,
ADP-ribosylation, amidation, covalent attachment of flavin, covalent
attachment of a heme
moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent
attachment of a
lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-
linking, cyclization,
disulfide bond formation, demethylation, formation of covalent cross-links,
formation of
cysteine, formation of pyroglutamate, formylation, gamma-carboxylation,
glycosylation, GPI
anchor formation, hydroxylation, iodination, methylation, myristoylation,
oxidation,
pegylation, proteolytic processing, phosphorylation, prenylation,
racemization, selenoylation,
sulfation, transfer-RNA mediated addition of amino acids to proteins such as
arginylation, and
ubiquitination. (See, for instance, PROTEINS - STRUCTURE AND MOLECULAR
PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York
(1993); POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.
2o Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al.,
Meth Enzymol
182:626-646 ( 1990); Rattan et al., Ann NY Acad Sci 663:48-62 ( 1992)).
As mentioned the TR14 polypeptides (proteins) of the invention may be 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 TR14
polypeptide. TR14 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
TR14 polypeptides may result from posttranslation natural prpcesses or may be
made by
synthetic methods. Modifications include acetylation, acylation, ADP-
ribosylation, amidation,
3o 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 phosphotidylinosi~tol, cross-linking, cyclization, disulfide
bond formation,
demethylation, formation of covalent cross-links, formation of cysteine,
formation of
pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor
formation,
hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation,
proteolytic
processing, phosphorylation, prenylation, racemization, selenoylation,
sulfation, transfer-RNA
mediated addition of amino acids to proteins such as arginylation, and
ubiquitination. (See, for
instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E.
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
99
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 182:626-646 (1990);
Rattan et al., Ann
NY Acad Sci 663:48-62 ( 1992)).
The TR 13 polypeptides (proteins) of the invention can be recovered and
purified from
chemical synthesis and recombinant cell cultures by standard methods which
include, but are
not limited to, ammonium sulfate or ethanol precipitation, acid extraction,
anion or canon
exchange chromatography, phosphocellulose chromatography, hydrophobic
interaction
chromatography, affinity chromatography, hydroxylapatite chromatography and
lectin
chromatography. Most preferably, high performance liquid chromatography
("HPLC") is
employed for purification. Well known techniques for refolding protein may be
employed to
regenerate active conformation when the polypeptide is denatured during
isolation and/or
purification.
The TR14 polypeptides (proteins) of the invention can be recovered and
purified from
chemical synthesis and recombinant cell cultures by standard methods which
include, but are
not limited to, ammonium sulfate or ethanol precipitation, acid extraction,
anion or canon
exchange chromatography, phosphocellulose chromatography, hydrophobic
interaction
chromatography, affinity chromatography, hydroxylapatite chromatography and
lectin
chromatography. Most preferably, high performance liquid chromatography
("HPLC") is
employed for purification. Well known techniques for refolding protein may be
employed to
regenerate active conformation when the polypeptide is denatured during
isolation and/or
purification.
TR13 polynucleotides and polypeptides of the present invention, and agonsits
or
antogonists thereof, may be used in accordance with the present invention for
a variety of
applications, particularly those that make use of the chemical and biological
properties of TR13.
Among these are, for example, applications in treatment of tumors; resistance
to parasites,
bacteria and viruses; to regulate (i.e., induce) proliferation of T-cells,
endothelial cells and
hematopoietic cells; to treat restenosis, and graft vs. host disease; to
regulate anti-viral
responses; and to prevent certain autoimmune diseases after stimulation of
TR13 by an
3o agonist. Additional applications relate to diagnosis and to treatment of
disorders of cells,
tissues and organisms. These aspects of the invention are discussed further
below.
TR14 polynucleotides and polypeptides of the present invention, and agonsits
or
antogonists thereof, may be used in accordance with the present invention for
a variety of
applications, particularly those that make use of the chemical and biological
properties of TR 14.
Among these are, for example, applications in treatment of tumors; resistance
to parasites,
bacteria and viruses; to regulate (i.e., induce) proliferation of T-cells,
endothelial cells and
hematopoietic cells; to treat restenosis, and graft vs. host disease; to
regulate anti-viral
responses; and to prevent certain autoimmune diseases after stimulation of
TR14 by an
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
100
agonist. Additional applications relate to diagnosis and to treatment of
disorders of cells,
tissues and organisms. These aspects of the invention are discussed further
below.
Transgenics and "knock-outs"
The TR13 polypeptides (proteins) of the invention can also be expressed in
transgenic
animals. Animals of any species, including, but not limited to, mice, rats,
rabbits, hamsters,
guinea pigs, pigs, micro-pigs, goats, sheep, cows and non-human primates,
e.g., baboons,
monkeys, and chimpanzees may be used to generate transgenic animals. In a
specific
embodiment, techniques described herein or otherwise known in the art, are
used to express
1o polypeptides of the invention in humans, as part of a gene therapy
protocol.
The TR14 polypeptides (proteins) of the invention can also be expressed in
transgenic
animals. Animals of any species, including, but not limited to, mice, rats,
rabbits, hamsters,
guinea pigs, pigs, micro-pigs, goats, sheep, cows and non-human primates,
e.g., baboons,
monkeys, and chimpanzees may be used to generate transgenic animals. In a
specific
~5 embodiment, techniques described herein or otherwise known in the art, are
used to express
polypeptides of the invention in humans, as part of a gene therapy protocol.
Any technique known in the art may be used to introduce the transgene (i.e.,
nucleic
acids of the invention) into animals to produce the founder lines of
transgenic animals. Such
techniques include, but are not limited to, pronuclear microinjection
(Paterson et al., Appl.
20 Microbiol. Biotechnol. 40:691-698 (1994); Carver et al., Biotechnology (NY)
11:1263-1270
( 1993); Wright et al., Biotechnology (NY) 9:830-834 ( 1991 ); and Hoppe et al
., US Patent
Number 4,873,191 (1989)); retrovirus mediated gene transfer into germ lines
(Van der Putten
et al., Proc. Natl. Acad. Sci., USA 82:6148-6152 (1985)), blastocysts or
embryos; gene
targeting in embryonic stem cells (Thompson et al., Cell 56:313-321 (1989));
electroporation of
25 cells or embryos (Lo, Mol Cell. Biol. 3:1803-1814 (1983)); introduction of
the polynucleotides
of the invention using a gene gun (see, e.g., Ulmer et al., Science 259:1745
(1993);
introducing nucleic acid constructs into embryonic pleuripotent stem cells and
transferring the
stem cells back into the blastocyst; and sperm-mediated gene transfer
(Lavitrano et al., Cell
57:717-723 (1989); etc. For a review of such techniques, see Gordon,
"Transgenic Animals,"
30 Intl. Rev. Cytol. 115:171-229 (1989), which is incorporated by reference
herein in its entirety.
Further, the contents of each of the documents recited in this paragraph is
herein incorporated
by reference in its entirety. See also, U.S. Patent No. 5,464,764 (Capecchi,
et al., Positive-
Negative Selection Methods and Vectors); U.S. Patent No. 5,631,153 (Capecchi,
et al., Cells
and Non-Human Organisms Containing Predetermined Genomic Modifications and
Positive-
35 Negative Selection Methods and Vectors for Making Same); U.S. Patent No.
4,736,866
(Leder, et al., Transgenic Non-Human Animals); and U.S. Patent No. 4,873,191
(Wagner, et
al., Genetic Transformation of Zygotes); each of which is hereby incorporated
by reference in
its entirety.
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
101
Any technique known in the art may be used to produce transgenic clones
containing
polynucleotides of the invention, for example, nuclear transfer into
enucleated oocytes of nuclei
from cultured embryonic, fetal, or adult cells induced to quiescence (Campell
et al., Nature
380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)), each of which is
herein
incorporated by reference in its entirety).
The present invention provides for transgenic animals that carry the transgene
in all their
cells, as well as animals which carry the transgene in some, but not all their
cells, i.e., mosaic
animals or chimeric animals. The transgene may be integrated as a single
transgene or as
multiple copies such as in concatamers, e.g., head-to-head tandems or head-to-
tail tandems.
The transgene may also be selectively introduced into and activated in a
particular cell type by
following, for example, the teaching of Lasko et al. (Proc. Natl. Acad. Sci.
USA 89:6232-
6236 ( 1992)). The regulatory sequences required for such a cell-type specific
activation will
depend upon the particular cell type of interest, and will be apparent to
those of skill in the art.
When it is desired that the polynucleotide transgene be integrated into the
chromosomal site of
~5 the endogenous gene, gene targeting is preferred. Briefly, when such a
technique is to be
utilized, vectors containing some nucleotide sequences homologous to the
endogenous gene are
designed for the purpose of integrating, via homologous recombination with
chromosomal
sequences, into and disrupting the function of the nucleotide sequence of the
endogenous gene.
The transgene may also be selectively introduced into a particular cell type,
thus inactivating the
2o endogenous gene in only that cell type, by following, for example, the
teaching of Gu et al.
(Science 265:103-106 (1994)). The regulatory sequences required for such a
cell-type specific
inactivation will depend upon the particular cell type of interest, and will
be apparent to those of
skill in the art. The contents of each of the documents recited in this
paragraph is herein
incorporated by reference in its entirety.
25 Once transgenic animals have been generated, the expression of the
recombinant gene
may be assayed utilizing standard techniques. Initial screening may be
accomplished by
Southern blot analysis or PCR techniques to analyze animal tissues to verify
that integration of
the transgene has taken place. The level of mRNA expression of the transgene
in the tissues of
the transgenic animals may also be assessed using techniques which include,
but are not limited
3o to, Northern blot analysis of tissue samples obtained from the animal, in
situ hybridization
analysis, and reverse transcriptase-PCR (rt-PCR). Samples of transgenic gene-
expressing
tissue may also be evaluated immunocytochemically or immunohistochemically
using
antibodies specific for the transgene product.
Once the founder animals are produced, they may be bred, inbred, outbred, or
35 crossbred to produce colonies of the particular animal. Examples of such
breeding strategies
include, but are not limited to: outbreeding of founder animals with more than
one integration
site in order to establish separate lines; inbreeding of separate lines in
order to produce
compound transgenics that express the transgene at higher levels because of
the effects of
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
102
additive expression of each transgene; crossing of heterozygous transgenic
animals to produce
animals homozygous for a given integration site in order to both augment
expression and
eliminate the need for screening of animals by DNA analysis; crossing of
separate homozygous
lines to produce compound heterozygous or homozygous lines; and breeding to
place the
transgene on a distinct background that is appropriate for an experimental
model of interest.
Transgenic and "knock-out" animals of the invention have uses which include,
but are
not limited to, animal model systems useful in elaborating the biological
function of TR13
polypeptides, studying conditions and/or disorders associated with aberrant
TR13 expression,
and in screening for compounds effective in ameliorating such conditions
and/or disorders.
Transgenic and "knock-out" animals of the invention have uses which include,
but are
not limited to, animal model systems useful in elaborating the biological
function of TR14
polypeptides, studying conditions and/or disorders associated with aberrant
TR14 expression,
and in screening for compounds effective in ameliorating such conditions
and/or disorders.
In further embodiments of the invention, cells that are genetically engineered
to express
the proteins of the invention, or alternatively, that are genetically
engineered not to express the
proteins of the invention (e.g., knockouts) are administered to a patient in
vivo. Such cells
may be obtained from the patient (i.e., animal, including human) or an MHC
compatible donor
and can include, but are not limited to fibroblasts, bone marrow cells, blood
cells (e.g.,
lymphocytes), adipocytes, muscle cells, endothelial cells, etc. The cells are
genetically
2o engineered in vitro using recombinant DNA techniques to introduce the
coding sequence of
polypeptides of the invention into the cells, or alternatively, to disrupt the
coding sequence
and/or endogenous regulatory sequence associated with the polypeptides of the
invention, e.g.,
by transduction (using viral vectors, and preferably vectors that integrate
the transgene into the
cell genome) or transfection procedures, including, but not limited to, the
use of clones,
cosmids, YACs, naked DNA, electroporation, liposomes, etc. The coding sequence
of the
polypeptides of the invention can be placed under the control of a strong
constitutive or
inducible promoter or promoter/enhancer to achieve expression, and preferably
secretion, of the
polypeptides of the invention. The engineered cells which express and
preferably secrete the
polypeptides of the invention can be introduced into the patient systemically,
e.g., in the
3o circulation, or intraperitoneally. Alternatively, the cells can be
incorporated into a matrix and
implanted in the body, e.g., genetically engineered fibroblasts can be
implanted as part of a
skin graft; genetically engineered endothelial cells can be implanted as part
of a lymphatic or
vascular graft. (See, for example, Anderson et al. US Patent Number 5,399,349;
and Mulligan
& Wilson, US Patent Number 5,460,959, each of which is incorporated by
reference herein in
its entirety).
When the cells to be administered are non-autologous or non-MHC compatible
cells,
they can be administered using well known techniques which prevent the
development of a host
immune response against the introduced cells. For example, the cells may be
introduced in an
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
I03
encapsulated form which, while allowing for an exchange of components with the
immediate
extracellular environment, does not allow the introduced cells to be
recognized by the host
immune system.
TRI3 Polypeptides
The TR13 proteins (polypeptides) of the invention may be in monomers or
multimers
(i.e., dimers, trimers, tetramers, and higher multimers). Accordingly, the
present invention
relates to monomers and multimers of the TRI3 proteins (polypeptides) of the
invention, their
preparation, and compositions (preferably, pharmaceutical compositions)
containing them. In
to specific embodiments, the polypeptides of the invention are monomers,
dimers, trimers or
tetramers. In additional embodiments, the multimers of the invention are at
least dimers, at
least trimers, or at least tetramers.
Multimers encompassed by the invention may be homomers or heteromers. As used
herein, the term TR13 homomer, refers to a multimer containing only TR13
proteins of the
invention (including TR13 fragments, variants, and fusion proteins, as
described herein).
These homomers may contain TR13 proteins having identical or different
polypeptide
sequences. In a specific embodiment, a homomer of the invention is a multimer
containing
only TR13 proteins having an identical polypeptide sequence. In another
specific embodiment,
a homomer of the invention is a multimer containing TR13 proteins having
different
polypeptide sequences. In specific embodiments, the multimer of the invention
is a homodimer
(e.g., containing TR13 proteins having identical or different polypeptide
sequences) or a
homotrimer (e.g., containing TR13 proteins having identical or different
polypeptide
sequences). In additional embodiments, the homomeric multimer of the invention
is at least a
homodimer, at least a homotrimer, or at least a homotetramer.
As used herein, the term TR13 heteromer refers to a multimer containing
heterologous
proteins (i.e., proteins containing only polypeptide sequences that do not
correspond to a
polypeptide sequences encoded by the TR13 gene) in addition to the TR13
proteins of the
invention. In a specific embodiment, the multimer of the invention is a
heterodimer, a
heterotrimer, or a heterotetramer. In additional embodiments, the heteromeric
multimer of the
3o invention is at least a heterodimer, at least a heterotrimer, or at least a
heterotetramer.
Multimers of the invention may be the result of hydrophobic, hydrophilic,
ionic and/or
covalent associations and/or may be indirectly linked, by for example,
liposome formation.
Thus, in one embodiment, multimers of the invention, such as, for example,
homodimers or
homotrimers, are formed when proteins of the invention contact one another in
solution. In
another embodiment, heteromultimers of the invention, such as, for example,
heterotrimers or
heterotetramers, are formed when proteins of the invention contact antibodies
to the
polypeptides of the invention (including antibodies to the heterologous
polypeptide sequence in
a fusion protein of the invention) in solution. In other embodiments,
multimers of the
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
104
invention are formed by covalent associations with and/or between the TR13
proteins of the
invention. Such covalent associations may involve one or more amino acid
residues contained
in the polypeptide sequence of the protein (e.g., the polypeptide sequence
recited in SEQ ID
N0:2 or SEQ ID N0:40 or the polypeptide encoded by the cDNA deposited in ATCC
Deposit
No. PTA-349 or ATCC Deposit No. PTA-507. 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
to contained in the heterologous polypeptide sequence in a TR13 fusion
protein. In one example,
covalent associations are between the heterologous sequence contained in a
fusion protein of
the invention (see, e.g., US Patent Number 5,478,925). In a specific example,
the covalent
associations are between the heterologous sequence contained in a TR13-Fc
fusion protein of
the invention (as described herein). In another specific example, covalent
associations of
fusion proteins of the invention are between heterologous polypeptide
sequences from another
TNF family ligand/receptor member that is capable of forming covalently
associated multimers,
such as for example, oseteoprotegerin (see, e.g., International Publication
No. WO 98/49305,
the contents of which are herein incorporated by reference in its entirety).
In another
embodiment, two or more TR13 polypeptides of the invention are joined through
synthetic
linkers (e.g., peptide, carbohydrate or soluble polymer linkers). Examples
include those
peptide linkers described in U.S. Pat. No. 5,073,627 (hereby incorporated by
reference).
Proteins comprising multiple TR13 polypeptides separated by peptide linkers
may be produced
using conventional recombinant DNA technology.
Another method for preparing multimer TR13 polypeptides of the invention
involves
use of TR13 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
3o naturally occurring peptides and derivatives thereof that dimerize or
trimerize. Examples of
leucine zipper domains suitable for producing soluble multimeric TR13 proteins
are those
described in PCT application WO 94/10308, hereby incorporated by reference.
Recombinant
fusion proteins comprising a soluble TR13 polypeptide fused to a peptide that
dimerizes or
trimerizes in solution are expressed in suitable host cells, and the resulting
soluble multimeric
TR13 is recovered from the culture supernatant using techniques known in the
art.
Certain members of the TNF family of proteins are believed to exist in
trimeric form
(Beutler and Huffel, Science 264:667, 1994; Banner et al., Cel173:431, 1993).
Thus, trimeric
TR13 may offer the advantage of enhanced biological activity. Preferred
leucine zipper
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
105
moieties are those that preferentially form trimers. One example is a leucine
zipper derived
from lung surfactant protein D (SPD), as described in Hoppe et al. (FEBS
Letters 344:191,
(1994)) and in U.S. patent application Ser. No. 08/446,922, hereby
incorporated by reference.
Other peptides derived from naturally occurring trimeric proteins may be
employed in preparing
trimeric TR13.
In further preferred embodiments, TR 13 polynucleotides of the invention are
fused to a
polynucleotide encoding a "FLAG" polypeptide. Thus, a TR13-FLAG fusion protein
is encompassed
by the present invention. The FLAG antigenic polypeptide may be fused to a
TR13 polypeptide of the
invention at either or both the amino or the carboxy terminus. In preferred
embodiments, a TR13-
to FLAG fusion protein is expressed from a pFLAG-CMV-Sa or a pFLAG-CMV-1
expression vector
(available from Sigma, St. Louis, MO, USA). See, Andersson, S., et al., J.
Biol. Chem. 264:8222-
29 (1989); Thomsen, D. R., et al., Proc. Natl. Acad. Sci. USA, 81:659-63
(1984); and Kozak, M.,
Nature 308:241 (1984) (each of which is hereby incorporated by reference). In
further preferred
embodiments, a TR13-FLAG fusion protein is detectable by anti-FLAG monoclonal
antibodies (also
available from Sigma).
In another example, proteins of the invention are associated by interactions
between
Flag~ polypeptide sequence contained in Flag~-TR13 fusion proteins of the
invention. In a
further embodiment, associated proteins of the invention are associated by
interactions between
heterologous polypeptide sequence contained in Flag~-TR13 fusion proteins of
the invention
and anti-Flag~ antibody.
The multimers of the invention may be generated using chemical techniques
known in
the art. For example, proteins desired to be contained in the multimers of the
invention may be
chemically cross-linked using linker molecules and linker molecule length
optimization
techniques known in the art (see, e.g., US Patent Number 5,478,925, which is
herein
incorporated by reference in its entirety). Additionally, multimers of the
invention may be
generated using techniques known in the art to form one or more inter-molecule
cross-links
between the cysteine residues located within the polypeptide sequence of the
proteins desired to
be contained in the multimer (see, e.g., US Patent Number 5,478,925, which is
herein
incorporated by reference in its entirety). Further, proteins of the invention
may be routinely
modified by the addition of cysteine or biotin to the C terminus or N-terminus
of the
polypeptide sequence of the protein and techniques known in the art may be
applied to generate
multimers containing one or more of these modified proteins (see, e.g., US
Patent Number
5,478,925, which is herein incorporated by reference in its entirety).
Additionally, techniques
known in the art may be applied to generate liposomes containing the protein
components
desired to be contained in the multimer of the invention (see, e.g., US Patent
Number
5,478,925, which is herein incorporated by reference in its entirety).
Alternatively, multimers of the invention may be generated using genetic
engineering
techniques known in the art. In one embodiment, proteins contained in
multimers of the
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
106
invention are produced recombinantly using fusion protein technology described
herein or
otherwise known in the art (see, e.g., US Patent Number 5,478,925, which is
herein
incorporated by reference in its entirety). In a specific embodiment,
polynucleotides coding for
a homodimer of the invention are generated by ligating a polynucleotide
sequence encoding a
polypeptide of the invention to a sequence encoding a linker polypeptide and
then further to a
synthetic polynucleotide encoding the translated product of the polypeptide in
the reverse
orientation from the original C-terminus to the N-terminus (lacking the leader
sequence) (see,
e.g., US Patent Number 5,478,925, which is herein incorporated by reference in
its entirety).
In another embodiment, recombinant techniques described herein or otherwise
known in the art
are applied to generate recombinant polypeptides of the invention which
contain a
transmembrane domain and which can be incorporated by membrane reconstitution
techniques
into liposomes (see, e.g., US Patent Number 5,478,925, which is herein
incorporated by
reference in its entirety).
The polypeptides (proteins) of the present invention are preferably provided
in an
isolated form. By "isolated polypeptide" is intended a polypeptide removed
from its native
environment. Thus, a polypeptide produced and/or contained within a
recombinant host cell is
considered isolated for purposes of the present invention. Also intended as an
"isolated
polypeptide" are polypeptides that have been purified, partially or
substantially, from a
recombinant host cell. For example, a recombinantly produced version of the
TR13
2o polypeptide can be substantially purified by the one-step method described
in Smith and
Johnson, Gene 67:31-40 (1988).
Accordingly, in one embodiment, the invention provides an isolated TR13
polypeptide
having the amino acid sequence encoded by the cDNA deposited in ATCC Deposit
No. PTA
349 or ATCC Deposit No. PTA-507, or the amino acid sequence in SEQ ID N0:2 or
SEQ ID
N0:40, or a peptide or polypeptide comprising a portion of the above
polypeptides.
Polypeptide fragments of the present invention include polypeptides comprising
or
alternatively, consisting of, an amino acid sequence contained in SEQ ID N0:2,
encoded by the
cDNA contained in the clone deposited as ATCC Deposit No. PTA-349, or encoded
by a
nucleic acid which hybridizes (e.g., under stringent hybridization conditions)
to the nucleotide
3o sequence contained in the deposited clone, or shown in Figures lA-C (SEQ ID
NO:1) or the
complementary strand thereto, or polynucleotide fragments thereof (e.g., as
disclosed herein).
Protein fragments may be "free-standing," or comprised within a larger
polypeptide of which
the fragment forms a part or region, most preferably as a single continuous
region.
Representative examples of polypeptide fragments of the invention, include,
for example,
fragments that comprise, or alternatively consist of, from about amino acid
residues: 1 to 50,
51 to 100, 101 to 150, 151 to 200, 201 to 250, 251 to 300, 301 to 350, 351 to
400, 401 to
450, 451 to 500, 501 to 550, 551 to 600, 601 to 650, 651 to 700, and/or 701 to
750 of SEQ
ID N0:2. Moreover, polypeptide fragments can be at least 10, 20, 30, 40, 50,
60, 70, 80, 90,
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
107
100, 110, 120, 130, 140, 150, 175 or 200 amino acids in length. In this
context "about"
includes the particularly recited ranges, larger or smaller by several (5, 4,
3, 2, or 1) amino
acids, at either extreme or at both extremes. Polynucleotides encoding these
polypeptide
fragments are also encompassed by the invention.
Polypeptide fragments of the present invention include polypeptides comprising
or
alternatively, consisting of, an amino acid sequence contained in SEQ ID
N0:40, encoded by
the cDNA contained in the clone deposited as ATCC Deposit No. PTA-507, or
encoded by a
nucleic acid which hybridizes (e.g., under stringent hybridization conditions)
to the nucleotide
sequence contained in the deposited clone, or shown in Figures 7A-D (SEQ ID
N0:40) or the
l0 complementary strand thereto or polynucleotide fragments thereof (e.g., as
disclosed herein).
Protein fragments may be "free-standing," or comprised within a larger
polypeptide of which
the fragment forms a part or region, most preferably as a single continuous
region.
Representative examples of polypeptide fragments of the invention, include,
for example,
fragments that comprise, or alternatively consist of, from about amino acid
residues: 1 to 50,
~5 51 to 100, 101 to 150, 151 to 200, 201 to 250, 251 to 300, 301 to 350, 351
to 400, 401 to
450, 451 to 500, 501 to 550, 551 to 600, 601 to 650, 651 to 700, 701 to 750,
751 to 800, 801
to 850, 851 to 900, 901 to 950, and/or 951 to 1001 of SEQ ID N0:2. Moreover,
polypeptide
fragments can be at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120,
130, 140, 150,
175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850,
900, 950, or
20 1001 amino acids in length. In this context "about" includes the
particularly recited ranges,
larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme
or at both extremes.
Polynucleotides encoding these polypeptide fragments are also encompassed by
the invention.
Polypeptide fragments of the present invention include polypeptides comprising
or
alternatively, consisting of from about amino acid residues: 105 to about 170,
from 251 to
25 about 265, from 331 to about 410, from 580 to about 610, from 139 to about
142, from 140 to
about 143, from 153 to about 156, from 293 to about 296, from 325 to about
328, from 421 to
about 424, from 466 to about 469, from 696 to about 699, from 728 to about
731, from 312 to
about 315, from 454 to about 461, from 458 to about 461, from 50 to about 53,
from 66 to
about 69, from 80 to about 83, from 276 to about 279, from 311 to about 314,
from 438 to
30 about 441, from 559 to about 562, from 564 to about 567, from 698 to about
701, from 725 to
about 728, from 80 to about 83, from 89 to about 92, from 180 to about 183,
from 198 to
about 201, from 214 to about 217, from 272 to about 275, from 306 to about
309, from 510 to
about 513, from 529 to about 532, from 584 to about 867, from 609 to about
612, from 642 to
about 645, from 698 to about 701, from 69 to about 74, from 149 to about 154,
from 154 to
35 about 159, from 163 to about 168, from 212 to about 217, from 248 to about
253, from 365 to
about 370, from 383 to about 388, from 393 to about 398, from 588 to about
593, from 623 to
about 628, from 661 to about 666, from 665 to about 670, and/or 456 to about
459 of SEQ ID
N0:2. In this context "about" includes the particularly recited ranges, larger
or smaller by
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
108
several (5, 4, 3, 2, or 1 ) amino acids, at either extreme or at both
extremes. Polynucleotides
encoding these polypeptide fragments are also encompassed by the invention.
Polypeptide fragments of the present invention include polypeptides comprising
or
alternatively, consisting of from about amino acid residues: 42 to about 906,
from 42 to about
1001, from 906 to 'about 931, from 932 to about 1001, from 271 to about 421,
from 271 to
about 286, from 290 to about 300, from 301 to about 320, from 329 to about
361, from 404 to
about 421, from 585 to about 595, from 661 to about 674, from 710 to about
744, from 980 to
about 991, from 45 to about 60, from 121 to about 135, from 145 to about 160,
from I to
about 262, from 264 to about 423, from 437 to about 789, from 791 to about
1001, from 310
to about 363, from 477 to about 519, from 769 to about 887, from 153 to about
156, from 11
to about 13, from about 18 to about 20, from 107 to about 109, from about 156
to about 158,
from about 224 to about 226, from about 301 to about 303, from about 317 to
about 319, from
about 331 to about 333, from about 527 to about 529, from about 562 to about
564, from
about 689 to about 691, from about 810 to about 812, from about 815 to about
817, from
about 949 to about 951, from about 976 to about 978, from 42 to about 45, from
about 59 to
about 62, from about 81 to about 84, from about 146 to about 149, from about
282 to about
285, from about 331 to about 334, from about 340 to about 343, from about 431
to about 434,
from about 449 to about 452, from about 465 to about 468, from about 523 to
about 526, from
about 557 to about 560, from about 761 to about 764, from about 780 to about
783, from
about 780 to about 783, from about 835 to about 838, from about 860 to about
863, from
about 893 to about 896, from about 949 to about 952, from from about 77 to
about 82, from
about 88 to about 93, from about 152 to about 157, from about 268 to about
273, from about
288 to about 293, from about 320 to about 325, from about 400 to about 405,
from about 414
to about 419, from about 463 to about 468, from about 599 to about 604, from
about 616 to
about 621, from about 634 to about 639, from about 644 to about 649, from
about 839 to
about 844, from about 874 to about 879, from about 912 to about 917, from
about 916 to
about 921, from from about 50 to about 56, from from about 109 to about 116,
from from
about 153 to about 156, from 390 to about 393, from 391 to about 394, from
about 404 to
about 407, from about 544 to about 547, from about 576 to about 579, from
about 672 to
3o about 675, from about 717 to about 720, from about 947 to about 950, from
and about 979 to
about 982 of SEQ ID N0:40. 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.
In additional embodiments, the polypeptide fragments of the invention
comprise, or
alternatively consist of, one or more domains of the TR13 polypeptide
disclosed in Figures 1A
C. Preferred polypeptide fragments of the present invention include a member
selected from the
group: (a) a polypeptide comprising or alternatively, consisting of, any
combination of one,
two, three, or all four of the TR13 cysteine rich domains disclosed in Figures
lA-C (predicted
to constitute amino acid residues from about 105 to about 170, about 251 to
about 265, about
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
109
331 to about 410, and about 580 to about 610 of SEQ ID N0:2); (b) a
polypeptide comprising,
or alternatively, consisting of, one, two, three, four or more, epitope
bearing portions of the
TR13 receptor protein disclosed in Figures lA-C (for example, those epitope
bearing portions
predicted to constitute amino acid residues from about 1 to about 170, or
about 210 to about
318, or about 343 to about 480, or about 548 to about 592, or about 632 to
about 742 of SEQ
ID N0:2); (c) any combination of polypeptides (a)-(c). Polynucleotides
encoding these
polypeptides are also encompassed by the invention. In this context "about"
includes the
particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1 )
amino acids, at either
extreme or at both extremes. Polynucleotides encoding these polypeptide
fragments are also
to encompassed by the invention.
In additional embodiments, the polypeptide fragments of the invention
comprise, or
alternatively consist of, one or more domains of the TR13 polypeptide
disclosed in Figure 7A-
D. Preferred polypeptide fragments of the present invention include a member
selected from the
group: (a) a polypeptide comprising, or alternatively consisting of, amino
acids 1 to about 41
of SEQ ID N0:40; (b) a polypeptide comprising, or alternatively consisting of,
amino acids 42
to about 906 of SEQ ID N0:40; (c) a polypeptide comprising, or alternatively
consisting of,
amino acids 907 to about 931 of SEQ ID N0:40; (d) a polypeptide comprising, or
alternatively
consisting of, amino acids 932 to about 1001 of SEQ ID N0:40; (e) a
polypeptide comprising
or alternatively, consisting of, any combination of one, two, three, four or
more of the TR13
2o cysteine rich domains disclosed in Figures 7A-D (predicted to constitute
amino acid residues
from about 271 to about 421, 271 to about 286, about 290 to about 300, about
301 to about
320, about 329 to about 361, about 404 to about 421, about 585 to about 595 of
SEQ ID
N0:40); (f) a polypeptide comprising, or alternatively, consisting of, one,
two, three, four or
more, epitope bearing portions of the TR13 receptor protein disclosed in
Figures 7A-D (for
example, these epitope bearing portions predicted to constitute amino acid
residues from about
1 to about 262, or about 264 to about 423, or about 437 to about 789, or about
791 to about
1001, of SEQ ID N0:40); and (g) any combination of polypeptides (a)-(f). 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. Polynucleotides encoding
these
3o polypeptides are also encompassed by the invention.
As discussed above, it is believed that the extracellular cysteine rich motifs
of TR13 are
important for interactions between TR13 and its ligands. Accordingly, in
preferred
embodiments, polypeptide fragments of the invention comprise, or alternatively
consist of
amino acid residues from about 105 to about 170, about 251 to about 265, about
331 to about
410 and/or about 580 to about 610 of the amino acid sequence disclosed in
Figures lA-C (SEQ
ID N0:2). In a specific embodiment the polypeptides of the invention comprise,
or alternatively
consist of any combination of one, two, three or all four extracellular
cysteine rich motifs
disclosed in Figures lA-C. In this context "about" includes the particularly
recited ranges,
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
110
larger or smaller by several (S, 4, 3, 2, or 1 ) amino acids, at either
extreme or at both extremes.
Polynucleotides encoding these polypeptides are also encompassed by the
invention.
As discussed above, it is believed that the extracellular cysteine rich motifs
of TR13 are
important for interactions between TR13 and its ligands. Accordingly, in
preferred
embodiments, polypeptide fragments of the invention comprise, or alternatively
consist of
amino acid residues from about 271 to about 421, or 271 to about 286, or about
290 to about
300, or about 301 to about 320, or about 329 to about 361, or about 404 to
about 421, or about
585 to about 595 of the amino acid sequence disclosed in Figures 7A-D (SEQ ID
N0:40). In a
specific embodiment the polypeptides of the invention comprise, or
alternatively consist of any
l0 combination of one, two, three, four or more of the extracellular cysteine
rich motifs disclosed
in Figures 7A-D. 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.
Polynucleotides encoding these polypeptides are also encompassed by the
invention.
Among the especially preferred fragments of the invention are fragments
characterized
by structural or functional attributes of TR13 (SEQ ID N0:2 or SEQ ID N0:40).
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
2o 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) TR13 (SEQ ID N0:2 or SEQ ID N0:40). Certain preferred regions are
those set out in
Figure 3 (Table I) and Figure 9 (Table III) and include, but are not limited
to, regions of the
aforementioned types identified by analysis of the amino acid sequence
depicted in Figures lA-
C (SEQ ID N0:2) or Figures 7A-D (SEQ ID N0:40), respectively. 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 and Hopp-Woods predicted hydrophobic regions; Eisenberg alpha and
beta
3o amphipathic regions; Emini surface-forming regions; and Jameson-Wolf high
antigenic index
regions, as predicted using the default parameters of these computer programs.
Polynucleotides encoding these polypeptides are also encompassed by the
invention.
As mentioned above, even if deletion of one or more amino acids from the N-
terminus
of a protein results in modification of loss of one or more biological
functions of the protein,
other functional activities (e.g., biological activities, ability to
multimerize, ability to bind TR13
ligand) may still be retained. For example, the ability of shortened TR13
muteins to induce
and/or bind to antibodies which recognize the complete or mature forms of the
polypeptides
generally will be retained when less than the majority of the residues of the
complete or mature
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
111
polypeptide are removed from the N-terminus. Whether a particular polypeptide
lacking N-
terminal residues of a complete polypeptide retains such immunologic
activities can readily be
determined by routine methods described herein and otherwise known in the art.
It is not
unlikely that an TR13 mutein with a large number of deleted N-terminal amino
acid residues
may retain some biological or immunogenic activities. In fact, peptides
composed of as few as
six TR13 amino acid residues may often evoke an immune response.
Accordingly, the present invention further provides polypeptides having one or
more
residues deleted from the amino terminus of the TR13 amino acid sequence shown
in Figures
lA-C, up to the aspartic acid residue at position number 745 and
polynucleotides encoding
such polypeptides. In particular, the present invention provides polypeptides
comprising, or
alternatively consisting of, the amino acid sequence of residues n'-750 of
Figures lA-C, where
n' is an integer from 2 to 745 corresponding to the position of the amino acid
residue in Figures
lA-C (which is identical to the sequence shown as SEQ ID N0:2). In a specific
embodiment,
the present invention provides polypeptides comprising, or alternatively
consisting of, the
amino acid sequence of residues n'-750 of Figures lA-C, where n' is an integer
from 2 to 610
corresponding to the position of the amino acid residue in Figures lA-C.
Polynucleotides
encoding these polypeptides are also encompassed.
In one embodiment, N-terminal deletions of the TR13 polypeptides of the
invention can
be described by the general formula n2-750, where n2 is a number from 2 to
745,
corresponding to the position of amino acid identified in Figures lA-C (SEQ ID
N0:2). N-
terminal deletions of the TR13 polypeptide of the invention shown as SEQ ID
N0:2 include
polypeptides comprising, or alternatively consisting of, the amino acid
sequence of residues: D-
2 to R-750; Q-3 to R-750; S-4 to R-750; T-5 to R-750; Q-6 to R-750; A-7 to R-
750; C-8 to 8-
750; A-9 to R-750; G-10 to R-750; E-11 to R-750; K-12 to R-750; H-13 to R-750;
C-14 to R-
750; H-15 to R-750; N-16 to R-750; R-17 to R-750; G-18 to R-750; G-19 to R-
750; L-20 to
R-750; H-21 to R-750; F-22 to R-750; R-23 to R-750; M-24 to R-750; L-25 to R-
750; P-26 to
R-750; L-27 to R-750; Q-28 to R-750; T-29 to R-750; W-30 to R-750; H-31 to R-
750; V-32 to
R-750; C-33 to R-750; R-34 to R-750; Q-35 to R-750; A-36 to R-750; G-37 to R-
750; L-38 to
R-750; L-39 to R-750; F-40 to R-750; L-41 to R-750; Q-42 to R-750; T-43 to R-
750; L-44 to
3o R-750; P-45 to R-750; S-46 to R-750; N-47 to R-750; S-48 to R-750; Y-49 to
R-750; S-50 to
R-750; N-51 to R-750; K-52 to R-750; G-53 to R-750; E-54 to R-750; T-55 to R-
750; S-56 to
R-750; C-57 to R-750; H-58 to R-750; Q-59 to R-750; C-60 to R-750; D-61 to R-
750; P-62 to
R-750; D-63 to R-750; K-64 to R-750; Y-65 to R-750; S-66 to R-750; E-67 to R-
750; K-68 to
R-750; G-69 to R-750; S-70 to R-750; S-71 to R-750; S-72 to R-750; C-73 to R-
750; N-74 to
R-750; V-75 to R-750; R-76 to R-750; P-77 to R-750; A-78 to R-750; C-79 to R-
750; T-80 to
R-750; D-81 to R-750; K-82 to R-750; D-83 to R-750; Y-84 to R-750; F-85 to R-
750; Y-86 to
R-750; T-87 to R-750; H-88 to R-750; T-89 to R-750; A-90 to R-750; C-91 to R-
750; D-92 to
R-750; A-93 to R-750; N-94 to R-750; G-95 to R-750; E-96 to R-750; T-97 to R-
750; Q-98 to
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
112
R-750; L-99 to R-750; M-100 to R-750; Y-101 to R-750; K-102 to R-750; W-103 to
R-750;
A-104 to R-750; K-105 to R-750; P-106 to R-750; K-107 to R-750; I-108 to R-
750; C-109 to
R-750; S-110 to R-750; E-111 to R-750; D-112 to R-750; L-113 to R-750; E-114
to R-750; 6-
115 to R-750; A-116 to R-750; V-117 to R-750; K-118 to R-750; L-119 to R-750;
P-120 to R-
750; A-121 to R-750; S-122 to R-750; G-123 to R-750; V-124 to R-750; K-125 to
R-750; T-
126 to R-750; H-127 to R-750; C-128 to R-750; P-129 to R-750; P-130 to R-750;
C-131 to R-
750; N-132 to R-750; P-133 to R-750; G-134 to R-750; F-135 to R-750; F-136 to
R-750; K-
137 to R-750; T-138 to R-750; N-139 to R-750; N-140 to R-750; S-141 to R-750;
T-142 to 8-
750; C-143 to R-750; Q-144 to R-750; P-145 to R-750; C-146 to R-750; P-147 to
R-750; Y-
148 to R-750; G-149 to R-750; S-150 to R-750; Y-151 to R-750; S-152 to R-750;
N-153 to R-
750; G-154 to R-750; S-155 to R-750; D-156 to R-750; C-157 to R-750; T-158 to
R-750; 8-
159 to R-750; C-160 to R-750; P-161 to R-750; A-162 to R-750; G-163 to R-750;
T-164 to R-
750; E-165 to R-750; P-166 to R-750; A-167 to R-750; V-168 to R-750; G-169 to
R-750; F-
170 to R-750; E-171 to R-750; Y-172 to R-750; K-173 to R-750; W-174 to R-750;
W-175 to
R-750; N-176 to R-750; T-177 to R-750; L-178 to R-750; P-179 to R-750; T-180
to R-750; N-
181 to R-750; M-182 to R-750; E-183 to R-750; T-184 to R-750; T-185 to R-750;
V-186 to R-
750; L-187 to R-750; S-188 to R-750; G-189 to R-750; I-190 to R-750; N-191 to
R-750; F-
192 to R-750; E-193 to R-750; Y-194 to R-750; K-195 to R-750; G-196 to R-750;
M-197 to
R-750; T-198 to R-750; G-199 to R-750; W-200 to R-750; E-201 to R-750; V-202
to R-750;
2o A-203 to R-750; G-204 to R-750; D-205 to R-750; H-206 to R-750; I-207 to R-
750; Y-208 to
R-750; T-209 to R-750; A-210 to R-750; A-211 to R-750; G-212 to R-750; A-213
to R-750;
S-214 to R-750; D-215 to R-750; N-216 to R-750; D-217 to R-750; F-218 to R-
750; M-219 to
R-750; I-220 to R-750; L-221 to R-750; T-222 to R-750; L-223 to R-750; V-224
to R-750; V-
225 to R-750; P-226 to R-750; G-227 to R-750; F-228 to R-750; R-229 to R-750;
P-230 to R-
750; P-231 to R-750; Q-232 to R-750; S-233 to R-750; V-234 to R-750; M-235 to
R-750; A-
236 to R-750; D-237 to R-750; T-238 to R-750; E-239 to R-750; N-240 to R-750;
K-241 to R-
750; E-242 to R-750; V-243 to R-750; A-244 to R-750; R-245 to R-750; I-246 to
R-750; T-
247 to R-750; F-248 to R-750; V-249 to R-750; F-250 to R-750; E-251 to R-750;
T-252 to 8-
750; L-253 to R-750; C-254 to R-750; S-255 to R-750; V-256 to R-750; N-257 to
R-750; C-
258 to R-750; E-259 to R-750; L-260 to R-750; Y-261 to R-750; F-262 to R-750;
M-263 to R-
750; V-264 to R-750; G-265 to R-750; V-266 to R-750; N-267 to R-750; S-268 to
R-750; R-
269 to R-750; T-270 to R-750; N-271 to R-750; T-272 to R-750; P-273 to R-750;
V-274 to 8-
750; E-275 to R-750; T-276 to R-750; W-277 to R-750; K-278 to R-750; G-279 to
R-750; S-
280 to R-750; K-281 to R-750; G-282 to R-750; K-283 to R-750; Q-284 to R-750;
S-285 to
R-750; Y-286 to R-750; T-287 to R-750; Y-288 to R-750; I-289 to R-750; I-290
to R-750; E-
291 to R-750; E-292 to R-750; N-293 to R-750; T-294 to R-750; T-295 to R-750;
T-296 to 8-
750; S-297 to R-750; F-298 to R-750; T-299 to R-750; W-300 to R-750; A-301 to
R-750; F-
302 to R-750; Q-303 to R-750; R-304 to R-750; T-305 to R-750; T-306 to R-750;
F-307 to R-
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
113
750; H-308 to R-750; E-309 to R-750; A-310 to R-750; S-311 to R-750; R-312 to
R-750; K-
313 to R-750; Y-314 to R-750; T-315 to R-750; N-316 to R-750; D-317 to R-750;
V-318 to 8-
750; A-319 to R-750; K-320 to R-750; I-321 to R-750; Y-322 to R-750; S-323 to
R-750; I-324
to R-750; N-325 to R-750; V-326 to R-750; T-327 to R-750; N-328 to R-750; V-
329 to R-750;
M-330 to R-750; N-331 to R-750; G-332 to R-750; V-333 to R-750; A-334 to R-
750; S-335 to
R-750; Y-336 to R-750; C-337 to R-750; R-338 to R-750; P-339 to R-750; C-340
to R-750;
A-341 to R-750; L-342 to R-750; E-343 to R-750; A-344 to R-750; S-345 to R-
750; D-346 to
R-750; V-347 to R-750; G-348 to R-750; S-349 to R-750; S-350 to R-750; C-351
to R-750; T-
352 to R-750; S-353 to R-750; C-354 to R-750; P-355 to R-750; A-356 to R-750;
G-357 to R-
l0 750; Y-358 to R-750; Y-359 to R-750; I-360 to R-750; D-361 to R-750; R-362
to R-750; D-
363 to R-750; S-364 to R-750; G-365 to R-750; T-366 to R-750; C-367 to R-750;
H-368 to R-
750; S-369 to R-750; C-370 to R-750; P-371 to R-750; P-372 to R-750; N-373 to
R-750; T-
374 to R-750; I-375 to R-750; L-376 to R-750; K-377 to R-750; A-378 to R-750;
H-379 to 8-
750; Q-380 to R-750; P-381 to R-750; Y-382 to R-750; G-383 to R-750; V-384 to
R-750; Q-
~5 385 to R-750; A-386 to R-750; C-387 to R-750; V-388 to R-750; P-389 to R-
750; C-390 to R-
750; G-391 to R-750; P-392 to R-750; G-393 to R-750; T-394 to R-750; K-395 to
R-750; N-
396 to R-750; N-397 to R-750; K-398 to R-750; I-399 to R-750; H-400 to R-750;
S-401 to R-
750; L-402 to R-750; C-403 to R-750; Y-404 to R-750; N-405 to R-750; D-406 to
R-750; C-
407 to R-750; T-408 to R-750; F-409 to R-750; S-410 to R-750; R-411 to R-750;
N-412 to R-
20 750; T-413 to R-750; P-414 to R-750; T-415 to R-750; R-416 to R-750; T-417
to R-750; F-
418 to R-750; N-419 to R-750; Y-420 to R-750; N-421 to R-750; F-422 to R-750;
S-423 to 8-
750; A-424 to R-750; L-425 to R-750; A-426 to R-750; N-427 to R-750; T-428 to
R-750; V-
429 to R-750; T-430 to R-750; L-431 to R-750; A-432 to R-750; G-433 to R-750;
G-434 to 8-
750; P-435 to R-750; S-436 to R-750; F-437 to R-750; T-438 to R-750; S-439 to
R-750; K-
25 440 to R-750; G-441 to R-750; L-442 to R-750; K-443 to R-750; Y-444 to R-
750; F-445 to 8-
750; H-446 to R-750; H-447 to R-750; F-448 to R-750; T-449 to R-750; L-450 to
R-750; S-
451 to R-750; L-452 to R-750; C-453 to R-750; G-454 to R-750; N-455 to R-750;
Q-456 to 8-
750; G-457 to R-750; R-458 to R-750; K-459 to R-750; M-460 to R-750; S-461 to
R-750; V-
462 to R-750; C-463 to R-750; T-464 to R-750; D-465 to R-750; N-466 to R-750;
V-467 to R-
30 750; T-468 to R-750; D-469 to R-750; L-470 to R-750; R-471 to R-750; I-472
to R-750; P-
473 to R-750; E-474 to R-750; G-475 to R-750; E-476 to R-750; S-477 to R-750;
G-478 to 8-
750; F-479 to R-750; S-480 to R-750; K-481 to R-750; S-482 to R-750; I-483 to
R-750; T-
484 to R-750; A-485 to R-750; Y-486 to R-750; V-487 to R-750; C-488 to R-750;
Q-489 to 8-
750; A-490 to R-750; V-491 to R-750; I-492 to R-750; I-493 to R-750; P-494 to
R-750; P-495
35 to R-750; E-496 to R-750; V-497 to R-750; T-498 to R-750; G-499 to R-750; Y-
500 to R-750;
K-501 to R-750; A-502 to R-750; G-503 to R-750; V-504 to R-750; S-505 to R-
750; S-506 to
R-750; Q-507 to R-750; P-508 to R-750; V-509 to R-750; S-510 to R-750; L-511
to R-750; A-
512 to R-750; D-513 to R-750; R-514 to R-750; L-515 to R-750; I-516 to R-750;
G-517 to R-
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
114
750; V-518 to R-750; T-519 to R-750; T-520 to R-750; D-521 to R-750; M-522 to
R-750; T-
523 to R-750; L-524 to R-750; D-525 to R-750; G-526 to R-750; I-527 to R-750;
T-528 to 8-
750; S-529 to R-750; P-530 to R-750; A-531 to R-750; E-532 to R-750; L-533 to
R-750; F-
534 to R-750; H-535 to R-750; L-536 to R-750; E-537 to R-750; S-538 to R-750;
L-539 to R-
750; G-540 to R-750; I-541 to R-750; P-542 to R-750; D-543 to R-750; V-544 to
R-750; I-545
to R-750; F-546 to R-750; F-547 to R-750; Y-548 to R-750; R-549 to R-750; S-
550 to R-750;
N-551 to R-750; D-552 to R-750; V-553 to R-750; T-554 to R-750; Q-555 to R-
750; S-556 to
R-750; C-557 to R-750; S-558 to R-750; S-559 to R-750; G-560 to R-750; R-561
to R-750; S-
562 to R-750; T-563 to R-750; T-564 to R-750; I-565 to R-750; R-566 to R-750;
V-567 to R-
l 0 750; R-568 to R-750; C-569 to R-750; S-570 to R-750; P-571 to R-750; Q-572
to R-750; K-
573 to R-750; T-574 to R-750; V-575 to R-750; P-576 to R-750; G-577 to R-750;
S-578 to 8-
750; L-579 to R-750; L-580 to R-750; L-581 to R-750; P-582 to R-750; G-583 to
R-750; T-
584 to R-750; C-585 to R-750; S-586 to R-750; D-587 to R-750; G-588 to R-750;
T-589 to 8-
750; C-590 to R-750; D-591 to R-750; G-592 to R-750; C-593 to R-750; N-594 to
R-750; F-
595 to R-750; H-596 to R-750; F-597 to R-750; L-598 to R-750; W-599 to R-750;
E-600 to R-
750; S-601 to R-750; A-602 to R-750; A-603 to R-750; A-604 to R-750; C-605 to
R-750; P-
606 to R-750; L-607 to R-750; C-608 to R-750; S-609 to R-750; V-610 to R-750;
A-611 to 8-
750; D-612 to R-750; Y-613 to R-750; H-614 to R-750; A-615 to R-750; I-616 to
R-750; V-
617 to R-750; S-618 to R-750; S-619 to R-750; C-620 to R-750; V-621 to R-750;
A-622 to R-
750; G-623 to R-750; I-624 to R-750; Q-625 to R-750; K-626 to R-750; T-627 to
R-750; T-
628 to R-750; Y-629 to R-750; V-630 to R-750; W-631 to R-750; R-632 to R-750;
E-633 to
R-750; P-634 to R-750; K-635 to R-750; L-636 to R-750; C-637 to R-750; S-638
to R-750;
G-639 to R-750; G-640 to R-750; I-641 to R-750; S-642 to R-750; L-643 to R-
750; P-644 to
R-750; E-645 to R-750; Q-64b to R-750; R-647 to R-750; V-648 to R-750; T-649
to R-750; I-
650 to R-750; C-651 to R-750; K-652 to R-750; T-653 to R-750; I-654 to R-750;
D-655 to 8-
750; F-656 to R-750; W-657 to R-750; L-658 to R-750; K-659 to R-750; V-660 to
R-750; G-
661 to R-750; I-662 to R-750; S-663 to R-750; A-664 to R-750; G-665 to R-750;
T-666 to 8-
750; C-667 to R-750; T-668 to R-750; A-669 to R-750; I-670 to R-750; L-671 to
R-750; L-
672 to R-750; T-673 to R-750; V-674 to R-750; L-675 to R-750; T-676 to R-750;
C-677 to R-
750; Y-678 to R-750; F-679 to R-750; W-680 to R-750; K-681 to R-750; K-682 to
R-750; N-
683 to R-750; Q-684 to R-750; K-685 to R-750; L-686 to R-750; E-687 to R-750;
Y-688 to 8-
750; K-689 to R-750; Y-690 to R-750; S-691 to R-750; K-692 to R-750; L-693 to
R-750; V-
694 to R-750; M-695 to R-750; N-696 to R-750; A-697 to R-750; T-698 to R-750;
L-699 to 8-
750; K-700 to R-750; D-701 to R-750; C-702 to R-750; D-703 to R-750; L-704 to
R-750; P-
705 to R-750; A-706 to R-750; A-707 to R-750; D-708 to R-750; S-709 to R-750;
C-710 to R-
750; A-711 to R-750; I-712 to R-750; M-713 to R-750; E-714 to R-750; G-715 to
R-750; E-
716 to R-750; D-717 to R-750; V-718 to R-750; E-719 to R-750; D-720 to R-750;
D-721 to 8-
750; L-722 to R-750; I-723 to R-750; F-724 to R-750; T-725 to R-750; S-726 to
R-750; K-
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
115
727 to R-750; N-728 to R-750; H-729 to R-750; S-730 to R-750; L-731 to R-750;
G-732 to 8-
750; R-733 to R-750; S-734 to R-750; N-735 to R-750; H-736 to R-750; L-737 to
R-750; P-
738 to R-750; P-739 to R-750; R-740 to R-750; G-741 to R-750; L-742 to R-750;
L-743 to 8-
750; M-744 to R-750; D-745 to R-750; of SEQ ID N0:2. Polynucleotides encoding
these
polypeptides are also encompassed by the invention.
Accordingly, the present invention further provides polypeptides having one or
more
residues deleted from the amino terminus of the TR13 amino acid sequence shown
in Figures
7A-D, up to the aspartic acid residue at position number 996 and
polynucleotides encoding
such polypeptides. In particular, the present invention provides polypeptides
comprising, or
1o alternatively consisting of, the amino acid sequence of residues n'-1001 of
Figures 7A-D,
. where n' is an integer from 2 to 996 corresponding to the position of the
amino acid residue in
Figures 7A-C (which is identical to the sequence shown as SEQ ID N0:40). In a
specific
embodiment, the present invention provides polypeptides comprising, or
alternatively
consisting of, the amino acid sequence of residues n'-906 of Figures 7A-D
where n' is an
integer from 42 to 595 corresponding to the position of the amino acid residue
in Figures 7A
D. Polynucleotides encoding these polypeptides are also encompassed by the
invention.
In another embodiment, N-terminal deletions of the TR13 polypeptide can be
described
by the general formula n2-1001, where n2 is a number from 2 to 996,
corresponding to the
position of amino acid identified in Figures 7A-D (SEQ ID N0:40). N-terminal
deletions of the
2o TR13 polypeptide of the invention shown as SEQ ID N0:40 include
polypeptides comprising,
or alternatively consisting of, the amino acid sequence of residues: A-2 to R-
1001; E-3 to 8-
1001; P-4 to R-1001; G-5 to R-1001; H-6 toR-1001; S-7 to R-1001; H-8 to R-
1001; H-9 to R-
1001; L-10 to R-1001; S-11 to R-1001; A-12 to R-1001; R-13 to R-1001;V-14 to R-
1001; 8-
15 to R-1001; G-16 to R-1001; R-17 to R-1001; T-18 to R-1001; E-19 to R-1001;
R-20 to 8-
1001;8-21 to R-1001; I-22 to R-1001; P-23 to R-1001; R-24 to R-1001; L-25 to R-
1001; W-
26 to R-1001; R-27 to R-1001; L-28to R-1001; L-29 to R-1001; L-3 0 to R-1001;
W-31 to 8-
1001; A-32 to R-1001; G-33 to R-1001; T-34 to R-1001; A-35 toR-1001; F-36 to R-
1001; Q-
37 to R-1001; V -3 8 to R-1001; T-39 to R-1001; Q-40 to R-1001; G -41 to R-
1001; T-42 toR-
1001; G-43 to R-1001; P-44 to R-1001; E-45 to R-1001; L-46 to R-1001; H-47 to
R-1001; A-
48 to R-1001; C-49 toR-1001; K-50 to R-1001; E-51 to R-1001; S-52 to R-1001; E-
53 to 8-
1001; Y-54 to R-1001; H-55 to R-1001; Y-56 toR-1001; E-57 to R-1001; Y-58 to R-
1001; T-
59 to R-1001; A-60 to R-1001; C-61 to R-1001; D-62 to R-1001; S-63 toR-1001; T-
64 to R-
1001; G-65 to R-1001; S-66 to R-1001; R-67 to R-1001; W-68 to R-1001; R-69 to
R-1001; V-
70 to R-1001; A-71 to R-1001; V -72 to R-1001; P-73 to R-1001; H-74 to R-1001;
T-75 to 8-
1001; P-76 to R-1001; G-77 toR-1001; L-78 to R-1001; C-79 to R-1001; T-80 to R-
1001; S-
81 to R-1001; L-82 to R-1001; P-83 to R-1001; D-84 toR-1001; P-85 to R-1001; V-
86 to R-
1001; K-87 to R-1001; G-8 8 to R-1001; T-89 to R-1001; E-90 to R-1001; C-91
toR-1001; S -
92 to R-1001; F-93 to R-1001; S-94 to R-1001; C-95 to R-1001; N-96 to R-1001;
A-97 to R-
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
116
I 001; G-98 toR-1001; E-99 to R-1001; F-100 to R-1001; L-1 O l to R-100 I ; D-
102 to R-1001;
M-103 to R-1001; K-104 to R-1001; D-1 OS to R-1001; Q-106 to R-1001; S-107 to
R- I 001; C-
108 to R-1001; K-109 to R-1001; P-110 to R-1001; C-111 to R- l 001;A-112 to R-
1001; E-113
to R-1001; G-114 to R-1001; R-115 to R-1001; Y-116 to R-1001; S-117 to R-1001;
L-118
toR-1001; G-119 to R-1001; T-120 to R-1001; G-121 to R-1001; I-122 to R-1001;
R-123 to
R-1001; F-124 to R- l 001;D- I 25 to R-1001; E-126 to R-1001; W-127 to R-1001;
D-128 to R-
1001; E-129 to R-1001; L-130 to R-1001; P-131 toR-1001; H-132 to R-1001; G-133
to R-
1001; F-134 to R-1001; A-135 to R-1001; S-136 to R-1001; L-137 to R-1001;5-138
to R-
1001; A-139 to R-1001; N-140 to R-1001; M-141 to R-1001; E-142 to R-1001; L-
143 to 8-
1001; D-144 toR-1001; D-145 to R-1001; S-146 to R-1001; A-147 to R-1001; A-148
to R-
1001; E-149 to R-1001; S-150 to R-1001sT-151 to R-1001; G-152 to R-1001; N-153
to R-
1001; C-154 to R-1001; T-155 to R-1001; S-156 to R-1001; S-157 toR-1001; K-158
to 8-
1001; W-159 to R-1001; V-160 to R-1001; P-161 to R-1001; R-162 to R-1001; G-
163 to R-
1001;D-164 to R-1001; Y-165 to R-1001; I-166 to R-1001; A-167 to R-1001; F-168
to R-
1001; N-169 to R-1001; T-170 toR-1001; D-171 to R-1001; E-172 to R-1001; C-173
to 8-
1001; T-174 to R-1001; A-175 to R-1001; T-176 to R-1001;L-177 to R-1001; M-178
to R-
1001; Y-179 to R-1001; A-180 to R-1001; V-181 to R-1001; N-182 to R-1001; L-
183 toR-
1001; K-184 to R- I 001; Q-185 to R-1001; S-186 to R-1001; G-187 to R-1001; T-
18 8 to 8-
1001; V -189 to R-1001; N-190 to R-1001; F-191 to R-1001; E-192 to R-1001; Y-
193 to R-
1001; Y-194 to R-1001; Y-195 to R- I 001; P- I 96 toR-1001; D-197 to R-1001; S-
198 to R-
I 001; S-199 to R-1001; I-200 to R-1001; I-201 to R-1001; F-202 to R-100 I ; E-
203 to R-1001;
F-204 to R-1001; F-205 to R-1001; V -206 to R-1001; Q-207 to R-1001; N-208 to
R-1001; D-
209 to R- l 001;Q-210 to R-1001; C-211 to R-1001; Q-212 to R-1001; P-213 to R-
1001; N-214
to R-1001; A-215 to R-1001; D-216 toR-1001; D-217 to R-1001; S-218 to R-1001;
R-219 to
R-1001; W-220 to R-1001; M-221 to R-1001; K-222 to R- l 001sT-223 to R-1001; T-
224 to R-
1001; E-225 to R-1001; K-226 to R-1001; G-227 to R-1001; W-228 to R-1001; E-
229 toR-
1001; F-230 to R-1001; H-231 to R-1001; S-232 to R-1001; V-233 to R-1001; E-
234 to R-
1001; L-235 to R-1001;N-236 to R-1001; R-237 to R-1001; G-238 to R-1001; N-239
to R-
1001; N-240 to R-1001; V -241 to R-1001; L-242 toR-1001; Y-243 to R-1001; W-
244 to 8-
1001; R-245 to R-1001; T-246 to R-1001; T-247 to R-1001; A-248 to R- l 001;F-
249 to 8-
1001; S-250 to R-1001; V-251 to R-1001; W-252 to R-1001; T-253 to R-1001; K-
254 to R-
1001; V-255 toR-1001; P-256 to R-1001; K-257 to R-1001; P-258 to R-1001; V-259
to R-
1001; L-260 to R-1001; V -261 to R-1001;8-262 to R-1001; N-263 to R-1001; I-
264 to R-
1001; A-265 to R-1001; I-266 to R-1001; T-267 to R-1001; G-268 toR-1001; V-269
to 8-
1001; A-270 to R-1001; Y-271 to R-1001; T-272 to R-1001; S-273 to R-1001; E-
274 to 8-
1001; C-275 to R-1001; F-276 to R- I 001; P-277 to R-1001; C-278 to R-1001; K-
279 to R-
1001; P-280 to R-1001; G-281 toR-1001; T-282 to R-1001; Y-283 to R-1001; A-284
to 8-
1001; D-285 to R-1001; K-286 to R-1001; Q-287 to R-1001;6-288 to R-1001; S-289
to R-
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
117
1001; S-290 to R-1001; F-291 to R-1001; C-292 to R-1001; K-293 to R-1001; L-
294 toR-
1001; C-295 to R-1001; P-296 to R-1001; A-297 to R-1001; N-298 to R-1001; S-
299 to R-
1001; Y-300 to R-1001;S-301 to R-1001; N-302 to R-1001; K-303 to R-1001; G-304
to R-
1001; E-305 to R-1001; T-306 to R-1001; S-307 toR-1001; C-308 to R-1001; H-309
to R-
1001; Q-310 to R-1001; C-311 to R-1001; D-312 to R-100 I ; P-313 to R-1001; D-
314 to 8-
1001; K-315 to R-1001; Y-316 to R-1001; S-317 to R-1001; E-318 to R-1001; K-
319 to 8-
1001; G-320 toR-1001; S-321 to R-1001; S-322 to R-1001; S-323 to R-1001; C-324
to R-
1001; N-325 to R-1001; V-326 to R-1001;8-327 to R-1001; P-328 to R-1001; A-329
to R-
1001; C-330 to R-1001; T-331 to R-1001; D-332 to R-1001; K-333 toR-1001; D-334
to R-
1001; Y-335 to R-1001; F-336 to R-1001; Y-337 to R-1001; T-338 to R-1001; H-
339 to R-
l 001sT-340 to R-1001; A-341 to R-1001; C-342 to R-1001; D-343 to R-1001; A-
344 to R-
1001; N-345 to R-1001; G-346 toR-1001; E-347 to R-1001; T-348 to R-1001; Q-349
to 8-
1001; L-350 to R-1001; M-351 to R-1001; Y-352 to R-1001;x-353 to R-1001; W-354
to 8-
1001; A-355 to R-1001; K-356 to R-1001; P-357 to R-1001; K-358 to R-1001; I-
359 toR-
1001; C-360 to R-1001; S-361 to R-1001; E-362 to R-1001; D-363 to R-1001; L-
364 to 8-
1001; E-365 to R-1001;6-366 to R-1001; A-367 to R-1001; V-368 to R-1001; K-369
to R-
1001; L-370 to R-1001; P-371 to R-1001; A-372 toR-1001; S-373 to R-1001; G-374
to R-
1001; V-375 to R-1001; K-376 to R-1001; T-377 to R-1001; H-378 to R-1001;C-379
to 8-
1001; P-380 to R-1001; P-381 to R-1001; C-382 to R-1001; N-383 to R-1001; P-
384 to R-
1001; G-385 toR-1001; F-386 to R-1001; F-387 to R-1001; K-388 to R-1001; T-389
to 8-
1001; N-390 to R-1001; N-391 to R-1001;S-392 to R-1001; T-393 to R-1001; C-394
to 8-
1001; Q-395 to R-1001; P-396 to R-1001; C-397 to R-1001; P-398 toR-1001; Y-399
to 8-
1001; G-400 to R-1001; S-401 to R-1001; Y-402 to R-1001; S-403 to R-1001; N-
404 to 8-
1001; G-405 to R-1001; S-406 to R-1001; D-407 to R-1001; C-408 to R-1001; T-
409 to 8-
1001; R-410 to R-1001; C-411 toR-1001; P-412 to R-1001; A-413 to R-1001; G-414
to R-
1001; T-415 to R-1001; E-416 to R-1001; P-417 to R- l 001;A-418 to R-1001; V -
419 to 8-
1001; G-420 to R- I 001; F-421 to R-1001; E-422 to R-1001; Y -423 to R-1001; K-
424 toR-
1001; W-425 to R-1001; W-426 to R-1001; N-427 to R-1001; T-428 to R-1001; L-
429 to R-
1001; P-430 to R- l 001sT-431 to R-1001; N-432 to R-1001; M-433 to R-1001; E-
434 to R-
1001; T-435 to R-1001; T-436 to R-1001; V-437 toR-1001; L-438 to R-1001; S-439
to 8-
1001; G-440 to R-1001; I-441 to R-1001; N-442 to R-1001; F-443 to R-1001; E-
444to 8-
1001; Y-445 to R-1001; K-446 to R-1001; G-447 to R-1001; M-448 to R-1001; T-
449 to R-
1001; G-450 to R-1001; W-451 to R-1001; E-452 to R- I 001; V -45 3 to R-1001;
A-454 to R-
1001; G-455 to R-I 001; D-456 to R-1001; H-457 toR-1001; I-458 to R-1001; Y-
459 to 8-
1001; T-460 to R-1001; A-461 to R-1001; A-462 to R-1001; G-4.63 to R-1001;A-
464 to 8-
1001; S-4.65 to R-1001; D-466 to R-1001; N-467 to R-1001; D-4.68 to R-1001; F-
469 to 8-
1001; M-470 toR-1001; I-471 to R-1001; L-472 to R-1001; T-473 to R-1001; L-474
to R-
1001; V -475 to R-1001; V -476 to R-1001; P-477 to R-1001; G-47 8 to R-1001; F-
479 to R-
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
118
1001; R-480 to R-1001; P-481 to R-1001; P-482 to R-1001; Q-483 to R-1001; S-
484 to 8-
1001; V -485 to R-1001; M-486 to R-1001; A-487 to R-1001; D-48 8 to R-1001; T-
489 to 8-
1001; E-490 toR-1001; N-491 to R-1001; K-492 to R-1001; E-493 to R-1001; V -
494 to 8-
1001; A-495 to R-1001; R-496 to R-1001; I-497 to R-1001; T-498 to R-1001; F-
499 to R-
1001; V-500 to R-1001; F-501 to R-1001; E-502 to R-1001; T-503 toR-1001; L-504
to R-
1001; C-505 to R-1001; S-506 to R-1001; V-507 to R-1001; N-508 to R-1001; C-
509 to R-
l 001;E-510 to R-1001; L-511 to R-1001; Y -512 to R-1001; F-513 to R-1001; M-
514 to 8-
1001; V-515 to R-1001; G-516 toR-1001; V-517 to R-1001; N-518 to R-1001; S-519
to 8-
1001; R-520 to R-1001; T-521 to R-1001; N-522 to R- l 001sT-523 to R-1001; P-
524 to R-
1001; V-525 to R-1001; E-526 to R-1001; T-527 to R-1001; W-528 to R-1001; K-
529 toR-
1001; G-530 to R-1001; S-531 to R-1001; K-532 to R-1001; G-533 to R-1001; K-
534 to 8-
1001; Q-535 to R-1001;5-536 to R-1001; Y-537 to R-1001; T-538 to R-1001; Y-539
to 8-
1001; I-540 to R-1001; I-541 to R-1001; E-542 toR-1001; E-543 to R-1001; N-544
to R-1001;
T-545 to R-1001; T-546 to R-1001; T-547 to R-1001; S-548 to R-1001; F-549 to R-
1001; T-
550 to R-1001; W-551 to R-1001; A-552 to R-1001; F-553 to R-1001; Q-554 to R-
1001; 8-
555 toR-1001; T-556 to R-1001; T-557 to R-1001; F-558 to R-1001; H-559 to R-
1001; E-560
to R-1001; A-561 to R-1001;S-562 to R-1001; R-563 to R-1001; K-564 to R-1001;
Y-565 to
R-1001; T-566 to R-1001; N-567 to R-1001; D-568 toR-1001; V-569 to R-1001; A-
570 to R-
1001; K-571 to R-1001; I-572 to R-1001; Y-573 to R-1001; S-574 to R-1001; I-
575to R-1001;
N-576 to R-1001; V-577 to R-1001; T-578 to R-1001; N-579 to R-1001; V-580 to R-
1001; M-
581 to R-1001;N-582 to R-1001; G-583 to R-1001; V-584 to R-1001; A-585 to R-
1001; S-586
to R-1001; Y-587 to R-1001; C-588 toR-1001; R-589 to R-1001; P-590 to R-1001;
C-591 to
R-1001; A-592 to R-1001; L-593 to R-1001; E-594 to R-1001;A-595 to R-1001; S-
596 to R-
1001; D-597 to R-1001; V-598 to R-1001; G-599 to R-1001; S-600 to R-1001; S-
601 toR-
1001; C-602 to R-1001; T-603 to R-1001; S-604 to R-1001; C-605 to R-1001; P-
606 to 8-
1001; A-607 to R-1001;6-608 to R-1001; Y-609 to R-1001; Y-610 to R-1001; I-611
to R-
1001; D-612 to R-1001; R-613 to R-1001; D-614 toR-1001; S-615 to R-1001; G-616
to R-
1001; T-617 to R-1001; C-618 to R-1001; H-619 to R-1001; S -620 to R- l 001;C-
621 to 8-
1001; P-622 to R-1001; P-623 to R-1001; N-624 to R-1001; T-625 to R-1001; I-
626 to R-
1001; L-627 toR-1001; K-628 to R-1001; A-629 to R-1001; H-63 0 to R-1001; Q-
631 to 8-
1001; P-632 to R-1001; Y-633 to R-1001;6-634 to R-1001; V-635 to R-1001; Q-636
to R-
1001; A-637 to R-1001; C-63 8 to R-1001; V -639 to R-1001; P-640 toR-1001; C-
641 to 8-
1001; G-642 to R-1001; P-643 to R-1001; G-644 to R-1001; T-645 to R-1001; K-
646 to R-
l 001;N-647 to R-1001; N-648 to R-1001; K-649 to R-1001; I-650 to R-1001; H-
651 to R-
1001; S-652 to R-1001; L-653 toR-1001; C-654 to R-1001; Y-655 to R-1001; N-656
to R-
1001; D-657 to R-1001; C-65 8 to R-1001; T-659 to R-1001; F-660 to R-1001; S-
661 to 8-
1001; R-662 to R-1001; N-663 to R-1001; T-664 to R-1001; P-665 to R-1001; T-
666 toR-
1001; R-667 to R-1001; T-668 to R-1001; F-669 to R-1001; N-670 to R-1001; Y-
671 to R-
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
119
1001; N-672 to R- l 001;F-673 to R-1001; S-674 to R-1001; A-675 to R-1001; L-
676 to 8-
1001; A-677 to R-1001; N-67 8 to R-1001; T-679 toR-1001; V -680 to R-1001; T-
681 to 8-
1001; L-682 to R-1001; A-683 to R-1001; G-684 to R-1001; G-685 to R-1001; P-
686 to 8-
1001; S-687 to R-1001; F-688 to R-1001; T-689 to R-1001; S-690 to R-1001; K-
691 to R-
1001; G-692 toR-1001; L-693 to R-1001; K-694 to R-1001; Y-695 to R-1001; F-696
to 8-
1001; H-697 to R-1001; H-698 to R- l 001;F-699 to R-1001; T-700 to R-1001; L-
701 to 8-
1001; S-702 to R-1001; L-703 to R-1001; C-704 to R-1001; G-705 toR-1001; N-706
to 8-
1001; Q-707 to R-1001; G-708 to R-1001; R-709 to R-1001; K-710 to R-1001; M-
711 to 8-
1001; S-712 to R-1001; V -713 to R-1001; C-7 I 4 to R-1001; T-715 to R-1001; D-
716 to 8-
1001; N-717 to R-1001; V -718 toR-1001; T-719 to R-1001; D-720 to R-1001; L-
721 to 8-
1001; R-722 to R-1001; I-723 to R-1001; P-724 to R-1001; E-725to R-1001; G-726
to 8-
100 I ; E-727 to R-1001; S-728 to R-1001; G-729 to R-1001; F-730 to R-1001; S-
731 to R-
1001;K-732 to R-1001; S-733 to R-1001; I-734 to R-1001; T-735 to R-1001; A-736
to R-
1001; Y-737 to R-1001; V-738 toR-1001; C-739 to R-1001; Q-740 to R-1001; A-741
to 8-
1001; V -742 to R-1001; I-743 to R-1001; I-744 to R-1001; P-745to R-1001; P-
746 to R-1001;
E-747 to R-1001; V-748 to R-1001; T-749 to R-1001; G-750 to R-1001; Y-751 to R-
1001;K-
752 to R-1001; A-753 to R-1001; G-754 to R-1001; V-755 to R-1001; S-756 to R-
1001; 5-
757 to R-1001; Q-758 toR-1001; P-759 to R-1001; V-760 to R-1001; S-761 to R-
1001; L-762
to R-1001; A-763 to R-1001; D-764 to R-1001;8-765 to R-1001; L-766 to R-1001;
I-767 to 8-
1001; G-768 to R-1001; V-769 to R-1001; T-770 to R-1001; T-771 toR-1001; D-772
to 8-
1001; M-773 to R-1001; T-774 to R-1001; L-775 to R-1001; D-776 to R-1001; G-
777 to R-
1001;I-778 to R-1001; T-779 to R-1001; S-780 to R-1001; P-781 to R-1001; A-782
to 8-
1001; E-783 to R-1001; L-784 toR-1001; F-785 to R-1001; H-786 to R-1001; L-787
to 8-
1001; E-788 to R-1001; S-789 to R-1001; L-790 to R-1001;6-791 to R-1001; I-792
to R-
1001; P-793 to R-1001; D-794 to R-1001; V -795 to R-1001; I-796 to R-1001; F-
797 toR-
1001; F-798 to R-1001; Y-799 to R-1001; R-800 to R-1001; S-801 to R-1001; N-
802 to 8-
1001; D-803 to R-1001;V-804 to R-1001; T-805 to R-1001; Q-806 to R-1001; S-807
to R-
1001; C-808 to R-1001; S-809 to R-1001; S-810 toR-1001; G-811 to R-1001; R-812
to 8-
1001; S-813 to R-1001; T-814 to R-1001; T-815 to R-1001; I-816 to R-1001; R-8
l7to 8-
1001; V-818 to R-1001; R-819 to R-1001; C-820 to R-1001; S-821 to R-1001; P-
822 to 8-
1001; Q-823 to R-1001;x-824 to R-1001; T-825 to R-1001; V-826 to R-1001; P-827
to 8-
1001; G-828 to R-1001; S-829 to R-1001; L-830 toR-1001; L-831 to R-1001; L-832
to 8-
1001; P-833 to R-1001; G-834 to R-1001; T-835 to R-1001; C-836 to R-1001;S-837
to 8-
1001; D-838 to R-1001; G-8 3 9 to R-1001; T-840 to R-1001; C-841 to R-1001; D-
842 to 8-
1001; G-843 toR-1001; C-844 to R-1001; N-845 to R-1001; F-846 to R- I 001; H-
847 to R-
1001; F-848 to R-1001; L-849 to R-1001; W-850 to R-1001; E-851 to R-1001; S-
852 to 8-
1001; A-853 to R-1001; A-854 to R-1001; A-855 to R-1001; C-856 toR-1001; P-857
to 8-
1001; L-858 to R-1001; C-859 to R-1001; S-860 to R-1001; V-861 to R-1001; A-
862 to R-
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
120
l 001;D-863 to R-1001; Y-864 to R-1001; H-865 to R-1001; A-866 to R-1001; I-
867 to 8-
1001; V -868 to R-1001; S-869 toR-1001; S-870 to R-1001; C-871 to R-1001; V-
872 to 8-
1001; A-873 to R-1001; G-874 to R-1001; I-875 to R- l 001;Q-876 to R- I 001; K-
877 to 8-
1001; T-878 to R-1001; T-879 to R-1001; Y-880 to R-1001; V-881 to R-1001; W-
882 toR-
1001; R-883 to R-1001; E-884 to R-1001; P-885 to R-1001; K-886 to R-1001; L-
887 to 8-
1001; C-888 to R-1001;S-889 to R-1001; G-890 to R-1001; G-89-1 to R-1001; I-
892 to 8-
1001; S-893 to R-1001; L-894 to R-1001; P-895 toR-1001; E-896 to R-1001; Q-897
to 8-
1001; R-898 to R-1001; V-899 to R-1001; T-900 to R-1001; I-901 to R-1001; C-
902to 8-
1001; K-903 to R-1001; T-904 to R-1001; I-905 to R-1001; D-906 to R-1001; F-
907 to R-
100 I ; W-908 to R-1001; L-909 to R-1001; K-910 to R-1001; V -911 to R-1001; G-
912 to 8-
1001; I-913 to R-1001; S-914 to R-1001; A-915 toR-1001; G-916 to R-1001; T-917
to 8-
1001; C-918 to R-1001; T-919 to R-1001; A-920 to R-1001; I-921 to R-1001; L-
922to 8-
1001; L-923 to R-1001; T-924 to R-1001; V -925 to R-1001; L-926 to R-1001; T-
927 to 8-
1001; C-928 to R- l 001;Y-929 to R-1001; F-93 0 to R-1001; W-931 to R-1001; K-
932 to R-
~5 1001; K-933 to R-1001; N-934 to R-1001; Q-935 toR-1001; K-936 to R-1001; L-
937 to 8-
1001; E-93 8 to R-1001; Y-939 to R-1001; K-940 to R-1001; Y-941 to R-1001; S-
942 to 8-
1001; K-943 to R-1001; L-944 to R-1001; V -945 to R-1001; M-946 to R-1001; N-
947 to 8-
1001; A-948 toR-1001; T-949 to R-1001; L-950 to R-1001; K-951 to R-1001; D-952
to 8-
1001; C-95 3 to R-1001; D-954 to R-1001; L-955 to R-1001; P-956 to R-1001; A-
957 to 8-
1001; A-958 to R-1001; D-959 to R-1001; S-960 to R-1001; C-961 toR-1001; A-962
to R-
1001; I-963 to R-1001; M-964 to R-1001; E-965 to R-1001; G -966 to R-1001; E-
967 to R-
l 001;D-968 to R-1001; V -969 to R-1001; E-970 to R-1001; D-971 to R-1001; D-
972 to 8-
1001; L-973 to R-1001; I-974 toR-1001; F-975 to R-1001; T-976 to R-1001; S-977
to R-1001;
K-978 to R-1001; N-979 to R-1001; H-980 to R-1001; S-981 to R-1001; L-982 to R-
1001; G-
983 to R-1001; R-984 to R-1001; S-985 to R-1001; N-986 to R-1001; H-987 toR-
1001; L-988
to R-1001; P-989 to R-1001; P-990 to R-1001; R-991 to R-1001; G-992 to R-1001;
L-993 to
R- l 001;L-994 to R-1001; M-995 to R-1001; D-996 to R-1001; of S EQ ID N0:40.
Polynucleotides encoding these polypeptides are also encompassed by the
invention.
Also as mentioned above, even if deletion of one or more amino acids from the
C-
3o 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 TR13 ligand) may still be retained. For example the ability of the
shortened TR13 mutein
to induce and/or bind to antibodies which recognize the complete or mature
forms of the
polypeptide generally will be retained when less than the majority of the
residues of the
complete or mature polypeptide are removed from the C-terminus. Whether a
particular
polypeptide lacking C-terminal residues of a complete polypeptide retains such
immunologic
activities can readily be determined by routine methods described herein and
otherwise known
in the art. It is not unlikely that an TR13 mutein with a large number of
deleted C-terminal
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
121
amino acid residues may retain some biological or immunogenic activities. In
fact, peptides
composed of as few as six TR13 amino acid residues may often evoke an immune
response.
Accordingly, the present invention further provides polypeptides having one or
more
residues deleted from the carboxy terminus of the amino acid sequence of the
TR13 polypeptide
shown in Figures lA-C (SEQ ID N0:2), up to the glutamine residue at position
number 6, and
polynucleotides encoding such polypeptides. In particular, the present
invention provides
polypeptides comprising, or alternatively consisting of, the amino acid
sequence of residues 1-
m' of Figures 1 A-C, where m' is an integer from 6 to 749 corresponding to the
position of the
amino acid residue in Figures lA-C.
1o Moreover, the invention provides TR13 polypeptides comprising, or
alternatively
consisting of, the amino acid sequence of residues: Q-6 to C-749; Q-6 to Q-
748; Q-6 to T-747;
Q-6 to L-746; Q-6 to D-745; Q-6 to M-744; Q-6to L-743; Q-6 to L-742; Q-6 to G-
741; Q-6 to
R-740; Q-6 to P-739; Q-6 to P-738; Q-6 to L-737; Q-6 to H-736; Q-6 to N-735; Q-
6to S-734;
Q-6 to R-733; Q-6 to G-732; Q-6 to L-731; Q-6 to S-730; Q-6 to H-729; Q-6 to N-
728; Q-6 to
K-727; Q-6 to S-726;Q-6 to T-725; Q-6 to F-724; Q-6 to I-723; Q-6 to L-722; Q-
6 to D-721;
Q-6 to D-720; Q-6 to E-719; Q-6 to V-718; Q-6 to D-717;Q-6 to E-716; Q-6 to G-
715; Q-6 to
E-714; Q-6 to M-713; Q-6 to I-712; Q-6 to A-711; Q-6 to C-710; Q-6 to S-709; Q-
6 to D-
708;Q-6 to A-707; Q-6 to A-706; Q-6 to P-705; Q-6 to L-704; Q-6 to D-703; Q-6
to C-702; Q-6
to D-701; Q-6 to K-700; Q-6 to L-699;Q-6 to T-698; Q-6 to A-697; Q-6 to N-696;
Q-6 to M-
695; Q-6 to V-694; Q-6 to L-693; Q-6 to K-692; Q-6 to S-691; Q-6 toY-690; Q-6
to K-689; Q-
6 to Y-688; Q-6 to E-687; Q-6 to L-686; Q-6 to K-685; Q-6 to Q-684; Q-6 to N-
683; Q-6 to K-
682; Q-6to K-681; Q-6 to W-680; Q-6 to F-679; Q-6 to Y-678; Q-6 to C-677; Q-6
to T-676; Q-
6 to L-675; Q-6 to V-674; Q-6 to T-673;Q-6 to L-672; Q-6 to L-671; Q-6 to I-
670; Q-6 to A-
669; Q-6 to T-668; Q-6 to C-667; Q-6 to T-666; Q-6 to G-665; Q-6 to A-664;Q-6
to S-663; Q-6
to I-662; Q-6 to G-661; Q-6 to V-660; Q-6 to K-659; Q-6 to L-658; Q-6 to W-
657; Q-6 to F-
656; Q-6 to D-655;Q-6 to I-654; Q-6 to T-653; Q-6 to K-652; Q-6 to C-651; Q-6
to I-650; Q-6
to T-649; Q-6 to V-648; Q-6 to R-647; Q-6 to Q-646;Q-6 to E-645; Q-6 to P-644;
Q-6 to L-
643; Q-6 to S-642; Q-6 to I-641; Q-6 to G-640; Q-6 to G-639; Q-6 to S-638; Q-6
to C-637;Q-6
to L-636; Q-6 to K-635; Q-6 to P-634; Q-6 to E-633; Q-6 to R-632; Q-6 to W-
631; Q-6 to V-
630; Q-6 to Y-629; Q-6 toT-628; Q-6 to T-627; Q-6 to K-626; Q-6 to Q-625; Q-6
to I-624; Q-6
to G-623; Q-6 to A-622; Q-6 to V-621; Q-6 to C-620; Q-6 toS-619; Q-6 to S-618;
Q-6 to V-
617; Q-6 to I-616; Q-6 to A-615; Q-6 to H-614; Q-6 to Y-613; Q-6 to D-612; Q-6
to A-611; Q-
6 toV-610; Q-6 to S-609; Q-6 to C-608; Q-6 to L-607; Q-6 to P-606; Q-6 to C-
605; Q-6 to A-
604; Q-6 to A-603; Q-6 to A-602; Q-6 toS-601; Q-6 to E-600; Q-6 to W-599; Q-6
to L-598; Q-
6 to F-597; Q-6 to H-596; Q-6 to F-595; Q-6 to N-594; Q-6 to C-593; Q-6to G-
592; Q-6 to D-
591; Q-6 to C-590; Q-6 to T-589; Q-6 to G-588; Q-6 to D-587; Q-6 to S-586; Q-6
to C-585; Q-
6 to T-584;Q-6 to G-583; Q-6 to P-582; Q-6 to L-581; Q-6 to L-580; Q-6 to L-
579; Q-6 to S-
578; Q-6 to G-577; Q-6 to P-576; Q-6 to V-575;Q-6 to T-574; Q-6 to K-573; Q-6
to Q-572; Q-
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
122
6 to P-571; Q-6 to S-570; Q-6 to C-569; Q-6 to R-568; Q-6 to V-567; Q-6 to R-
566;Q-6 to I-
565; Q-6 to T-564; Q-6 to T-563; Q-6 to S-562; Q-6 to R-561; Q-6 to G-560; Q-6
to S-559; Q-
6 to S-558; Q-6 to C-557;Q-6 to S-556; Q-6 to Q-555; Q-6 to T-554; Q-6 to V-
553; Q-6 to D-
552; Q-6 to N-551; Q-6 to S-550; Q-6 to R-549; Q-6 to Y-548;Q-6 to F-547; Q-6
to F-546; Q-6
to I-545; Q-6 to V-544; Q-6 to D-543; Q-6 to P-542; Q-6 to I-541; Q-6 to G-
540; Q-6 to L-
539;Q-6 to S-538; Q-6 to E-537; Q-6 to L-536; Q-6 to H-535; Q-6 to F-534; Q-6
to L-533; Q-6
to E-532; Q-6 to A-531; Q-6 to P-530;Q-6 to S-529; Q-6 to T-528; Q-6 to I-527;
Q-6 to G-526;
Q-6 to D-525; Q-6 to L-524; Q-6 to T-523; Q-6 to M-522; Q-6 to D-521;Q-6 to T-
520; Q-6 to
T-519; Q-6 to V-518; Q-6 to G-517; Q-6 to I-516; Q-6 to L-515; Q-6 to R-514; Q-
6 to D-513;
1o Q-6 to A-512;Q-6 to L-511; Q-6 to S-510; Q-6 to V-509; Q-6 to P-508; Q-6 to
Q-507; Q-6 to
S-506; Q-6 to S-505; Q-6 to V-504; Q-6 to G-503;Q-6 to A-502; Q-6 to K-501; Q-
6 to Y-500;
Q-6 to G-499; Q-6 to T-498; Q-6 to V-497; Q-6 to E-496; Q-6 to P-495; Q-6 to P-
494;Q-6 to I-
493; Q-6 to I-492; Q-6 to V-491; Q-6 to A-490; Q-6 to Q-489; Q-6 to C-488; Q-6
to V-487; Q-
6 to Y-486; Q-6 to A-485;Q-6 to T-484; Q-6 to I-483; Q-6 to S-482; Q-6 to K-
481; Q-6 to S-
480; Q-6 to F-479; Q-6 to G-478; Q-6 to S-477; Q-6 to E-476;Q-6 to G-475; Q-6
to E-474; Q-6
to P-473; Q-6 to I-472; Q-6 to R-471; Q-6 to L-470; Q-6 to D-469; Q-6 to T-
468; Q-6 to V-
467;Q-6 to N-466; Q-6 to D-465; Q-6 to T-464; Q-6 to C-463; Q-6 to V-462; Q-6
to S-461; Q-
6 to M-460; Q-6 to K-459; Q-6 toR-458; Q-6 to G-457; Q-6 to Q-456; Q-6 to N-
455; Q-6 to 6-
454; Q-6 to C-453; Q-6 to L-452; Q-6 to S-451; Q-6 to L-450; Q-6to T-449; Q-6
to F-448; Q-6
to H-447; Q-6 to H-446; Q-6 to F-445; Q-6 to Y-444; Q-6 to K-443; Q-6 to L-
442; Q-6 to G-
441;Q-6 to K-440; Q-6 to S-439; Q-6 to T-438; Q-6 to F-437; Q-6 to S-436; Q-6
to P-435; Q-6
to G-434; Q-6 to G-433; Q-6 to A-432;Q-6 to L-431; Q-6 to T-430; Q-6 to V-429;
Q-6 to T-
428; Q-6 to N-427; Q-6 to A-426; Q-6 to L-425; Q-6 to A-424; Q-6 to S-423;Q-6
to F-422; Q-6
to N-421; Q-6 to Y-420; Q-6 to N-419; Q-6 to F-418; Q-6 to T-417; Q-6 to R-
416; Q-6 to T-
415; Q-6 to P-414;Q-6 to T-413; Q-6 to N-412; Q-6 to R-411; Q-6 to S-410; Q-6
to F-409; Q-6
to T-408; Q-6 to C-407; Q-6 to D-406; Q-6 to N-405;Q-6 to Y-404; Q-6 to C-403;
Q-6 to L-
402; Q-6 to S-401; Q-6 to H-400; Q-6 to I-399; Q-6 to K-398; Q-6 to N-397; Q-6
to N-396;Q-
6 to K-395; Q-6 to T-394; Q-6 to G-393; Q-6 to P-392; Q-6 to G-391; Q-6 to C-
390; Q-6 to P-
389; Q-6 to V-388; Q-6 toC-387; Q-6 to A-386; Q-6 to Q-385; Q-6 to V-384; Q-6
to G-383; Q-
6 to Y-382; Q-6 to P-381; Q-6 to Q-380; Q-6 to H-379; Q-6to A-378; Q-6 to K-
377; Q-6 to L-
376; Q-6 to I-375; Q-6 to T-374; Q-6 to N-373; Q-6 to P-372; Q-6 to P-371; Q-6
to C-370; Q-
6to S-369; Q-6 to H-368; Q-6 to C-367; Q-6 to T-366; Q-6 to G-365; Q-6 to S-
364; Q-6 to D-
363; Q-6 to R-362; Q-6 to D-361;Q-6 to I-360; Q-6 to Y-359; Q-6 to Y-358; Q-6
to G-357; Q-6
to A-356; Q-6 to P-355; Q-6 to C-354; Q-6 to S-353; Q-6 to T-352;Q-6 to C-351;
Q-6 to S-
350; Q-6 to S-349; Q-6 to G-348; Q-6 to V-347; Q-6 to D-346; Q-6 to S-345; Q-6
to A-344; Q-
6 to E-343;Q-6 to L-342; Q-6 to A-341; Q-6 to C-340; Q-6 to P-339; Q-6 to R-
338; Q-6 to C-
337; Q-6 to Y-336; Q-6 to S-335; Q-6 to A-334;Q-6 to V-333; Q-6 to G-332; Q-6
to N-331; Q-
6 to M-330; Q-6 to V-329; Q-6 to N-328; Q-6 to T-327; Q-6 to V-326; Q-6 toN-
325; Q-6 to I-
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
123
324; Q-6 to S-323; Q-6 to Y-322; Q-6 to I-321; Q-6 to K-320; Q-6 to A-319; Q-6
to V-318; Q-
6 to D-317; Q-6 toN-316; Q-6 to T-315; Q-6 to Y-314; Q-6 to K-313; Q-6 to R-
312; Q-6 to S-
311; Q-6 to A-310; Q-6 to E-309; Q-6 to H-308; Q-6to F-307; Q-6 to T-306; Q-6
to T-305; Q-6
to R-304; Q-6 to Q-303; Q-6 to F-302; Q-6 to A-301; Q-6 to W-300; Q-6 to T-
299; Q-6to F-
298; Q-6 to S-297; Q-6 to T-296; Q-6 to T-295; Q-6 to T-294; Q-6 to N-293; Q-6
to E-292; Q-
6 to E-291; Q-6 to I-290; Q-6to I-289; Q-6 to Y-288; Q-6 to T-287; Q-6 to Y-
286; Q-6 to S-
285; Q-6 to Q-284; Q-6 to K-283; Q-6 to G-282; Q-6 to K-281; Q-6to S-280; Q-6
to G-279; Q-
6 to K-278; Q-6 to W-277; Q-6 to T-276; Q-6 to E-275; Q-6 to V-274; Q-6 to P-
273; Q-6 to T-
272;Q-6 to N-271; Q-6 to T-270; Q-6 to R-269; Q-6 to S-268; Q-6 to N-267; Q-6
to V-266; Q-
l0 6 to G-265; Q-6 to V-264; Q-6 toM-263; Q-6 to F-262; Q-6 to Y-261; Q-6 to L-
260; Q-6 to E-
259; Q-6 to C-258; Q-6 to N-257; Q-6 to V-256; Q-6 to S-255; Q-6to C-254; Q-6
to L-253; Q-
6 to T-252; Q-6 to E-251; Q-6 to F-250; Q-6 to V-249; Q-6 to F-248; Q-6 to T-
247; Q-6 to I-
246; Q-6to R-245; Q-6 to A-244; Q-6 to V-243; Q-6 to E-242; Q-6 to K-241; Q-6
to N-240; Q-
6 to E-239; Q-6 to T-238; Q-6 to D-237;Q-6 to A-236; Q-6 to M-235; Q-6 to V-
234; Q-6 to S-
233; Q-6 to Q-232; Q-6 to P-231; Q-6 to P-230; Q-6 to R-229; Q-6 to F-228;Q-6
to G-227; Q-6
to P-226; Q-6 to V -225 ; Q-6 to . V -224; Q-6 to L-223 ; Q-6 to T-222; Q-6 to
L-221; Q-6 to I-220;
Q-6 to M-219;Q-6 to F-218; Q-6 to D-217; Q-6 to N-216; Q-6 to D-215; Q-6 to S-
214; Q-6 to
A-213; Q-6 to G-212; Q-6 to A-211; Q-6 toA-210; Q-6 to T-209; Q-6 to Y-208; Q-
6 to I-207;
Q-6 to H-206; Q-6 to D-205; Q-6 to G-204; Q-6 to A-203; Q-6 to V-202; Q-6 toE-
201; Q-6 to
2o W-200; Q-6 to G-199; Q-6 to T-198; Q-6 to M-197; Q-6 to G-196; Q-6 to K-
195; Q-6 to Y-
194; Q-6 to E-193; Q-6to F-192; Q-6 to N-191; Q-6 to I-190; Q-6 to G-189; Q-6
to S-188; Q-6
to L-187; Q-6 to V-186; Q-6 to T-185; Q-6 to T-184; Q-6to E-183; Q-6 to M-182;
Q-6 to N-
181; Q-6 to T-180; Q-6 to P-179; Q-6 to L-178; Q-6 to T-177; Q-6 to N-176; Q-6
to W-175;Q-
6 to W-174; Q-6 to K-173; Q-6 to Y-172; Q-6 to E-171; Q-6 to F-170; Q-6 to G-
169; Q-6 to V-
168; Q-6 to A-167 ; Q-6 toP-166; Q-6 to E-165 ; Q-6 to T-164; Q-6 to G-163 ; Q-
6 to A-162; Q-6
to P-161; Q-6 to C-160; Q-6 to R-159; Q-6 to T-158; Q-6 toC-157; Q-6 to D-156;
Q-6 to S-
155; Q-6 to G-154; Q-6 to N-153; Q-6 to S-152; Q-6 to Y-151; Q-6 to S-150; Q-6
to G-149; Q-
6to Y -148 ; Q-6 to P-147 ; Q-6 to C-146; Q-6 to P-145 ; Q-6 to Q-144; Q-6 to
C-143 ; Q-6 to T-
142; Q-6 to S-141; Q-6 to N-140; Q-6to N-139; Q-6 to T-138; Q-6 to K-137; Q-6
to F-136; Q-
6 to F-135; Q-6 to G-134; Q-6 to P-133; Q-6 to N-132; Q-6 to C-131;Q-6 to P-
130; Q-6 to P-
129; Q-6 to C-128; Q-6 to H-127; Q-6 to T-126; Q-6 to K-125; Q-6 to V-124; Q-6
to G-123;
Q-6 toS-122; Q-6 to A-121; Q-6 to P-120; Q-6 to L-119; Q-6 to K-118; Q-6 to V-
117; Q-6 to
A-116; Q-6 to G-115; Q-6 to E- I 14; Q-6 toL-113; Q-6 to D-112; Q-6 to E-111;
Q-6 to S-110;
Q-6 to C-109; Q-6 to I-108; Q-6 to K-107; Q-6 to P-106; Q-6 to K-105; Q-6 toA-
104; Q-6 to
W-103; Q-6 to K-102; Q-6 to Y-101; Q-6 to M-100; Q-6 to L-99; Q-6 to Q-98; Q-6
to T-97; Q-
6 to E-96; Q-6 toG-95; Q-6 to N-94; Q-6 to A-93; Q-6 to D-92; Q-6 to C-91; Q-6
to A-90; Q-6
to T-89; Q-6 to H-88; Q-6 to T-87; Q-6 to Y-86; Q-6to F-85; Q-6 to Y-84; Q-6
to D-83; Q-6 to
K-82; Q-6 to D-81; Q-6 to T-80; Q-6 to C-79; Q-6 to A-78; Q-6 to P-77; Q-6 to
R-76;Q-6 to V-
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
124
75; Q-6 to N-74; Q-6 to C-73; Q-6 to S-72; Q-6 to S-71; Q-6 to S-70; Q-6 to G-
69; Q-6 to K-
68; Q-6 to E-67; Q-6 toS-66; Q-6 to Y-65; Q-6 to K-64; Q-6 to D-63; Q-6 to P-
62; Q-6 to D-61;
Q-6 to C-60; Q-6 to Q-59; Q-6 to H-58; Q-6 to C-57; Q-6to S-56; Q-6 to T-55; Q-
6 to E-54; Q-
6 to G-53; Q-6 to K-52; Q-6 to N-51; Q-6 to S-50; Q-6 to Y-49; Q-6 to S-48; Q-
6 to N-47;Q-6
to S-46; Q-6 to P-45; Q-6 to L-44; Q-6 to T-43; Q-6 to Q-42; Q-6 to L-41; Q-6
to F-40; Q-6 to
L-39; Q-6 to L-38; Q-6 toG-37; Q-6 to A-36; Q-6 to Q-35; Q-6 to R-34; Q-6 to C-
33; Q-6 to V-
32; Q-6 to H-31; Q-6 to W-30; Q-6 to T-29; Q-6 to Q-28;Q-6 to L-27; Q-6 to P-
26; Q-6 to L-
25; Q-6 to M-24; Q-6 to R-23; Q-6 to F-22; Q-6 to H-21; Q-6 to L-20; Q-6 to G-
19; Q-6 toG-
18 ; Q-6 to R-17 ; Q-6 to N-16; Q-6 to H- I 5 ; Q-6 to C-14; Q-6 to H-13 ; Q-6
to K-12; of S EQ ID
N0:2. Polynucleotides encoding these polypeptides are also encompassed by the
invention.
The present invention further provides polypeptides having one or more
residues
deleted from the carboxy terminus of the amino acid sequence of the TR13
polypeptide shown
in Figures 7A-D (SEQ ID N0:40), up to the histidine residue at position number
6, and
polynucleotides encoding such polypeptides. In particular, the present
invention provides
polypeptides comprising, or alternatively consisting of, the amino acid
sequence of residues 1-
m' of Figures 7A-D, where m' is an integer from 6 to 1001 corresponding to the
position of the
amino acid residue in Figures 7A-D.
Moreover, the invention provides TR13 polypeptides comprising, or
alternatively
consisting of, the amino acid sequence of residues: H-6 to C-1000; H-6 to Q-
999; H-6 to T-
998;H-6 to L-997; H-6 to D-996; H-6 to M-995; H-6 to L-994; H-6 to L-993; H-6
to G-992;
H-6 to R-991; H-6 toP-990; H-6 to P-989; H-6 to L-988; H-6 to H-987; H-6 to N-
986; H-6 to
S-985; H-6 to R-984; H-6 to G-983;H-6 to L-982; H-6 to S-981; H-6 to H-980; H-
6 to N-979;
H-6 to K-978; H-6 to S-977; H-6 to T-976; H-6 toF-975; H-6 to I-974; H-6 to L-
973; H-6 to
D-972; H-6 to D-971; H-6 to E-970; H-6 to V-969; H-6 to D-968;H-6 to E-967; H-
6 to G-966;
H-6 to E-965; H-6 to M-964; H-6 to I-963; H-6 to A-962; H-6 to C-961; H-6 toS-
960; H-6 to
D-959; H-6 to A-958; H-6 to A-957; H-6 to P-956; H-6 to L-955; H-6 to D-954; H-
6 to C-
953;H-6 to D-952; H-6 to K-951; H-6 to L-950; H-6 to T-949; H-6 to A-948; H-6
to N-947;
H-6 to M-946; H-6 toV-945; H-6 to L-944; H-6 to K-943; H-6 to S-942; H-6 to Y-
941; H-6 to
K-940; H-6 to Y-939; H-6 to E-938;H-6 to L-937; H-6 to K-936; H-6 to Q-935; H-
6 to N-934;
3o H-6 to K-933; H-6 to K-932; H-6 to W-931; H-6to F-930; H-6 to Y-929; H-6 to
C-928; H-6
to T-927; H-6 to L-926; H-6 to V-925; H-6 to T-924; H-6 toL-923; H-6 to L-922;
H-6 to I-
921; H-6 to A-920; H-6 to T-919; H-6 to C-918; H-6 to T-917; H-6 to G-916;H-6
to A-915;
H-6 to S-914; H-6 to I-913; H-6 to G-912; H-6 to V-911; H-6 to K-910; H-6 to L-
909; H-6
toW-908; H-6 to F-907; H-6 to D-906; H-6 to I-905; H-6 to T-904; H-6 to K-903;
H-6 to C-
902; H-6 to I-901;H-6 to T-900; H-6 to V-899; H-6 to R-898; H-6 to Q-897; H-6
to E-896; H-
6 to P-895; H-6 to L-894; H-6 toS-893; H-6 to I-892; H-6 to G-891; H-6 to G-
890; H-6 to S-
889; H-6 to C-888; H-6 to L-887; H-6 to K-886;H-6 to P-885; H-6 to E-884; H-6
to R-883;
H-6 to W-882; H-6 to V-881; H-6 to Y-880; H-6 to T-879; H-6 toT-878; H-6 to K-
877; H-6 to
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
125
Q-876; H-6 to I-875; H-6 to G-874; H-6 to A-873; H-6 to V-872; H-6 to C-871;H-
6 to S-870;
H-6 to S-869; H-6 to V-868; H-6 to I-867; H-6 to A-866; H-6 to H-865; H-6 to Y-
864; H-6
toD-863; H-6 to A-862; H-6 to V-861; H-6 to S-860; H-6 to C-859; H-6 to L-858;
H-6 to P-
857; H-6 to C-856;H-6 to A-855; H-6 to A-854; H-6 to A-853; H-6 to S-852; H-6
to E-851;
H-6 to W-850; H-6 to L-849; H-6 toF-848; H-6 to H-847; H-6 to F-846; H-6 to N-
845; H-6 to
C-844; H-6 to G-843; H-6 to D-842; H-6 to C-841;H-6 to T-840; H-6 to G-839; H-
6 to D-838;
H-6 to S-837; H-6 to C-836; H-6 to T-835; H-6 to G-834; H-6 toP-833; H-6 to L-
832; H-6 to
L-831; H-6 to L-830; H-6 to S-829; H-6 to G-828; H-6 to P-827; H-6 to V-826;H-
6 to T-825;
H-6 to K-824; H-6 to Q-823; H-6 to P-822; H-6 to S-821; H-6 to C-820; H-6 to R-
819; H-6
toV-818; H-6 to R-817; H-6 to I-816; H-6 to T-815; H-6 to T-814; H-6 to S-813;
H-6 to 8-
812; H-6 to G-811;H-6 to S-810; H-6 to S-809; H-6 to C-808; H-6 to S-807; H-6
to Q-806;
H-6 to T-805; H-6 to V-804; H-6 toD-803; H-6 to N-802; H-6 to S-801; H-6 to R-
800; H-6 to
Y-799; H-6 to F-798; H-6 to F-797; H-6 to I-796;H-6 to V-795; H-6 to D-794; H-
6 to P-793;
H-6 to I-792; H-6 to G-791; H-6 to L-790; H-6 to S-789; H-6 toE-788; H-6 to L-
787; H-6 to
t5 H-786; H-6 to F-785; H-6 to L-784; H-6 to E-783; H-6 to A-782; H-6 to P-
781;H-6 to S-780;
H-6 to T-779; H-6 to I-778; H-6 to G-777; H-6 to D-776; H-6 to L-775; H-6 to T-
774; H-6
toM-773; H-6 to D-772; H-6 to T-771; H-6 to T-770; H-6 to V-769; H-6 to G-768;
H-6 to I-
767; H-6 to L-766;H-6 to R-765; H-6 to D-764; H-6 to A-763; H-6 to L-762; H-6
to S-761; H-
6 to V-760; H-6 to P-759; H-6 toQ-758; H-6 to S-757; H-6 to S-756; H-6 to V-
755; H-6 to G-
754; H-6 to A-753; H-6 to K-752; H-6 to Y-751;H-6 to G-750; H-6 to T-749; H-6
to V-748;
H-6 to E-747; H-6 to P-746; H-6 to P-745; H-6 to I-744; H-6 toI-743; H-6 to V-
742; H-6 to
A-741; H-6 to Q-740; H-6 to C-739; H-6 to V-738; H-6 to Y-737; H-6 to A-736;H-
6 to T-735;
H-6 to I-734; H-6 to S-733; H-6 to K-732; H-6 to S-731; H-6 to F-730; H-6 to G-
729; H-6
toS-728; H-6 to E-727; H-6 to G-726; H-6 to E-725; H-6 to P-724; H-6 to I-723;
H-6 to R-
722; H-6 to L-721;H-6 to D-720; H-6 to T-719; H-6 to V-718; H-6 to N-717; H-6
to D-716;
H-6 to T-715; H-6 to C-714; H-6 toV-713; H-6 to S-712; H-6 to M-711; H-6 to K-
710; H-6 to
R-709; H-6 to G-708; H-6 to Q-707; H-6 toN-706; H-6 to G-705; H-6 to C-704; H-
6 to L-
703; H-6 to S-702; H-6 to L-701; H-6 to T-700; H-6 to F-699;H-6 to H-698; H-6
to H-697;
H-6 to F-696; H-6 to Y-695; H-6 to K-694; H-6 to L-693; H-6 to G-692; H-6 toK-
691; H-6 to
3o S-690; H-6 to T-689; H-6 to F-688; H-6 to S-687; H-6 to P-686; H-6 to G-
685; H-6 to G-
684;H-6 to A-683; H-6 to L-682; H-6 to T-681; H-6 to V-680; H-6 to T-679; H-6
to N-678;
H-6 to A-677; H-6 toL-676; H-6 to A-675; H-6 to S-674; H-6 to F-673; H-6 to N-
672; H-6 to
Y-671; H-6 to N-670; H-6 to F-669;H-6 to T-668; H-6 to R-667; H-6 to T-666; H-
6 to P-665;
H-6 to T-664; H-6 to N-663; H-6 to R-662; H-6 toS-661; H-6 to F-660; H-6 to T-
659; H-6 to
C-658; H-6 to D-657; H-6 to N-656; H-6 to Y-655; H-6 to C-654;H-6 to L-653; H-
6 to S-652;
H-6 to H-651; H-6 to I-650; H-6 to K-649; H-6 to N-648; H-6 to N-647; H-6 toK-
646; H-6 to
T-645; H-6 to G-644; H-6 to P-643; H-6 to G-642; H-6 to C-641; H-6 to P-640; H-
6 to V-
639;H-6 to C-638; H-6 to A-637; H-6 to Q-636; H-6 to V-635; H-6 to G-634; H-6
to Y-633;
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
126
H-6 to P-632; H-6 toQ-631; H-6 to H-630; H-6 to A-629; H-6 to K-628; H-6 to L-
627; H-6 to
I-626; H-6 to T-625; H-6 to N-624;H-6 to P-623; H-6 to P-622; H-6 to C-621; H-
6 to S-620;
H-6 to H-619; H-6 to C-618; H-6 to T-617; H-6 toG-616; H-6 to S-615; H-6 to D-
614; H-6 to
R-613; H-6 to D-612; H-6 to I-61 l; H-6 to Y-610; H-6 to Y-609;H-6 to G-608; H-
6 to A-607;
s H-6 to P-606; H-6 to C-605; H-6 to S-604; H-6 to T-603; H-6 to C-602; H-6
toS-601; H-6 to
S-600; H-6 to G-599; H-6 to V-598; H-6 to D-597; H-6 to S-596; H-6 to A-595; H-
6 to E-
594;H-6 to L-593; H-6 to A-592; H-6 to C-591; H-6 to P-590; H-6 to R-589; H-6
to C-588;
H-6 to Y-587; H-6 toS-586; H-6 to A-585; H-6 to V-584; H-6 to G-583; H-6 to N-
582; H-6 to
M-581; H-6 to V-580; H-6 toN-579; H-6 to T-578; H-6 to V-577; H-6 to N-576; H-
6 to I-575;
1o H-6 to S-574; H-6 to Y-573; H-6 to I-572;H-6 to K-571; H-6 to A-570; H-6 to
V-569; H-6 to
D-568; H-6 to N-567; H-6 to T-566; H-6 to Y-565; H-6 toK-564; H-6 to R-563; H-
6 to S-562;
H-6 to A-561; H-6 to E-560; H-6 to H-559; H-6 to F-558; H-6 to T-557;H-6 to T-
556; H-6 to
R-555; H-6 to Q-554; H-6 to F-553; H-6 to A-552; H-6 to W-551; H-6 to T-550; H-
6 toF-549;
H-6 to S-548; H-6 to T-547; H-6 to T-546; H-6 to T-545; H-6 to N-544; H-6 to E-
543; H-6 to
15 E-542;H-6 to I-541; H-6 to I-540; H-6 to Y-539; H-6 to T-538; H-6 to Y-537;
H-6 to S-536;
H-6 to Q-535; H-6 toK-534; H-6 to G-533; H-6 to K-532; H-6 to S-531; H-6 to G-
530; H-6 to
K-529; H-6 to W-528; H-6 toT-527; H-6 to E-526; H-6 to V-525; H-6 to P-524; H-
6 to T-523;
H-6 to N-522; H-6 to T-521; H-6 to R-520;H-6 to S-519; H-6 to N-518; H-6 to V-
517; H-6 to
G-516; H-6 to V-515; H-6 to M-514; H-6 to F-513; H-6 toY-512; H-6 to L-511; H-
6 to E-510;
20 H-6 to C-509; H-6 to N-508; H-6 to V-507; H-6 to S-506; H-6 to C-SOS;H-6 to
L-504; H-6 to
T-503; H-6 to E-502; H-6 to F-501; H-6 to V-500; H-6 to F-499; H-6 to T-498; H-
6 toI-497;
H-6 to R-496; H-6 to A-495; H-6 to V-494; H-6 to E-493; H-6 to K-492; H-6 to N-
491; H-6
to E-490;H-6 to T-489; H-6 to D-488; H-6 to A-487; H-6 to M-486; H-6 to V-485;
H-6 to 5-
484; H-6 to Q-483; H-6 toP-482; H-6 to P-481; H-6 to R-480; H-6 to F-479; H-6
to G-478;
2s H-6 to P-477; H-6 to V-476; H-6 to V-475;H-6 to L-474; H-6 to T-473; H-6 to
L-472; H-6 to
I-471; H-6 to M-470; H-6 to F-469; H-6 to D-468; H-6 toN-467; H-6 to D-466; H-
6 to S-465;
H-6 to A-464; H-6 to G-463; H-6 to A-462; H-6 to A-461; H-6 to T-460;H-6 to Y-
459; H-6 to
I-458; H-6 to H-457; H-6 to D-456; H-6 to G-455; H-6 to A-454; H-6 to V-453; H-
6 toE-452;
H-6 to W-451; H-6 to G-450; H-6 to T-449; H-6 to M-448; H-6 to G-447; H-6 to K-
446; H-6
30 toY-445; H-6 to E-444; H-6 to F-443; H-6 to N-442; H-6 to 1-441; H-6 to G-
440; H-6 to S-
439; H-6 to L-438;H-6 to V-437; H-6 to T-436; H-6 to T-435; H-6 to E-434; H-6
to M-433;
H-6 to N-432; H-6 to T-431; H-6 toP-430; H-6 to L-429; H-6 to T-428; H-6 to N-
427; H-6 to
W-426; H-6 to W-425; H-6 to K-424; H-6 toY-423; H-6 to E-422; H-6 to F-421; H-
6 to 6-
420; H-6 to V-419; H-6 to A-418; H-6 to P-417; H-6 to E-416;H-6 to T-415; H-6
to G-414;
3s H-6 to A-413; H-6 to P-412; H-6 to C-411; H-6 to R-410; H-6 to T-409; H-6
toC-408; H-6 to
D-407; H-6 to S-406; H-6 to G-405; H-6 to N-404; H-6 to S-403; H-6 to Y-402; H-
6 to S-
401;H-6 to G-400; H-6 to Y-399; H-6 to P-398; H-6 to C-397; H-6 to P-396; H-6
to Q-395;
H-6 to C-394; H-6 toT-393; H-6 to S-392; H-6 to N-391; H-6 to N-390; H-6 to T-
389; H-6 to
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
127
K-388; H-6 to F-387; H-6 to F-386;H-6 to G-385; H-6 to P-384; H-6 to N-383; H-
6 to C-382;
H-6 to P-381; H-6 to P-380; H-6 to C-379; H-6 toH-378; H-6 to T-377; H-6 to K-
376; H-6 to
V-375; H-6 to G-374; H-6 to S-373; H-6 to A-372; H-6 to P-371;H-6 to L-370; H-
6 to K-369;
H-6 to V-368; H-6 to A-367; H-6 to G-366; H-6 to E-365; H-6 to L-364; H-6 toD-
363; H-6 to
s E-362; H-6 to S-361; H-6 to C-360; H-6 to I-359; H-6 to K-358; H-6 to P-357;
H-6 to K-
356;H-6 to A-355; H-6 to W-354; H-6 to K-353; H-6 to Y-352; H-6 to M-351; H-6
to L-350;
H-6 to Q-349; H-6 toT-348; H-6 to E-347; H-6 to G-346; H-6 to N-345; H-6 to A-
344; H-6 to
D-343; H-6 to C-342; H-6 to A-341;H-6 to T-340; H-6 to H-339; H-6 to T-338; H-
6 to Y-337;
H-6 to F-336; H-6 to Y-335; H-6 to D-334; H-6 toK-333; H-6 to D-332; H-6 to T-
331; H-6 to
C-330; H-6 to A-329; H-6 to P-328; H-6 to R-327; H-6 to V-326;H-6 to N-325; H-
6 to C-324;
H-6 to S-323; H-6 to S-322; H-6 to S-321; H-6 to G-320; H-6 to K-319; H-6 toE-
318; H-6 to
S-317; H-6 to Y-316; H-6 to K-315; H-6 to D-314; H-6 to P-313; H-6 to D-312; H-
6 to C-
311;H-6 to Q-310; H-6 to H-309; H-6 to C-308; H-6 to S-307; H-6 to T-306; H-6
to E-305;
H-6 to G-304; H-6 toK-303; H-6 to N-302; H-6 to S-301; H-6 to Y-300; H-6 to S-
299; H-6 to
N-298; H-6 to A-297; H-6 to P-296;H-6 to C-295; H-6 to L-294; H-6 to K-293; H-
6 to C-292;
H-6 to F-291; H-6 to S-290; H-6 to S-289; H-6 toG-288; H-6 to Q-287; H-6 to K-
286; H-6 to
D-285; H-6 to A-284; H-6 to Y-283; H-6 to T-282; H-6 to G-281;H-6 to P-280; H-
6 to K-279;
H-6 to C-278; H-6 to P-277; H-6 to F-276; H-6 to C-275; H-6 to E-274; H-6 toS-
273; H-6 to
T-272; H-6 to Y-271; H-6 to A-270; H-6 to V-269; H-6 to G-268; H-6 to T-267; H-
6 to I-
266;H-6 to A-265; H-6 to I-264; H-6 to N-263; H-6 to R-262; H-6 to V-261; H-6
to L-260; H-
6 to V-259; H-6 toP-258; H-6 to K-257; H-6 to P-256; H-6 to V-255; H-6 to K-
254; H-6 to T-
253; H-6 to W-252; H-6 to V-251;H-6 to S-250; H-6 to F-249; H-6 to A-248; H-6
to T-247;
H-6 to T-246; H-6 to R-245; H-6 to W-244; H-6 toY-243; H-6 to L-242; H-6 to V-
241; H-6 to
N-240; H-6 to N-239; H-6 to G-238; H-6 to R-237; H-6 to N-236;H-6 to L-235; H-
6 to E-
234; H-6 to V-233; H-6 to S-232; H-6 to H-231; H-6 to F-230; H-6 to E-229; H-6
toW-228;
H-6 to G-227; H-6 to K-226; H-6 to E-225; H-6 to T-224; H-6 to T-223; H-6 to K-
222; H-6
toM-221; H-6 to W-220; H-6 to R-219; H-6 to S-218; H-6 to D-217; H-6 to D-216;
H-6 to A-
215; H-6 toN-214; H-6 to P-213; H-6 to Q-212; H-6 to C-211; H-6 to Q-210; H-6
to D-209;
H-6 to N-208; H-6 to Q-207;H-6 to V-206; H-6 to F-205; H-6 to F-204; H-6 to E-
203; H-6 to
3o F-202; H-6 to I-201; H-6 to I-200; H-6 toS-199; H-6 to S-198; H-6 to D-197;
H-6 to P-196;
H-6 to Y-195; H-6 to Y-194; H-6 to Y-193; H-6 to E-192;H-6 to F-191; H-6 to N-
190; H-6 to
V-189; H-6 to T-188; H-6 to G-187; H-6 to S-186; H-6 to Q-185; H-6 toK-184; H-
6 to L-183;
H-6 to N-182; H-6 to V-181; H-6 to A-180; H-6 to Y-179; H-6 to M-178; H-6 to L-
177;H-6 to
T-176; H-6 to A-175; H-6 to T-174; H-6 to C-173; H-6 to E-172; H-6 to D-171; H-
6 to T-170;
H-6 toN-169; H-6 to F-168; H-6 to A-167; H-6 to I-166; H-6 to Y-165; H-6 to D-
164; H-6 to
G-163; H-6 to R-162;H-6 to P-161; H-6 to V-160; H-6 to W-159; H-6 to K-158; H-
6 to S-
157; H-6 to S-156; H-6 to T-155; H-6 toC-154; H-6 to N-153; H-6 to G-152; H-6
to T-151;
H-6 to S-150; H-6 to E-149; H-6 to A-148; H-6 to A-147;H-6 to S-146; H-6 to D-
145; H-6 to
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
128
D-144; H-6 to L-143 ; H-6 to E-142; H-6 to M-141; H-6 to N-140; H-6 toA-139; H-
6 to S-13 8;
H-6 to L-137; H-6 to S-136; H-6 to A-135; H-6 to F-134; H-6 to G-133; H-6 to H-
132;H-6 to
P-131; H-6 to L-130; H-6 to E-129; H-6 to D-128; H-6 to W-127; H-6 to E-126; H-
6 to D-
125; H-6 toF-124; H-6 to R-123; H-6 to I-122; H-6 to G-121; H-6 to T-120; H-6
to G-119; H-
6 to L-118; H-6 to S-117;H-6 to Y-116; H-6 to R-115; H-6 to G-114; H-6 to E-
113; H-6 to A-
112; H-6 to C-111; H-6 to P-110; H-6 toK-109; H-6 to C-108; H-6 to S-107; H-6
to Q-106;
H-6 to D-105; H-6 to K-104; H-6 to M-103; H-6 to D-102;H-6 to L-101; H-6 to F-
100; H-6 to
E-99; H-6 to G-98; H-6 to A-97; H-6 to N-96; H-6 to C-95; H-6 to S-94;H-6 to F-
93; H-6 to
S-92; H-6 to C-91; H-6 to E-90; H-6 to T-89; H-6 to G-88; H-6 to K-87; H-6 to
V-86;H-6 to
to P-85; H-6 to D-84; H-6 to P-83; H-6 to L-82; H-6 to S-81; H-6 to T-80; H-6
to C-79; H-6 to
L-78; H-6to G-77; H-6 to P-76; H-6 to T-75; H-6 to H-74; H-6 to P-73; H-6 to V-
72; H-6 to
A-71; H-6 to V-70; H-6 toR-69; H-6 to W-68; H-6 to R-67; H-6 to S-66; H-6 to G-
65; H-6 to
T-64; H-6 to S-63; H-6 to D-62; H-6 toC-61; H-6 to A-60; H-6 to T-59; H-6 to Y-
58; H-6 to
E-57; H-6 to Y-56; H-6 to H-55; H-6 to Y-54; H-6 toE-53; H-6 to S-52; H-6 to E-
51; H-6 to
t5 K-50; H-6 to C-49; H-6 to A-48; H-6 to H-47; H-6 to L-46; H-6 toE-45; H-6
to P-44; H-6 to
G-43; H-6 to T-42; H-6 to G-41; H-6 to Q-40; H-6 to T-39; H-6 to V-38; H-6 toQ-
37; H-6 to
F-36; H-6 to A-35; H-6 to T-34; H-6 to G-33; H-6 to A-32; H-6 to W-31; H-6 to
L-30; H-6
toL-29; H-6 to L-28; H-6 to R-27; H-6 to W-26; H-6 to L-25; H-6 to R-24; H-6
to P-23; H-6
to I-22; H-6 toR-21; H-6 to R-20; H-6 to E-19; H-6 to T-18; H-6 to R-17; H-6
to G-16; H-6 to
2o R-15; H-6 to V-14; H-6 toR-13; H-6 to A-12; of SEQ ID N0:4~0.
Polynucleotides encoding
these polypeptides are also encompassed by the invention.
In another embodiment, N-terminal deletions of the predicted extracellular
domain of
the predicted mature TR13 protein, with the amino acid sequence shown in
Figures 7A-D, can
be described by the general formula n2-906, where n2 is a number from 2 to
900,
25 corresponding to the position of amino acid identified in Figures 7A-D (SEQ
ID N0:40). N-
terminal deletions of the TR13 polypeptide of the invention shown as SEQ ID
N0:4~0 include
polypeptides comprising, or alternatively consisting of, the amino acid
sequence of residues: T-
42 to D-906; G-43 to D-906; P-44 to D-906; E-45 to D-906; L-46 to D-906; H-47
to D-906; A-
48 to D-906; C-49 to D-906; K-50 to D-906; E-51 to D-906; S-52 to D-906; E-53
to D-906; Y-
3o 54 to D-906; H-55 to D-906; Y-56 toD-906; E-57 to D-906; Y-58 to D-906; T-
59 to D-906; A-
60 to D-906; C-61 to D-906; D-62 to D-906; S-63 toD-906; T-64 to D-906; G-65
to D-906; S-
66 to D-906; R-67 to D-906; W-68 to D-906; R-69 to D-906; V -70 toD-906; A-71
to D-906; V-
72 to D-906; P-73 to D-906; H-74 to D-906; T-75 to D-906; P-76 to D-906; G-77
toD-906; L-
78 to D-906; C-79 to D-906; T-80 to D-906; S-81 to D-906; L-82 to D-906; P-83
to D-906; D-
35 84 to D-906; P-85 to D-906; V-86 to D-906; K-87 to D-906; G-88 to D-906; T-
89 to D-906; E-
90 to D-906; C-91 toD-906; S-92 to D-906; F-93 to D-906; S-94 to D-906; C-95
to D-906; N-
96 to D-906; A-97 to D-906; G-98 toD-906; E-99 to D-906; F-100 to D-906; L-101
to D-906;
D-102 to D-906; M-103 to D-906; K-104 to D-906; D-105to D-906; Q-106 to D-906;
S-107 to
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
129
D-906; C-108 to D-906; K-109 to D-906; P-110 to D-906; C-111 to D-906;A-112 to
D-906; E-
113 to D-906; G-114 to D-906; R-115 to D-906; Y-116 to D-906; S-117 to D-906;
L-118 toD-
906; G-119 to D-906; T-120 to D-906; G-121 to D-906; I-122 to D-906; R-123 to
D-906; F-
124 to D-906;D-125 to D-906; E-126 to D-906; W-127 to D-906; D-128 to D-906; E-
129 to D-
906; L-130 to D-906; P-131 toD-906; H-132 to D-906; G-133 to D-906; F-134 to D-
906; A-
135 to D-906; S-136 to D-906; L-137 to D-906;S-138 to D-906; A-139 to D-906; N-
140 to D-
906; M-141 to D-906; E-142 to D-906; L-143 to D-906; D-144 toD-906; D-145 to D-
906; S -
146 to D-906; A-147 to D-906; A-148 to D-906; E-149 to D-906; S-150 to D-906;T-
151 to D-
906; G-152 to D-906; N-153 to D-906; C-154 to D-906; T-155 to D-906; S-156 to
D-906; S-
157 toD-906; K-158 to D-906; W-159 to D-906; V-160 to D-906; P-161 to D-906; R-
162 to D-
906; G-163 to D-906;D-164 to D-906; Y-165 to D-906; I-166 to D-906; A-167 to D-
906; F-
168 to D-906; N-169 to D-906; T-170 toD-906; D-171 to D-906; E-172 to D-906; C-
173 to D-
906; T-174 to D-906; A-175 to D-906; T-176 to D-906;L-177 to D-906; M-178 to D-
906; Y-
179 to D-906; A-180 to D-906; V-181 to D-906; N-182 to D-906; L-183 toD-906; K-
184 to D-
906; Q-185 to D-906; S-186 to D-906; G-187 to D-906; T-188 to D-906; V-189 to
D-906;N-
190 to D-906; F-191 to D-906; E-192 to D-906; Y-193 to D-906; Y-194 to D-906;
Y-195 to D-
906; P-196 toD-906; D-197 to D-906; S-198 to D-906; S-199 to D-906; I-200 to D-
906; I-201
to D-906; F-202 to D-906; E-203to D-906; F-204 to D-906; F-205 to D-906; V-206
to D-906;
Q-207 to D-906; N-208 to D-906; D-209 to D-906;Q-210 to D-906; C-211 to D-906;
Q-212 to
2o D-906; P-213 to D-906; N-214 to D-906; A-215 to D-906; D-216 toD-906; D-217
to D-906; 5-
218 to D-906; R-219 to D-906; W-220 to D-906; M-221 to D-906; K-222 to D-906;T-
223 to
D-906; T-224 to D-906; E-225 to D-906; K-226 to D-906; G-227 to D-906; W-228
to D-906;
E-229 toD-906; F-230 to D-906; H-231 to D-906; S-232 to D-906; V-233 to D-906;
E-234 to
D-906; L-235 to D-906;N-236 to D-906; R-237 to D-906; G-238 to D-906; N-239 to
D-906;
N-240 to D-906; V-241 to D-906; L-242 toD-906; Y-243 to D-906; W-244 to D-906;
R-245 to
D-906; T-246 to D-906; T-247 to D-906; A-248 to D-906;F-249 to D-906; S-250 to
D-906; V-
251 to D-906; W-252 to D-906; T-253 to D-906; K-254 to D-906; V-255 toD-906; P-
256 to D-
906; K-257 to D-906; P-258 to D-906; V-259 to D-906; L-260 to D-906; V-261 to
D-906;R-
262 to D-906; N-263 to D-906; I-264 to D-906; A-265 to D-906; I-266 to D-906;
T-267 to D-
906; G-268 toD-906; V-269 to D-906; A-270 to D-906; Y-271 to D-906; T-272 to D-
906; S-
273 to D-906; E-274 to D-906;C-275 to D-906; F-276 to D-906; P-277 to D-906; C-
278 to D-
906; K-279 to D-906; P-280 to D-906; G-281 toD-906; T-282 to D-906; Y-283 to D-
906; A-
284 to D-906; D-285 to D-906; K-286 to D-906; Q-287 to D-906;6-288 to D-906; S-
289 to D-
906; S-290 to D-906; F-291 to D-906; C-292 to D-906; K-293 to D-906; L-294 toD-
906; C-
295 to D-906; P-296 to D-906; A-297 to D-906; N-298 to D-906; S-299 to D-906;
Y-300 to D-
906;S-301 to D-906; N-302 to D-906; K-303 to D-906; G-304 to D-906; E-305 to D-
906; T-
306 to D-906; S-307 toD-906; C-308 to D-906; H-309 to D-906; Q-310 to D-906; C-
311 to D-
906; D-312 to D-906; P-313 to D-906;D-314 to D-906; K-315 to D-906; Y-316 to D-
906; S-
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
130
317 to D-906; E-318 to D-906; K-319 to D-906; G-320 toD-906; S-321 to D-906; S-
322 to D-
906; S-323 to D-906; C-324 to D-906; N-325 to D-906; V-326 to D-906;8-327 to D-
906; P-
328 to D-906; A-329 to D-906; C-330 to D-906; T-331 to D-906; D-332 to D-906;
K-333 toD-
906; D-334 to D-906; Y-335 to D-906; F-336 to D-906; Y-337 to D-906; T-338 to
D-906; H-
339 to D-906;T-340 to D-906; A-341 to D-906; C-342 to D-906; D-343 to D-906; A-
344 to D-
906; N-345 to D-906; G-346 toD-906; E-347 to D-906; T-348 to D-906; Q-349 to D-
906; L-
350 to D-906; M-351 to D-906; Y-352 to D-906;x-353 to D-906; W-354 to D-906; A-
355 to
D-906; K-356 to D-906; P-357 to D-906; K-358 to D-906; I-359 toD-906; C-360 to
D-906; S-
361 to D-906; E-362 to D-906; D-363 to D-906; L-364 to D-906; E-365 to D-906;6-
366 to D-
906; A-367 to D-906; V-368 to D-906; K-369 to D-906; L-370 to D-906; P-371 to
D-906; A-
372 toD-906; S-373 to D-906; G-374 to D-906; V-375 to D-906; K-376 to D-906; T-
377 to D-
906; H-378 to D-906;C-379 to D-906; P-380 to D-906; P-381 to D-906; C-382 to D-
906; N-
383 to D-906; P-384 to D-906; G-385 toD-906; F-386 to D-906; F-387 to D-906; K-
388 to D-
906; T-389 to D-906; N-390 to D-906; N-391 to D-906;S-392 to D-906; T-393 to D-
906; C-
394 to D-906; Q-395 to D-906; P-396 to D-906; C-397 to D-906; P-398 toD-906; Y-
399 to D-
906; G-400 to D-906; S-401 to D-906; Y-402 to D-906; S-403 to D-906; N-404 to
D-906;G-
405 to D-906; S-406 to D-906; D-407 to D-906; C-408 to D-906; T-409 to D-906;
R-410 to D-
906; C-411 toD-906; P-412 to D-906; A-413 to D-906; G-414 to D-906; T-415 to D-
906; E-
416 to D-906; P-417 to D-906;A-418 to D-906; V-419 to D-906; G-420 to D-906; F-
421 to D-
906; E-422 to D-906; Y-423 to D-906; K-424 toD-906; W-425 to D-906; W-426 to D-
906; N-
427 to D-906; T-428 to D-906; L-429 to D-906; P-430 to D-906;T-431 to D-906; N-
432 to D-
906; M-433 to D-906; E-434 to D-906; T-435 to D-906; T-436 to D-906; V-437 toD-
906; L-
438 to D-906; S-439 to D-906; G-440 to D-906; I-441 to D-906; N-442 to D-906;
F-443 to D-
906; E-444to D-906; Y-445 to D-906; K-446 to D-906; G-447 to D-906; M-448 to D-
906; T-
449 to D-906; G-450 to D-906;W-451 to D-906; E-452 to D-906; V-453 to D-906; A-
454 to D-
906; G-455 to D-906; D-456 to D-906; H-457 toD-906; I-458 to D-906; Y-459 to D-
906; T-
460 to D-906; A-461 to D-906; A-4b2 to D-906; G-463 to D-906;A-464 to D-906; S-
465 to D-
906; D-466 to D-906; N-467 to D-906; D-468 to D-906; F-469 to D-906; M-470 toD-
906; I-
471 to D-906; L-472 to D-906; T-473 to D-906; L-474 to D-906; V-475 to D-906;
V-476 to D-
906; P-477to D-906; G-478 to D-906; F-479 to D-906; R-480 to D-906; P-481 to D-
906; P-
482 to D-906; Q-483 to D-906;5-484 to D-906; V-485 to D-906; M-486 to D-906; A-
487 to D-
906; D-488 to D-906; T-489 to D-906; E-490 toD-906; N-491 to D-906; K-492 to D-
906; E-
493 to D-906; V-494 to D-906; A-495 to D-906; R-496 to D-906;I-497 to D-906; T-
498 to D-
906; F-499 to D-906; V-500 to D-906; F-501 to D-906; E-502 to D-906; T-503 toD-
906; L-504
to D-906; C-505 to D-906; S-506 to D-906; V-507 to D-906; N-508 to D-906; C-
509 to D-
906;E-510 to D-906; L-511 to D-906; Y-512 to D-906; F-513 to D-906; M-514 to D-
906; V-
S 15 to D-906; G-516 toD-906; V-517 to D-906; N-518 to D-906; S-519 to D-906;
R-520 to D-
906; T-521 to D-906; N-522 to D-906;T-523 to D-906; P-524 to D-906; V-525 to D-
906; E-
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
131
526 to D-906; T-527 to D-906; W-528 to D-906; K-529 toD-906; G-530 to D-906; S-
531 to D-
906; K-532 to D-906; G-533 to D-906; K-534 to D-906; Q-535 to D-906;S-536 to D-
906; Y-
537 to D-906; T-538 to D-906; Y-539 to D-906; I-540 to D-906; I-541 to D-906;
E-542 toD-
906; E-543 to D-906; N-544 to D-906; T-545 to D-906; T-546 to D-906; T-547 to
D-906; S-
548 to D-906;F-549 to D-906; T-550 to D-906; W-551 to D-906; A-552 to D-906; F-
553 to D-
906; Q-554 to D-906; R-555 toD-906; T-556 to D-906; T-557 to D-906; F-558 to D-
906; H-
559 to D-906; E-560 to D-906; A-561 to D-906;S-562 to D-906; R-563 to D-906; K-
564 to D-
906; Y-565 to D-906; T-566 to D-906; N-567 to D-906; D-568 toD-906; V-569 to D-
906; A-
570 to D-906; K-571 to D-906; I-572 to D-906; Y-573 to D-906; S-574 to D-906;
I-575to D-
906; N-576 to D-906; V-577 to D-906; T-578 to D-906; N-579 to D-906; V-580 to
D-906; M-
581 to D-906;N-582 to D-906; G-583 to D-906; V-584 to D-906; A-585 to D-906; S-
586 to D-
906; Y-587 to D-906; C-588 toD-906; R-589 to D-906; P-590 to D-906; C-591 to D-
906; A-
592 to D-906; L-593 to D-906; E-594 to D-906;A-595 to D-906; S-596 to D-906; D-
597 to D-
906; V-598 to D-906; G-599 to D-906; S-600 to D-906; S-601 toD-906; C-602 to D-
906; T-
603 to D-906; S-604 to D-906; C-605 to D-906; P-606 to D-906; A-607 to D-906;6-
608 to D-
906; Y-609 to D-906; Y-610 to D-906; I-611 to D-906; D-612 to D-906; R-613 to
D-906; D-
614 toD-906; S-615 to D-906; G-616 to D-906; T-617 to D-906; C-618 to D-906; H-
619 to D-
906; S-620 to D-906;C-621 to D-906; P-622 to D-906; P-623 to D-906; N-624 to D-
906; T-
625 to D-906; I-626 to D-906; L-627 toD-906; K-628 to D-906; A-629 to D-906; H-
630 to D-
906; Q-631 to D-906; P-632 to D-906; Y-633 to D-906;6-634 to D-906; V-635 to D-
906; Q-
636 to D-906; A-637 to D-906; C-638 to D-906; V-639 to D-906; P-640 toD-906; C-
641 to D-
906; G-642 to D-906; P-643 to D-906; G-644 to D-906; T-645 to D-906; K-646 to
D-906;N-
647 to D-906; N-648 to D-906; K-649 to D-906; I-650 to D-906; H-651 to D-906;
S-652 to D-
906; L-653 toD-906; C-654 to D-906; Y-655 to D-906; N-656 to D-906; D-657 to D-
906; C-
658 to D-906; T-659 to D-906;F-660 to D-906; S-661 to D-906; R-662 to D-906; N-
663 to D-
906; T-664 to D-906; P-665 to D-906; T-666 toD-906; R-667 to D-906; T-668 to D-
906; F-669
to D-906; N-670 to D-906; Y-671 to D-906; N-672 to D-906;F-673 to D-906; S-674
to D-906;
A-675 to D-906; L-676 to D-906; A-677 to D-906; N-678 to D-906; T-679 toD-906;
V-680 to
D-906; T-681 to D-906; L-682 to D-906; A-683 to D-906; G-684 to D-906; G-685
to D-906;P-
686 to D-906; S-687 to D-906; F-688 to D-906; T-689 to D-906; S-690 to D-906;
K-691 to D-
906; G-692 toD-906; L-693 to D-906; K-694 to D-906; Y-695 to D-906; F-696 to D-
906; H-
697 to D-906; H-698 to D-906;F-699 to D-906; T-700 to D-906; L-701 to D-906; S-
702 to D-
906; L-703 to D-906; C-704 to D-906; G-705 toD-906; N-706 to D-906; Q-707 to D-
906; 6-
708 to D-906; R-709 to D-906; K-710 to D-906; M-711 to D-906;S-712 to D-906; V-
713 to D-
906; C-714 to D-906; T-715 to D-906; D-716 to D-906; N-717 to D-906; V-718 toD-
906; T-
719 to D-906; D-720 to D-906; L-721 to D-906; R-722 to D-906; I-723 to D-906;
P-724 to D-
906; E-725to D-906; G-726 to D-906; E-727 to D-906; S-728 to D-906; G-729 to D-
906; F-
730 to D-906; S-731 to D-906;x-732 to D-906; S-733 to D-906; I-734 to D-906; T-
735 to D-
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
132
906; A-736 to D-906; Y-737 to D-906; V-738 toD-906; C-739 to D-906; Q-740 to D-
906; A-
741 to D-906; V-742 to D-906; I-743 to D-906; I-744 to D-906; P-745to D-906; P-
746 to D-
906; E-747 to D-906; V-748 to D-906; T-749 to D-906; G-750 to D-906; Y-751 to
D-906;K-
752 to D-906; A-753 to D-906; G-754 to D-906; V-755 to D-906; S-756 to D-906;
S-757 to D-
906; Q-758 toD-906; P-759 to D-906; V-760 to D-906; S-761 to D-906; L-762 to D-
906; A-
763 to D-906; D-764 to D-906;8-765 to D-906; L-766 to D-906; I-767 to D-906; G-
768 to D-
906; V-769 to D-906; T=770 to D-906; T-771 toD-906; D-772 to D-906; M-773 to D-
906; T-
774 to D-906; L-775 to D-906; D-776 to D-906; G-777 to D-906;I-778 to D-906; T-
779 to D-
906; S-780 to D-906; P-781 to D-906; A-782 to D-906; E-783 to D-906; L-784 toD-
906; F-785
to to D-906; H-786 to D-906; L-787 to D-906; E-788 to D-906; S-789 to D-906; L-
790 to D-
906;6-791 to D-906; I-792 to D-906; P-793 to D-906; D-794 to D-906; V-795 to D-
906; I-796
to D-906; F-797 toD-906; F-798 to D-906; Y-799 to D-906; R-800 to D-906; S-801
to D-906;
N-802 to D-906; D-803 to D-906;V-804 to D-906; T-805 to D-906; Q-806 to D-906;
S-807 to
D-906; C-808 to D-906; S-809 to D-906; S-810 toD-906; G-811 to D-906; R-812 to
D-906; S-
t5 813 to D-906; T-814 to D-906; T-815 to D-906; I-816 to D-906; R-817to D-
906; V-818 to D-
906; R-819 to D-906; C-820 to D-906; S-821 to D-906; P-822 to D-906; Q-823 to
D-906;K-
824 to D-906; T-825 to D-906; V-826 to D-906; P-827 to D-906; G-828 to D-906;
S-829 to D-
906; L-830 toD-906; L-831 to D-906; L-832 to D-906; P-833 to D-906; G-834 to D-
906; T-
835 to D-906; C-836 to D-906;S-837 to D-906; D-838 to D-906; G-839 to D-906; T-
840 to D-
20 906; C-841 to D-906; D-842 to D-906; G-843 toD-906; C-844 to D-906; N-845
to D-906; F-
846 to D-906; H-847 to D-906; F-848 to D-906; L-849 to D-906;W-850 to D-906; E-
851 to D-
906; S-852 to D-906; A-853 to D-906; A-854 to D-906; A-855 to D-906; C-856 toD-
906; P-
857 to D-906; L-858 to D-906; C-859 to D-906; S-860 to D-906; V-861 to D-906;
A-862 to D-
906;D-863 to D-906; Y-864 to D-906; H-865 to D-906; A-866 to D-906; I-867 to D-
906; V-
25 868 to D-906; S-869 toD-906; S-870 to D-906; C-871 to D-906; V-872 to D-
906; A-873 to D-
906; G-874 to D-906; I-875 to D-906;Q-876 to D-906; K-877 to D-906; T-878 to D-
906; T-
879 to D-906; Y-880 to D-906; V-881 to D-906; W-882 toD-906; R-883 to D-906; E-
884 to D-
906; P-885 to D-906; K-886 to D-906; L-887 to D-906; C-888 to D-906;5-889 to D-
906; G-
890 to D-906; G-891 to D-906; I-892 to D-906; S-893 to D-906; L-894 to D-906;
P-895 toD-
30 906; E-896 to D-906; Q-897 to D-906; R-898 to D-906; V-899 to D-906; and T-
900 to D-906
of SEQ ID N0:40. Polynucleotides encoding these polypeptides are also
encompassed by the
mvent~on.
The present invention further provides polypeptides having one or more
residues
deleted from the carboxy terminus of the predicted extracellular domain of the
predicted mature
35 TR13 protein, with the amino acid sequence shown in Figures 7A-D (SEQ ID
N0:40), up to
the alanine residue at position number 48, and polynucleotides encoding such
polypeptides. In
particular, the present invention provides polypeptides comprising, or
alternatively consisting
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
133
of, the amino acid sequence of residues 42-m' of Figures 7A-D, where m' is an
integer from
48 to 906 corresponding to the position of the amino acid residue in Figures
7A-D.
Thus, the invention provides TR13 polypeptides comprising, or alternatively
consisting
of, the amino acid sequence of residues: T-42 to D-906; T-42 to I-905; T-42 to
T-904; T-42 to
K-903; T-42 to C-902; T-42 to I-901sT-42 to T-900; T-42 to V-899; T-42 to R-
898; T-42 to Q
897; T-42 to E-896; T-42 to P-895; T-42 to L-894; T-42 toS-893; T-42 to I-892;
T-42 to 6-
891; T-42 to G-890; T-42 to S-889; T-42 to C-888; T-42 to L-887; T-42 to K-
886;T-42 to P-
885; T-42 to E-884; T-42 to R-883; T-42 to W-882; T-42 to V-881; T-42 to Y-
880; T-42 to T-
879; T-42 toT-878; T-42 to K-877; T-42 to Q-876; T-42 to I-875; T-42 to G-874;
T-42 to A-
t0 873; T-42 to V-872; T-42 to C-871sT-42 to S-870; T-42 to S-869; T-42 to V-
868; T-42 to I-
867; T-42 to A-866; T-42 to H-865; T-42 to Y-864; T-42 toD-863; T-42 to A-862;
T-42 to V-
861; T-42 to S-860; T-42 to C-859; T-42 to L-858; T-42 to P-857; T-42 to C-
856;T-42 to A-
855; T-42 to A-854; T-42 to A-853; T-42 to S-852; T-42 to E-851; T-42 to W-
850; T-42 to L-
849; T-42 toF-848; T-42 to H-847; T-42 to F-846; T-42 to N-845; T-42 to C-844;
T-42 to G-
843; T-4.2 to D-842; T-42 to C-841sT-42 to T-840; T-42 to G-839; T-42 to D-
838; T-42 to S-
837; T-42 to C-836; T-42 to T-835; T-42 to G-834; T-42 toP-833; T-42 to L-832;
T-42 to L-
831; T-42 to L-830; T-42 to S-829; T-42 to G-828; T-42 to P-827; T-42 to V-
826;T-42 to T-
825; T-42 to K-824; T-42 to Q-823; T-42 to P-822; T-42 to S-821; T-42 to C-
820; T-42 to 8-
819; T-42 toV-818; T-42 to R-817; T-42 to I-816; T-42 to T-815; T-42 to T-814;
T-42 to S-
813; T-42 to R-812; T-42 to G-811;T-42 to S-810; T-42 to S-809; T-42 to C-808;
T-42 to S-
807; T-42 to Q-806; T-42 to T-805; T-42 to V-804; T-42 toD-803; T-42 to N-802;
T-42 to S-
801; T-42 to R-800; T-42 to Y-799; T-42 to F-798; T-42 to F-797; T-42 to I-
796;T-42 to V-
795; T-42 to D-794; T-42 to P-793; T-42 to I-792; T-42 to G-791; T-42 to L-
790; T-42 to S-
789; T-42 toE-788; T-42 to L-787; T-42 to H-786; T-42 to F-785; T-42 to L-784;
T-42 to E-
783; T-42 to A-782; T-42 to P-781sT-42 to S-780; T-42 to T-779; T-42 to I-778;
T-42 to 6-
777; T-42 to D-776; T-42 to L-775; T-42 to T-774; T-42 toM-773; T-42 to D-772;
T-42 to T-
771; T-42 to T-770; T-42 to V-769; T-42 to G-768; T-42 to I-767; T-42 to L-
766;T-42 to R-
765; T-42 to D-764; T-42 to A-763; T-42 to L-762; T-42 to S-761; T-42 to V-
760; T-42 to P-
759; T-42 toQ-758; T-42 to S-757; T-42 to S-756; T-42 to V-755; T-42 to G-754;
T-42 to A-
753; T-42 to K-752; T-42 to Y-751sT-42 to G-750; T-42 to T-749; T-42 to V-748;
T-42 to ~
747; T-4.2 to P-746; T-42 to P-745; T-42 to I-744; T-42 toI-743; T-42 to V-
742; T-42 to A-
741; T-42 to Q-740; T-42 to C-739; T-42 to V-738; T-42 to Y-737; T-42 to A-
736;T-42 to T-
735; T-42 to I-734; T-42 to S-733; T-42 to K-732; T-42 to S-731; T-42 to F-
730; T-42 to 6-
729; T-42 toS-728; T-42 to E-727; T-42 to G-726; T-42 to E-725; T-42 to P-724;
T-42 to I-
723; T-42 to R-722; T-42 to L-721sT-42 to D-720; T-42 to T-719; T-42 to V-718;
T-42 to N-
717; T-42 to D-716; T-42 to T-715; T-42 to C-714; T-42 toV-713; T-42 to S-712;
T-42 to M-
711; T-42 to K-710; T-42 to R-709; T-42 to G-708; T-42 to Q-707; T-42 toN-706;
T-42 to 6-
705; T-42 to C-704; T-42 to L-703; T-42 to S-702; T-42 to L-701; T-42 to T-
700; T-42 to F-
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
134
699;T-42 to T-4298; T-42 to T-4297; T-42 to F-696; T-42 to Y-695; T-42 to K-
694; T-42 to L-
693; T-42 to G-692; T-42 toK-691; T-42 to S-690; T-42 to T-689; T-42 to F-688;
T-42 to S-
687; T-42 to P-686; T-42 to G-685; T-42 to G-684;T-42 to A-683; T-42 to L-682;
T-42 to T-
681; T-42 to V-680; T-42 to T-679; T-42 to N-678; T-42 to A-677; T-42 toL-676;
T-42 to A-
675; T-42 to S-674; T-42 to F-673; T-42 to N-672; T-42 to Y-671; T-42 to N-
670; T-42 to F-
669;T-42 to T-668; T-42 to R-667; T-42 to T-666; T-42 to P-665; T-42 to T-664;
T-42 to N-
663; T-42 to R-662; T-42 toS-661; T-42 to F-660; T-42 to T-659; T-42 to C-658;
T-42 to D-
657; T-42 to N-656; T-42 to Y-655; T-42 to C-654;T-42 to L-653; T-42 to S-652;
T-42 to T-
4251; T-42 to I-650; T-42 to K-649; T-42 to N-648; T-42 to N-647; T-42 toK-
646; T-42 to T-
645; T-42 to G-644; T-42 to P-643; T-42 to G-642; T-42 to C-641; T-42 to P-
640; T-42 to V-
639;T-42 to C-638; T-42 to A-637; T-42 to Q-636; T-42 to V-635; T-42 to G-634;
T-42 to Y-
633; T-42 to P-632; T-42 toQ-631; T-42 to T-4230; T-42 to A-629; T-42 to K-
628; T-42 to L-
627; T-42 to I-626; T-42 to T-625; T-42 to N-624;T-42 to P-623; T-42 to P-622;
T-42 to C-
621; T-42 to S-620; T-42 to T-4219; T-42 to C-618; T-42 to T-617; T-42 toG-
616; T-42 to S-
615; T-42 to D-614; T-42 to R-613; T-42 to D-612; T-42 to I-611; T-42 to Y-
610; T-42 to Y-
609;T-42 to G-608; T-42 to A-607; T-42 to P-606; T-42 to C-605; T-42 to S-604;
T-42 to T-
603; T-42 to C-602; T-42 toS-601; T-42 to S-600; T-42 to G-599; T-42 to V-598;
T-42 to D-
597; T-42 to S-596; T-42 to A-595; T-42 to E-594;T-42 to L-593; T-42 to A-592;
T-42 to C-
591; T-42 to P-590; T-42 to R-589; T-42 to C-588; T-42 to Y-587; T-42 toS-586;
T-42 to A-
585; T-42 to V-584; T-42 to G-583; T-42 to N-582; T-42 to M-581; T-42 to V-
580; T-42 toN-
579; T-42 to T-578; T-42 to V-577; T-42 to N-576; T-42 to I-575; T-42 to S-
574; T-42 to Y-
573; T-42 to I-572;T~2 to K-571; T-42 to A-570; T-42 to V-569; T-42 to D-568;
T-42 to N-
567; T-42 to T-566; T-42 to Y-565; T-42 toK-564; T-42 to R-563; T-42 to S-562;
T-42 to A-
561; T-42 to E-560; T-42 to H-559; T-42 to F-558; T-42 to T-557;T-4.2 to T-
556; T-42 to R-
555; T-42 to Q-554; T-42 to F-553; T-42 to A-552; T-42 to W-551; T-42 to T-
550; T-42 toF-
549; T-42 to S-548; T-42 to T-547; T-42 to T-546; T-42 to T-545; T-42 to N-
544; T-42 to E-
543; T-42 to E-542;T-42 to I-541; T-42 to I-540; T-42 to Y-539; T-42 to T-538;
T-42 to Y-
537; T-42 to S-536; T-42 to Q-535; T-42 toK-534; T-42 to G-533; T-42 to K-532;
T-42 to S-
531; T-42 to G-530; T-42 to K-529; T-42 to W-528; T-42 toT-527; T-42 to E-526;
T-42 to V-
525; T-42 to P-524; T-42 to T-523; T-42 to N-522; T-42 to T-521; T-42 to R-
520;T-42 to S-
S 19; T-42 to N-518; T-42 to V-517; T-42 to G-516; T-42 to V-S 15; T-42 to M-
514; T-42 to F-
513; T-42 toY-512; T-42 to L-511; T-42 to E-510; T-42 to C-509; T-42 to N-508;
T-42 to V-
507; T-42 to S-506; T-42 to C-SOS;T-42 to L-504; T-42 to T-503; T-42 to E-502;
T-42 to F-
501; T-42 to V-500; T-42 to F-499; T-42 to T-498; T-42 toI-497; T-42 to R-496;
T-42 to A-
495; T-42 to V-494; T-42 to E-493; T-42 to K-492; T-42 to N-491; T-42 to E-
490;T-42 to T-
489; T-42 to D-488; T-42 to A-487; T-42 to M-486; T-42 to V-485; T-42 to S-
484; T-42 to Q-
483; T-42 toP-482; T-42 to P-481; T-42 to R-480; T-42 to F-479; T-42 to G-478;
T-42 to P-
477; T-42 to V-476; T-42 to V-475;T-42 to L-474; T-42 to T-473; T-42 to L-472;
T-42 to I-
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
135
471; T-42 to M-470; T-42 to F-469; T-42 to D-468; T-42 toN-467; T-42 to D-466;
T-42 to S-
465; T-42 to A-464; T-42 to G-463; T-42 to A-462; T-42 to A-461; T~2 to T-
460;T-42 to Y-
459; T-42 to I-458; T-42 to H-457; T-42 to D-456; T-42 to G-455; T-42 to A-
454; T-42 to V-
453; T-42 toE-452; T-42 to W-451; T-42 to G-450; T-42 to T-449; T-42 to M~48;
T-42 to G-
447; T-42 to K-446; T-42 toY~45; T-42 to E-444; T-42 to F-443; T-42 to N-442;
T-42 to I-
441; T-42 to G-440; T-42 to S-439; T-42 to L-438;T-42 to V-437; T-42 to T-436;
T-42 to T-
435; T-42 to E-434; T-42 to M-433; T-42 to N-432; T-42 to T-431; T-42 toP-430;
T-42 to L-
429; T-42 to T-428; T-42 to N-427; T-42 to W-426; T-42 to W-425; T-42 to K-
424; T-42 toY-
423; T-42 to E-422; T-42 to F-421; T-42 to G-420; T-42 to V-419; T-42 to A-
4.18; T-42 to P-
417; T-42 to E-416;T-42 to T-415; T-42 to G-414; T-42 to A-413; T-42 to P-412;
T-42 to C-
411; T-42 to R-410; T-42 to T-409; T-42 toC-408; T-42 to D-407; T-42 to S-406;
T-42 to G-
405; T-42 to N-404; T-42 to S-403; T-42 to Y-402; T-42 to S-4.O1;T-42 to G-
400; T-42 to Y-
399; T-42 to P-398; T-42 to C-397; T-42 to P-396; T-42 to Q-395; T-42 to C-
394; T-42 toT-
393; T-42 to S-392; T-42 to N-391; T-42 to N-390; T-42 to T-389; T-42 to K-
388; T-42 to F-
387; T-42 to F-386;T-42 to G-385; T-42 to P-384; T-42 to N-383; T-42 to C-382;
T-42 to P-
381; T-42 to P-380; T-42 to C-379; T-42 toH-378; T-42 to T-377; T-42 to K-376;
T-42 to V-
375; T-42 to G-374; T-42 to S-373; T-42 to A-372; T-42 to P-371sT-42 to L-370;
T-42 to K-
369; T-42 to V-368; T-42 to A-367; T-42 to G-366; T-42 to E-365; T-42 to L-
364; T-42 toD-
363; T-42 to E-362; T-42 to S-361; T-42 to C-360; T-42 to I-359; T-42 to K-
358; T-42 to P-
357; T-42 to K-356;T-42 to A-355; T-42 to W-354; T-42 to K-353; T-42 to Y-352;
T-42 to M-
351; T-42 to L-350; T-42 to Q-349; T-42 toT-348; T-42 to E-347; T-42 to G-34b;
T-42 to N-
345; T-42 to A-344; T-42 to D-343; T-42 to C-342; T-42 to A-341sT-42 to T-340;
T-42 to H-
339; T-42 to T-338; T-42 to Y-337; T-42 to F-336; T-42 to Y-335; T-42 to D-
334; T-42 toK-
333; T-42 to D-332; T-42 to T-331; T-42 to C-330; T-42 to A-329; T-42 to P-
328; T-42 to R-
327; T-42 to V-326;T-42 to N-325; T-42 to C-324; T-42 to S-323; T-42 to S-322;
T-42 to S-
321; T-42 to G-320; T-42 to K-319; T-42 toE-318; T-42 to S-317; T-42 to Y-316;
T-42 to K-
315; T-42 to D-314; T-42 to P-313; T-42 to D-312; T-42 to C-311;T-42 to Q-310;
T-42 to H-
309; T-42 to C-308; T-42 to S-307; T-42 to T-306; T-42 to E-305; T-42 to G-
304; T-42 toK-
303; T-42 to N-302; T-42 to S-301; T-42 to Y-300; T-42 to S-299; T-42 to N-
298; T-42 to A-
297; T-42 to P-296;T-42 to C-295; T-42 to L-294; T-42 to K-293; T-42 to C-292;
T-42 to F-
291; T-42 to S-290; T-42 to S-289; T-42 toG-288; T-42 to Q-287; T-42 to K-286;
T-42 to D-
285; T-42 to A-284; T-42 to Y-283; T-42 to T-282; T-42 to G-281sT-42 to P-280;
T-42 to K-
279; T-42 to C-278; T-42 to P-277; T-42 to F-276; T-42 to C-275; T-42 to E-
274; T-42 toS-
273; T-42 to T-272; T-42 to Y-271; T-42 to A-270; T-42 to V-269; T-42 to G-
268; T-42 to T-
267; T-42 to I-266;T-42 to A-265; T-42 to I-264; T-42 to N-263; T-42 to R-262;
T-42 to V-
261; T-42 to L-260; T-42 to V-259; T-42 toP-258; T-42 to K-257; T-42 to P-256;
T-42 to V-
255; T-42 to K-254; T-42 to T-253; T-42 to W-252; T-42 to V-251sT-42 to S-250;
T-42 to F-
249; T-42 to A-248; T-42 to T-247; T-42 to T-24b; T-4.2 to R-245; T-42 to W-
244; T-42 toY-
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
136
243; T-42 to L-242; T-42 to V-241; T-42 to N-240; T-42 to N-239; T-42 to G-
238; T-42 to 8-
237; T-42 to N-236;T-42 to L-235; T-42 to E-234; T-42 to V-233; T-42 to S-232;
T-42 to H-
231; T-42 to F-230; T-42 to E-229; T-42 toW-228; T-42 to G-227; T-42 to K-226;
T-42 to E-
225; T-42 to T-224; T-42 to T-223 ; T-42 to K-222; T-42 toM-221; T-42 to W-
220; T-42 to R-
219; T-42 to S-218; T-42 to D-217; T-42 to D-216; T-4.2 to A-215; T-42 toN-
214; T-42 to P-
213; T-42 to Q-212; T-42 to C-211; T-42 to Q-210; T-42 to D-209; T-42 to N-
208; T-42 to Q-
207;T-42 to V-206; T-42 to F-205; T-42 to F-204; T-42 to E-203; T-42 to F-202;
T-42 to I-
201; T-42 to I-200; T-42 toS-199; T-42 to S-198; T-42 to D-197; T-42 to P-196;
T-42 to Y-
195; T-42 to Y-194; T-42 to Y-193; T-42 to E-192;T-42 to F-191; T-42 to N-190;
T-42 to V-
189; T-42 to T-188; T-42 to G-187; T-42 to S-186; T-42 to Q-185; T-42 toK-184;
T-42 to L-
183; T-42 to N-182; T-42 to V-181; T-42 to A-180; T-42 to Y-179; T-42 to M-
178; T-42 to L-
177;T-42 to T-176; T-42 to A-175; T-42 to T-174; T-42 to C-173; T-42 to E-172;
T-42 to D-
171; T-42 to T-170; T-42 toN-169; T-42 to F-168; T-42 to A-167; T-42 to I-166;
T-42 to Y-
165 ; T-42 to D-164; T-42 to G-163 ; T-42 to R-162;T-42 to P-161; T-42 to V -
160; T-42 to W-
159; T-42 to K-158; T-42 to S-157; T-42 to S-156; T-42 to T-155; T-42 toC-154;
T-42 to N-
153; T-42 to G-152; T-42 to T-151; T-42 to S-150; T-42 to E-149; T-42 to A-
148; T-42 to A-
147;T-42 to S-146; T-42 to D-145; T-42 to D-144; T-42 to L-143; T-42 to E-142;
T-42 to M-
141; T-42 to N-140; T-42 toA-139; T-42 to S-138; T-42 to L-137; T-42 to S-136;
T-42 to A-
135 ; T-42 to F-134; T-42 to G-13 3 ; T-42 to H-132;T-42 to P-131; T-42 to L-
130; T-42 to E-
129; T-42 to D-128; T-42 to W-127; T-42 to E-126; T-42 to D-125; T-42 toF-124;
T-42 to 8-
123; T-42 to I-122; T-42 to G-121; T-42 to T-120; T-42 to G-119; T-42 to L-
118; T-42 to S-
117;T-42 to Y-116; T-42 to R-115; T-42 to G-114; T-42 to E-113; T-42 to A-112;
T-42 to C-
111; T-42 to P-110; T-42 toK-109; T-42 to C-108; T-42 to S-107; T-42 to Q-106;
T-42 to D-
105; T-42 to K-104; T-42 to M-103 ; T-42 to D-102;T-42 to L-1 O 1; T-42 to F-
100; T-42 to E-
99; T-42 to G-98; T-42 to A-97; T-42 to N-96; T-42 to C-95; T-42 to S-94;T-42
to F-93; T-42
to S-92; T-42 to C-91; T-42 to E-90; T-42 to T-89; T-42 to G-88; T-42 to K-87;
T-42 to V-
86;T-42 to P-85; T-42 to D-84; T-42 to P-83; T-42 to L-82; T-42 to S-81; T-42
to T-80; T-42
to C-79; T-42 to L-78; T-42to G-77; T-42 to P-76; T-42 to T-75; T-42 to H-74;
T-42 to P-73;
T-42 to V-72; T-42 to A-71; T-42 to V-70; T-42 toR-69; T-42 to W-68; T-42 to R-
67; T-42 to
S-66; T-42 to G-65; T-42 to T-64; T-42 to S-63; T-42 to D-62; T-42 toC-61; T-
42 to A-60; T-
42 to T-59; T-42 to Y-58; T-42 to E-57; T-42 to Y-56; T-4.2 to H-55; T-42 to Y-
54; T-42 toE-
53; T-42 to S-52; T-42 to E-51; T-42 to K-50; T-42 to C-49; and T-42 to A-48
of SEQ ID
N0:40. Polynucleotides encoding these polypeptides are also encompassed by the
invention.
The invention also provides polypeptides having one or more amino acids
deleted from
both the amino and the carboxyl termini, which may be described generally as
having residues
n'- m' and/or n2- m' of Figures lA-C (i.e., SEQ ID N0:2), where n', n2, and m'
are integers
as described above. Thus, any of the above listed N- or C-terminal deletions
can be combined
to produce an N- and C-terminal deleted TR13 polypeptide.
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
137
The invention also provides polypeptides having one or more amino acids
deleted from
both the amino and the carboxyl termini, which may be described generally as
having residues
n'- m' and/or n2- m' of Figures 7A-D (SEQ ID N0:40), where n', n2, and m' are
integers as
described above. Thus, any of the above listed N- or C-terminal deletions can
be combined to
produce an N- and C-terminal deleted TR13 polypeptide.
It will be recognized in the art that some amino acid sequences of TR13
polypeptides
can be varied without significant effect on the structure or function of the
protein. If such
differences in sequence are contemplated, it should be remembered that there
will be critical
areas on the protein which determine activity. Thus, the invention further
includes variations of
to the TR13 polypeptide, which show substantial TR13 activity or which include
regions of
TR13 polypeptides, such as the polypeptide portions discussed herein. Such
mutants include
deletions, insertions, inversions, repeats, and type substitutions. As
indicated above, guidance
concerning which amino acid changes are likely to be phenotypically silent can
be found in
J.U. Bowie et al., Science 247:1306-1310 (1990).
Thus, the fragment, derivative, or analog of the polypeptide of SEQ ID N0:2 or
SEQ
ID N0:40, or that encoded by the cDNA deposited as ATCC Deposit No. PTA-349 or
ATCC
Deposit No. PTA-507, 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
2o residues) and such substituted amino acid residue may or may not be one
encoded by the
genetic code, or (ii) one in which one or more of the amino acid residues
includes a substituent
group, or (iii) one in which the mature polypeptide is fused with another
compound, such as a
compound to increase the half-life of the polypeptide (for example,
polyethylene glycol), or (iv)
one in which the additional amino acids are fused to the mature polypeptide,
such as an IgG Fc
fusion region peptide or leader or secretory sequence or a sequence which is
employed for
purification of the mature polypeptide or a proprotein sequence. Such
fragments, derivatives
and analogs are deemed to be within the scope of those skilled in the art from
the teachings
herein.
Of particular interest are substitutions of charged amino acids with another
charged
3o 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 TR13
polypeptide. The
prevention of aggregation is highly desirable. Aggregation of proteins not
only results in a loss
of activity but can also be problematic when preparing pharmaceutical
formulations, because
they can be immunogenic. (Pinckard et al., Clin Exp. Immunol. 2:331-340
(1967); Robbins et
al., Diabetes 36:838-845 (1987); Cleland et al. Crit. Rev. Therapeutic Drug
Carrier Systems
10:307-377 (1993)).
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
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
138
selective binding of TNF-a to only one of the two known types of TNF
receptors. Thus, the
TR13 polypeptides of the present invention may include one or more amino acid
substitutions,
deletions, or additions, either from natural mutations or human manipulation.
As indicated, changes are preferably of a minor nature, such as conservative
amino acid
substitutions that do not significantly affect the folding or activity of the
protein (see Table V).
TABLE V. 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
Seri ne
Threonine
Methionine
G1 cine
In specific embodiments, the number of substitutions, additions or deletions
in the
amino acid sequence of Figures lA-C or Figures 7A-D and/or any of the
polypeptide fragments
described herein (e.g., one or more of the cysteine rich domains, the mature
extracellular
domain, etc.) is 75, 70, 60, 50, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, S, 4,
3, 2, 1 or 30-20,
20-15, 20-10, 15-10, 10-1, 5-10, 1-5, 1-3 or 1-2.
Amino acids in the TR13 polypeptides 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
2o 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
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
139
photoaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904 (1992) and de
Vos et al. Science
255:306-312 (1992)).
To improve or alter the characteristics of TR13 polypeptides, protein
engineering may
be employed. Recombinant DNA technology known to those skilled in the art can
be used to
create novel mutant proteins or "muteins including single or multiple amino
acid substitutions,
deletions, additions or fusion proteins. Such modified polypeptides can show,
e.g., enhanced
activity or increased stability. In addition, they may be purified in higher
yields and show
better solubility than the corresponding natural polypeptide, at least under
certain purification
and storage conditions.
Non-naturally occurring variants may be produced using art-known mutagenesis
techniques, which include, but are not limited to oligonucleotide mediated
mutagenesis, alanine
scanning, PCR mutagenesis, site directed mutagenesis (see e.g., Carter et al.,
Nucl. Acids
Res. 13:4331 ( 1986); and Zoller et al., Nucl. Acids Res. 10:6487 ( 1982)),
cassette
mutagenesis (see e.g., Wells et al., Gene 34:315 (1985)), restriction
selection mutagenesis
(see e.g., Wells et al., Philos. Traps. R. Soc. London SerA 317:415 (1986)).
Thus, the invention also encompasses TR13 derivatives and analogs that have
one or
more amino acid residues deleted, added, or substituted to generate TR13
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
2o 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 TR13 polypeptides of
the invention,
and/or an amino acid deletion at the second position of any one or more such
recognition
sequences will prevent glycosylation of the TR13 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 the polypeptides of the invention (e.g., arginine and lysine
residues) may be deleted
or substituted with another residue to eliminate undesired processing by
proteases such as, for
3o example, furins or kexins.
The polypeptides of the present invention include a polypeptide comprising, or
alternatively, consisting of the polypeptide encoded by the cDNA deposited as
ATCC Deposit
No. PTA-349, including the leader; a polypeptide comprising, or alternatively,
consisting of
the mature polypeptide encoded by the deposited cDNA minus the leader (i.e.,
the mature
protein); a polypeptide comprising, or alternatively, consisting of amino
acids from about 1 to
about 750 of SEQ ID N0:2; a polypeptide comprising, or alternatively,
consisting of amino
acids from about 2 to about 750 of SEQ ID N0:2; a polypeptide comprising, or
alternatively,
consisting of amino acids from about 1 to about 331 in SEQ ID N0:2; a
polypeptide
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
140
comprising, or alternatively, consisting of any one or more of the four
cysteine rich domains
disclosed in Figures lA-C (predicted to constitute amino acids from about 105
to about 170,
251 to about 265, about 331 to about 410, and about 580 to about 610 of SEQ ID
N0:2); as
well as polypeptides which are at least 80% identical, more preferably at
least 90% or 95%
identical, still more preferably at least 96%, 97%, 98%, or 99% identical to
the polypeptides
described above (e.g., the polypeptide encoded by the deposited cDNA clone,
the polypeptide
of Figures lA-C (SEQ ID N0:2) and polypeptide fragments thereof such as
disclosed herein),
and also include portions of such polypeptides with at least 30 amino acids
and more preferably
at least 50 amino acids. 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.
Polynucleotides encoding these polypeptides are also encompassed by the
invention.
The polypeptides of the present invention include a polypeptide comprising, or
alternatively, consisting of the polypeptide encoded by the cDNA deposited as
ATCC Deposit
No. PTA-507, including the leader; a polypeptide comprising, or alternatively,
consisting of
the mature polypeptide encoded by the deposited cDNA minus the leader (i.e.,
the mature
protein); a polypeptide comprising, or alternatively, consisting of amino
acids from about 1 to
about 1001 of SEQ ID N0:40; a polypeptide comprising, or alternatively,
consisting of amino
acids from about 2 to about 1001 of SEQ ID N0:40; a polypeptide comprising, or
alternatively,
consisting of amino acids from about 1 to about 906 in SEQ ID N0:40; a
polypeptide
2o comprising, or alternatively, consisting of amino acids from about 42 to
about 1001 in SEQ ID
N0:40; a polypeptide comprising, or alternatively, consisting of amino acids
from about 42 to
about 906 in SEQ ID N0:40; a polypeptide comprising, or alternatively,
consisting of amino
acids from about 907 to about 931 in SEQ ID N0:40; a polypeptide comprising,
or
alternatively, consisting of amino acids from about 932 to about 1001 in SEQ
ID N0:40; a
polypeptide comprising, or alternatively, consisting of any of the seven
cysteine rich domains
disclosed in Figures 7A-D (predicted to constitute amino acids from about 271
to about 421,
271 to about 286, about 290 to about 300, about 301 to about 320, about 329 to
about 361,
about 404 to about 421, and about 585 to about 595 of SEQ ID N0:40); as well
as
polypeptides which are at least 80% identical, more preferably at least 90% or
95% identical,
3o 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
polypeptide of Figures
7A-D (SEQ ID N0:40) and polypeptide fragments thereof, such as those disclosed
herein), and
also include portions of such polypeptides with at least 30 amino acids and
more preferably at
least 50 amino acids. 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.
Polynucleotides encoding these polypeptides are also encompassed by the
invention.
By a polypeptide (protein) comprising, or alternatively, consisting of, an
amino acid
sequence at least, for example, 95% "identical" to a reference amino acid
sequence of a TR13
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
141
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 TR13
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
to positions, interspersed either individually among residues in the reference
sequence or in one or
more contiguous groups within the reference sequence.
As a practical matter, whether any particular polypeptide is at least 90%,
95%, 96%,
97%, 98%, or 99% identical to, for instance, the amino acid sequence shown in
SEQ ID N0:2
or SEQ ID N0:40, or to the amino acid sequence encoded by the cDNA clone
deposited as
~5 ATCC Deposit No. PTA-349 or ATCC Deposit No. PTA-507, 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%
20 identical to a reference sequence according to the present invention, the
parameters are set, of
course, such that the percentage of identity is calculated over the full-
length of the reference
amino acid sequence and that gaps in homology of up to 5% of the total number
of amino acid
residues in the reference sequence are allowed.
In a specific embodiment, the identity between a reference (query) sequence (a
sequence
25 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
30 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
35 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
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
142
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
to 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
~5 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
2o the N- and C-terminal ends of the subject sequence, as displayed in the
FASTDB alignment,
which are not matched/aligned with the query sequence are manually corrected
for. No other
manual corrections are made for the purposes of this embodiment.
The present application is also directed to proteins comprising, or
alternatively
consisting of, a polypeptide sequence at least 90%, 95%, 96%, 97%, 98% or 99%
identical to
25 the TR13 polypeptide sequence set forth as n'-m', and/or n2- ml for
polypeptide sequence
shown in Figure lA-C or Figure 7A-D herein. In preferred embodiments, the
application is
directed to proteins comprising, or alternatively consisting of, a polypeptide
sequence at least
90%, 95%, 96%, 97%, 98% or 99% identical to polypeptides having the amino acid
sequence
of the specific TR13 N- and C-terminal deletions recited herein. Additional
preferred
3o embodiments are directed to fusion proteins comprising these polypeptide
sequences.
Polynucleotides encoding these polypeptides are also encompassed by the
invention.
In another aspect, the invention provides a peptide or polypeptide comprising
an
epitope-bearing portion of a polypeptide of the invention. The epitope of this
polypeptide
portion is an immunogenic or antigenic epitope of a polypeptide described
herein. An
35 "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
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
143
epitopes of a protein generally is less than the number of antigenic epitopes.
See, for instance,
Geysen et al., Proc. Natl. Acad. Sci. USA 87:3998- 4002 (1983).
As to the selection of peptides or polypeptides bearing an antigenic epitope
(i.e., that
contain a region of a protein molecule to which an antibody can bind), it is
well known in that
art that relatively short synthetic peptides that mimic part of a protein
sequence are routinely
capable of eliciting an antiserum that reacts with the partially mimicked
protein. See, for
instance, J.G. Sutcliffe et al., "Antibodies That React With Predetermined
Sites on Proteins,"
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
1o chemical rules, and are confined neither to immunodominant regions of
intact proteins (i.e.,
immunogenic epitopes) nor to the amino or carboxyl terminals.
Antigenic epitope-bearing peptides and polypeptides of the invention are
therefore
useful, for example, to raise antibodies, including monoclonal antibodies,
that bind specifically
to a polypeptide of the invention. See, for instance, Wilson et al., Cell
37:767-778 (1984) at
777. Antigenic epitope-bearing peptides and polypeptides of the invention
preferably contain a
sequence of at least seven, more preferably at least 9, at least 20, at least
25, at least 30, at least
40, at least 50 and most preferably between at least about 55 to about 100
amino acids
contained within the amino acid sequence of a polypeptide of the invention. In
this context
"about" includes the particularly recited ranges, larger or smaller by several
(5, 4, 3, 2,or 1)
amino acids, at either extreme or at both extremes.
Non-limiting examples of predicted antigenic polypeptides that can be used to
generate
TR13 receptor-specific antibodies include: a polypeptide comprising, or
alternatively consisting
of, amino acid residues from about 1 to about 170 in Figures lA-C
(corresponding to about
amino acid 1 to about 170 in SEQ ID N0:2); a polypeptide comprising amino acid
residues
from about 210 to about 318 in Figures lA-C (corresponding to about amino acid
210 to about
318 in SEQ ID N0:2); a polypeptide comprising amino acid residues from about
343 to about
480 in Figures lA-C (corresponding to about amino acid 343 to about 480 in SEQ
ID N0:2); a
polypeptide comprising amino acid residues from about 548 to about 592 in
Figures lA-C
(corresponding to about amino acid 548 to about 592 in SEQ ID N0:2); and a
polypeptide
comprising amino acid residues from about 632 to about 742 in Figures lA-C
(corresponding
to about amino acid 632 to about 742 in SEQ ID N0:2). As indicated above, the
inventors have
determined that the above polypeptide fragments are antigenic regions of the
TR13 receptor
protein. 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.
Polynucleotides
encoding these polypeptides are also encompassed by the invention.
Additional non-limiting examples of predicted antigenic polypeptides that can
be used to
generate TR13 receptor-specific antibodies include: a polypeptide comprising,
or alternatively
consisting of, amino acid residues from about about M1 to about A9, about K12
to about L20,
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
144
about N47 to about T55, about H58 to about S66, about D63 to about S71, about
P77 to about
F85, about A90 to about Q98, about F136 to about Q144, about S152 to about
C160, about
8159 to about A167, about A211 to about M219, about M235 to about V243, about
V266 to
about V274, about W277 to about S285, about I290 to about F298, about A310 to
about
V318, about E343 to about C351, about I360 to about H368, about 6391 to about
I399, about
F409 to about T417, about S436 to about Y444, about C453 to about S461, about
I472 to
about 5480, about Y548 to about S556, about C557 to about I565, about V567 to
about V575,
about T584 to about 6592, about 8632 to about 6640, about W680 to about Y688,
about
Q684 to about K692, about T698 to about A706, about S726 to about S734, and
about S734 to
to about L742 of SEQ ID N0:2 (Figures lA-C) correspond to the highly antigenic
regions of the
TR13 protein, predicted using the Jameson-Wolf antigenic index (See Figure 3
and Table I).
These highly antigenic fragments correspond to the amino acid residues
illustrated in Figure
lA-C and in SEQ ID N0:2. As indicated above, the inventors have determined
that the above
polypeptide fragments are antigenic regions of the TR13 receptor protein. 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. Polynucleotides encoding
these
polypeptides are also encompassed by the invention.
Non-limiting examples of predicted antigenic polypeptides that can be used to
generate
TR13-specific antibodies include: a polypeptide comprising, or alternatively
consisting of,
amino acid residues from about 1 to about 262 in Figures 7A-D (corresponding
to about amino
acid 1 to about 262 in SEQ ID N0:40); a polypeptide comprising amino acid
residues from
about 264 to about 423 in Figures 7A-D (corresponding to about amino acid 264
to about 423
in SEQ ID N0:40); a polypeptide comprising amino acid residues from about 437
to about 789
in Figures 7A-D (corresponding to about amino acid 437 to about 789 in SEQ ID
N0:40); and
a polypeptide comprising amino acid residues from about 791 to about 1001 in
Figures 7A-D
(corresponding to about amino acid 791 to about 1001 in SEQ ID N0:40). As
indicated above,
the inventors have determined that the above polypeptide fragments are
antigenic regions of the
TR13 receptor protein. 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.
3o Polynucleotides encoding these polypeptides are also encompassed by the
invention.
Additional non-limiting examples of predicted antigenic polypeptides that can
be used to
generate TR13 receptor-specific antibodies include: a polypeptide comprising,
or alternatively
consisting of, amino acid residues from about about Ml to about H9, about V 14
to about I22,
about H47 to about H55, about C61 to about R69, about L82 to about E90, about
D102 to
about P110, about K109 to about S117, about F124 to about H132, about M141 to
about
E149, about S146 to about C154, about S157 to about W165, about F168 to about
T176,
about N 182 to about N 190, about Q207 to about A215 , about P213 to about
M221, about
M221 to about E229, about V233 to about V241, about T253 to about V261, about
T282 to
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
145
about S290, about N298 to about T306, about C308 to about Y316, about K315 to
about
S323, about P328 to about F336, about A341 to about Q349, about F387 to about
Q395, about
S403 to about C411, about T409 to about P417, about F443 to about N451, about
W451 to
about Y459, about A462 to about M470, about 6478 to about M486, about A487 to
about
A495, about V517 to about V525, about T527 to about Q535, about I541 to about
F549, about
A561 to about V569, about E594 to about C602, about 1611 to about H619, about
6643 to
about I650, about P686 to about K694, about C704 to about 5712, about 8722 to
about 1730,
about E727 to about T735, about P746 to about 6754, about D776 to about L784,
about Y799
to about 5807, about C808 to about I816, about V818 to about V826, about T835
to about
6843, about 8883 to about 6891, about K932 to about K940, about Q935 to about
K943,
about T949 to about A957, about S977 to about S985, about 5981 to about P989,
and about
N986 to about L994 of SEQ ID N0:40 (Figures 7A-D) correspond to the highly
antigenic
regions of the TR13 protein, predicted using the Jameson-Wolf antigenic index
(See Figure 9
and Table III). These highly antigenic fragments correspond to the amino acid
residues
illustrated in Figure 7A-D and in SEQ ID N0:40. As indicated above, the
inventors have
determined that the above polypeptide fragments are antigenic regions of the
TR13 receptor
protein. 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.
Polynucleotides
encoding these polypeptides are also encompassed by the invention.
The epitope-bearing peptides and polypeptides of the invention may be produced
by any
conventional means. R.A. Houghten, "General Method for the Rapid Solid-phase
Synthesis of
Large Numbers of Peptides: Specificity of Antigen-Antibody Interaction at the
Level of
Individual Amino Acids," Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985). This
"Simultaneous Multiple Peptide Synthesis (SMPS)" process is further described
in U.S. Patent
No. 4,631,211 to Houghten et al. (1986).
As one of skill in the art will appreciate, TR13 receptor polypeptides of the
present
invention 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
105 to about
170, about 251 to about 265, about 331 to about 410, and/or about 580 to about
610 of SEQ
ID N0:2), can be combined with heterologous polypeptide sequences, for
example, the
polypeptides of the present invention may be fused with the constant domain of
immunoglobulins (IgA, IgE, IgG, IgM) or portions thereof (CHI, CH2, CH3, and
any
combination thereof, including both entire domains and portions thereof),
resulting in chimeric
polypeptides. These fusion proteins facilitate purification and show an
increased half-life 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
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
146
also be more efficient in binding and neutralizing other molecules than the
monomeric TR13
protein or protein fragment alone (Fountoulakis et al., J. Biochem. 270:3958-
3964 (1995)). 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.
As one of skill in the art will appreciate, TR13 receptor polypeptides of the
present
invention 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 about 262,
about 264 to about 423, about 437 to about 789, about 271 to about 421, and/or
about 585 to
599 of SEQ ID N0:40), can be combined with heterologous polypeptide sequences,
for
to example, the polypeptides of the present invention may be fused with the
constant domain of
immunoglobulins (IgA, IgE, IgG, IgM) or portions thereof (CH1, CH2, CH3, and
any
combination thereof, including both entire domains and portions thereof),
resulting in chimeric
polypeptides. These fusion proteins facilitate purification and show an
increased half-life 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 TR13
protein or protein fragment alone (Fountoulakis et al., J. Biochem. 270:3958-
3964 (1995)). In
2o 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.
Preferred TR13 Fc fusions of the present invention include, but are not
limited to
constructs comprising, or alternatively consisting of, amino acid residues 1
to 750, 10 to 750,
to 750, 30 to 750, 40 to 750, 1 to 740, 1 to 730, 1 to 720, 1 to 710, 10 to
740, 10 to 730,
and/or 10 to 720 of SEQ ID N0:2. Polynucleotides encoding these TR13 fusions
are also
encompassed by the invention.
Additional preferred TR13 Fc fusions of the present invention include, but are
not
limited to constructs comprising, or alternatively consisting of, amino acid
residues 1 to 906,
42 to 906, 271 to 421, 585 to 595, 1 to 1001, 10 to 1001, 20 to 1001, 30 to
1001, 42 to
1001, 42 to 906, 1 to 990, 1 to 980, 1 to 970, 1 to 960, 10 to 990, 10 to 980,
and/or 10 to
970 of SEQ ID N0:2. Polynucleotides encoding these TR13 fusions are also
encompassed by
the invention.
The polypeptides of the present invention have uses which include, but are not
limited
to, as sources for generating antibodies that bind the polypeptides of the
invention, and as
molecular weight markers on SDS-PAGE gels or on molecular sieve gel filtration
columns
using methods well known to those of skill in the art.
TRl4 Polypeptides
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
147
The TR14 proteins (polypeptides) of the invention may be in monomers or
multimers
(i.e., dimers, trimers, tetramers, and higher multimers). Accordingly, the
present invention
relates to monomers and multimers of the TR14 proteins (polypeptides) of the
invention, their
preparation, and compositions (preferably, pharmaceutical compositions)
containing them. In
specific embodiments, the polypeptides of the invention are monomers, dimers,
trimers or
tetramers. In additional embodiments, the multimers of the invention are at
least dimers, at
least trimers, or at least tetramers.
Multimers encompassed by the invention may be homomers or heteromers. As used
herein, the term TR14 homomer, refers to a multimer containing only TR14
proteins of the
1o invention (including TR14 fragments, variants, and fusion proteins, as
described herein).
These homomers may contain TR14 proteins having identical or different
polypeptide
sequences. In a specific embodiment, a homomer of the invention is a multimer
containing
only TR14 proteins having an identical polypeptide sequence. In another
specific embodiment,
a homomer of the invention is a multimer containing TR14 proteins having
different
polypeptide sequences. In specific embodiments, the multimer of the invention
is a homodimer
(e.g., containing TR14 proteins having identical or different polypeptide
sequences) or a
homotrimer (e.g., containing TR14 proteins having identical or different
polypeptide
sequences). In additional embodiments, the homomeric multimer of the invention
is at least a,
homodimer, at least a homotrimer, or at least a homotetramer.
2o As used herein, the term TR14 heteromer refers to a multimer containing
heterologous
proteins (i.e., proteins containing only polypeptide sequences that do not
correspond to a
polypeptide sequences encoded by the TR14 gene) in addition to the TR14
proteins of the
invention. In a specific embodiment, the multimer of the invention is a
heterodimer, a
heterotrimer, or a heterotetramer. In additional embodiments, the heteromeric
multimer of the
invention is at least a heterodimer, at least a heterotrimer, or at least a
heterotetramer.
Multimers of the invention may be the result of hydrophobic, hydrophilic,
ionic and/or
covalent associations and/or may be indirectly linked, by for example,
liposome formation.
Thus, in one embodiment, multimers of the invention, such as, for example,
homodimers or
homotrimers, are formed when proteins of the invention contact one another in
solution. In
3o another embodiment, heteromultimers of the invention, such as, for example,
heterotrimers or
heterotetramers, are formed when proteins of the invention contact antibodies
to the
polypeptides of the invention (including antibodies to the heterologous
polypeptide sequence in
a fusion protein of the invention) in solution. In other embodiments,
multimers of the
invention are formed by covalent associations with and/or between the TR14
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 preferably in
SEQ ID N0:61 or, alternatively, in SEQ ID NO:S or the polypeptide encoded by
the deposited
cDNA clone). In one instance, the covalent associations are cross-linking
between cysteine
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
148
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 TR14 fusion protein. In one example, covalent
associations are
between the heterologous sequence contained in a fusion protein of the
invention (see, e.g., US
Patent Number 5,478,925). In a specific example, the covalent associations are
between the
heterologous sequence contained in a TR14-Fc fusion protein of the invention
(as described
herein). In another specific example, covalent associations of fusion proteins
of the invention
l0 are between heterologous polypeptide sequences from another TNF family
ligand/receptor
member that is capable of forming covalently associated multimers, such as for
example,
oseteoprotegerin (see, e.g., International Publication No. WO 98/49305, the
contents of which
are herein incorporated by reference in its entirety). In another embodiment,
two or more TR14
polypeptides of the invention are joined through synthetic linkers (e.g.,
peptide, carbohydrate
or soluble polymer linkers). Examples include those peptide linkers described
in U.S. Pat.
No. 5,073,627 (hereby incorporated by reference). Proteins comprising multiple
TR14
polypeptides separated by peptide linkers may be produced using conventional
recombinant
DNA technology.
Another method for preparing multimer TR14 polypeptides of the invention
involves
2o use of TR14 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 TR14 proteins
are those
described in PCT application WO 94/10308, hereby incorporated by reference.
Recombinant
fusion proteins comprising a soluble TR14 polypeptide fused to a peptide that
dimerizes or
trimerizes in solution are expressed in suitable host cells, and the resulting
soluble multimeric
TR14 is recovered from the culture supernatant using techniques known in the
art.
Certain members of the TNF family of proteins are believed to exist in
trimeric form
(Beutler and Huffel, Science 264:667, 1994; Banner et al., Cel173:431, 1993).
Thus, trimeric
TR14 may offer the advantage of enhanced biological activity. Preferred
leucine zipper
moieties are those that preferentially form trimers. One example is a leucine
zipper derived
from lung surfactant protein D (SPD), as described in Hoppe et al. (FEBS
Letters 344:191,
(1994)) and in U.S. patent application Ser. No. 08/446,922, hereby
incorporated by reference.
Other peptides derived from naturally occurnng trimeric proteins may be
employed in preparing
trimeric TR14.
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
149
In further preferred embodiments, TR14 polynucleotides of the invention are
fused to a
polynucleotide encoding a "FLAG" polypeptide. Thus, a TR14-FLAG fusion protein
is
encompassed by the present invention. The FLAG antigenic polypeptide may be
fused to a
TR14 polypeptide of the invention at either or both the amino or the carboxy
terminus. In
preferred embodiments, a TR14-FLAG fusion protein is expressed from a pFLAG-
CMV-Sa or
a pFLAG-CMV-1 expression vector (available from Sigma, St. Louis, MO, USA).
See,
Andersson, S., et al., J. Biol. Chem. 264:8222-29 (1989); Thomsen, D. R., et
al., Proc. Natl.
Acad. Sci. USA, 81:659-63 (1984); and Kozak, M., Nature 308:241 (1984) (each
of which is
hereby incorporated by reference). In further preferred embodiments, a TR14-
FLAG fusion
protein is detectable by anti-FLAG monoclonal antibodies (also available from
Sigma).
In another example, proteins of the invention are associated by interactions
between
Flag~ polypeptide sequence contained in Flag~-TR14 fusion proteins of the
invention. In a
further embodiment, associated proteins of the invention are associated by
interactions between
heterologous polypeptide sequence contained in Flag~-TR14 fusion proteins of
the invention
and anti-Flag~ antibody.
The multimers of the invention may be generated using chemical techniques
known in
the art. For example, proteins desired to be contained in the multimers of the
invention may be
chemically cross-linked using linker molecules and linker molecule length
optimization
techniques known in the art (see, e.g., US Patent Number 5,478,925, which is
herein
2o incorporated by reference in its entirety). Additionally, multimers of the
invention may be
generated using techniques known in the art to form one or more inter-molecule
cross-links
between the cysteine residues located within the polypeptide sequence of the
proteins desired to
be contained in the multimer (see, e.g., US Patent Number 5,478,925, which is
herein
incorporated by reference in its entirety). Further, proteins of the invention
may be routinely
modified by the addition of cysteine or biotin to the C terminus or N-terminus
of the
polypeptide sequence of the protein and techniques known in the art may be
applied to generate
multimers containing one or more of these modified proteins (see, e.g., US
Patent Number
5,478,925, which is herein incorporated by reference in its entirety).
Additionally, techniques
known in the art may be applied to generate liposomes containing the protein
components
3o desired to be contained in the multimer of the invention (see, e.g., US
Patent Number
5,478,925, which is herein incorporated by reference in its entirety).
Alternatively, multimers of the invention may be generated using genetic
engineering
techniques known in the art. In one embodiment, proteins contained in
multimers of the
invention are produced recombinantly using fusion protein technology described
herein or
otherwise known in the art (see, e.g., US Patent Number 5,478,925, which is
herein
incorporated by reference in its entirety). In a specific embodiment,
polynucleotides coding for
a homodimer of the invention are generated by ligating a polynucleotide
sequence encoding a
polypeptide of the invention to a sequence encoding a linker polypeptide and
then further to a
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
150
synthetic polynucleotide encoding the translated product of the polypeptide in
the reverse
orientation from the original C-terminus to the N-terminus (lacking the leader
sequence) (see,
e.g., US Patent Number 5,478,925, which is herein incorporated by reference in
its entirety).
In another embodiment, recombinant techniques described herein or otherwise
known in the art
are applied to generate recombinant polypeptides of the invention which
contain a
transmembrane domain and which can be incorporated by membrane reconstitution
techniques
into liposomes (see, e.g., US Patent Number 5,478,925, which is herein
incorporated by
reference in its entirety).
The polypeptides (proteins) of the present invention are preferably provided
in an
1o isolated form. By "isolated polypeptide" is intended a polypeptide removed
from its native
environment. Thus, a polypeptide produced and/or contained within a
recombinant host cell is
considered isolated for purposes of the present invention. Also intended as an
"isolated
polypeptide" are polypeptides that have been purified, partially or
substantially, from a
recombinant host cell. For example, a recombinantly produced version of the
TR14
polypeptide can be substantially purified by the one-step method described in
Smith and
Johnson, Gene 67:31-40 (1988).
Accordingly, in one embodiment, the invention provides an isolated TR14
polypeptide
having the amino acid sequence encoded by the cDNA deposited as ATCC Deposit
No. PTA
348, or the amino acid sequence shown preferably in SEQ ID N0:61 or,
alternatively, in SEQ
2o ID NO:S, or a polypeptide comprising a portion of the above polypeptides.
The polypeptides of the present invention are preferably provided in an
isolated form.
By "isolated polypeptide" is intended a polypeptide removed from its native
environment.
Thus, a polypeptide produced and/or contained within a recombinant host cell
is considered
isolated for purposes of the present invention. Also intended as an "isolated
polypeptide" are
polypeptides that have been purified, partially or substantially, from a
recombinant host cell.
For example, a recombinantly produced version of the TR14 polypeptide can be
substantially
purified by the one-step method described in Smith and Johnson, Gene 67:31-40
(1988).
Polypeptide fragments of the present invention include polypeptides comprising
or
alternatively, consisting of, an amino acid sequence contained preferably in
SEQ ID N0:61 or,
alternatively, in SEQ ID NO:S, encoded by the cDNA contained in the clone
deposited as
ATCC Deposit No. PTA-348, or encoded by a nucleic acid which hybridizes (e.g.,
under
stringent hybridization conditions) to the nucleotide sequence contained in
the deposited clone,
or shown preferably in Figures l0A-H (SEQ ID N0:61) or, alternatively, in
Figures 4A-D
(SEQ ID N0:4) or the complementary strand thereto, or polynucleotide fragments
thereof (e.g.,
as disclosed herein). 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. Preferred representative examples of polypeptide fragments
of the
invention, include, for example, fragments that comprise, or alternatively
consist of, from
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
151
about amino acid residues: 1 to 50, 51 to 100, 101 to 150, 151 to 200, 201 to
231 of SEQ >D
N0:61. Alternative, less preferred representative examples of polypeptide
fragments of the
invention, include, for example, fragments that comprise, or alternatively
consist of, from
about amino acid residues: 1 to 50, 51 to 100, 101 to 150, 151 to 200, 201 to
226 of SEQ ID
NO:S, and the corresponding amino acid residues of SEQ ID N0:61 (as the
sequence of amino
acid residues T-78 to M-231 of SEQ ID N0:61 is identical to the sequence of
amino acid
residues T-73 to M-226 of SEQ ID NO:S). Moreover, polypeptide fragments can be
at least
10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 175 or 200
amino acids in
length. In this context "about" includes the particularly recited ranges,
larger or smaller by
to several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both
extremes. Polynucleotides
encoding these polypeptides are also encompassed by the invention.
Polypeptide fragments of the present invention include polypeptides comprising
or
alternatively, consisting of amino acid residues from about: 178 to about 180,
118 to about
121, 178 to about 181, 193 to about 196, 9 to about 14, and/or 65 to about 85
of SEQ >D
N0:2. 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.
Polynucleotides
encoding the polypeptide fragments are also encompassed by the invention.
In specific embodiments, polypeptide fragments of the invention comprise, or
alternatively consist of, amino acid residues from about: 1 to about 138, 139
to about 155,
2o and/or 156 to about 231 as depicted in SEQ ID N0:61; or, alternativley,
about 1 to about 133,
134 to about 150, and/or 151 to about 226 as depicted in SEQ ID NO:S. 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. Polynucleotides encoding
these
polypeptides are also encompassed by the invention.
In additional embodiments, the polypeptide fragments of the invention
comprise, or
alternatively consist of, one or more TR14 domains. Preferred polypeptide
fragments of the
present invention include a member selected from the group: (a) a polypeptide
comprising or
alternatively, consisting of, the TR14 extracellular domain (predicted to
constitute preferably
amino acid residues from about 1 to about 138 in Figures l0A-H and SEQ ID
N0:61, or,
3o alternatively, from about 1 to about 133 of SEQ ID NO:S and Figures 4A-D,
or from about 1 to
about 133 of SEQ ID NO:S); (b) a polypeptide comprising or alternatively,
consisting of, the
TR14 cysteine rich domain (predicted to constitute preferably amino acids Cys-
31 to Cys-104
of SEQ ID N0:61, or, alternatively, amino acid residues from about 65 to about
88 of Figures
4A-D, or from about 65 to about 85 in SEQ ID NO:S); (c) a polypeptide
comprising or
alternatively, consisting of, the TR14 transmembrane domain (predicted to
constitute amino
acid residues from about 139 to about 155 of Figures l0A-H and SEQ ID N0:61 or
from about
134 to about 150 of Figures 4A-D and SEQ ID NO:S); (d) a polypeptide
comprising or
alternatively, consisting of, the TR14 intracellular domain (predicted to
constitute amino acid
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
152
residues from about 155 to about 231 of Figures l0A-H and SEQ ID N0:61 or
amino acid
residues from about 151 to about 226 of Figures 4A-D and SEQ ID NO:S); (e) a
polypeptide
comprising, or alternatively, consisting of, one, two, three, four or more,
epitope bearing
portions of the TR14 polypeptide (predicted to constitute preferably Asp-2 to
Asp-10, Thr-17
to Asp-38, Pro-45 to Ser-52, Pro-88 to Arg-95, Thr-108 to Glu-I15, Thr-131 to
Glu-136,
Phe-166 to Gly-174, Ala-180 to Ala-200, and Gln-224 to Met-231 of SEQ ID
N0:61, or the
corresponding amino acid sequences in SEQ ID NO:S, as the sequence of amino
acid residues
T-78 to M-231 of SEQ ID N0:61 is identical to the sequence of amino acid
residues T-73 to M-
226 of SEQ ID NO:S. Additional epitope bearing TR14 polypeptides comprise or,
1o alternatively, consist of amino acid residues from about 2 to about 24, 42
to about 52, 80 to
about 115, and I55 to about 226 of SEQ ID NO:S (or the corresponding amino
acid sequences
in SEQ ID N0:61, as the sequence of amino acid residues T-78 to M-231 of SEQ
ID N0:61 is
identical to the sequence of amino acid residues T-73 to M-226 of SEQ ID
NO:S); and (f) any
combination of polypeptides (a)-(e). 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. Polynucleotides encoding these polypeptides are also encompassed by
the invention.
As discussed above, it is believed that the extracellular cysteine rich motifs
of TR14 is
important for interactions between TR14 and its ligands. Accordingly, in a
specific
embodiment, polypeptide fragments of the invention comprise, or alternatively
consist of amino
2o acid residues 31 to 104 of SEQ ID N0:61 or 65 to 85 of SEQ ID NO:S.
Polynucleotides
encoding these polypeptides are also encompassed by the invention.
Among the especially preferred fragments of the invention are fragments
characterized
by structural or functional attributes of TR14 (preferably SEQ ID N0:61 or,
alternatively, SEQ
ID NO:S). 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
3o equal to 1.5, as identified using the default parameters of the Jameson-
Wolf program) of
complete (i.e., full-length) TR14 (preferably SEQ ID N0:61 or, alternatively,
SEQ ID NO:S).
Certain preferred regions are those set out in Figure 6 and Table II and
include, but are not
limited to, regions of the aforementioned types identified by analysis of the
amino acid
sequence depicted preferably Figures l0A-H (SEQ ID N0:61) or, alternatively,
in Figures 4A-
D (SEQ ID NO:S), 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 and Hopp-Woods
predicted
hydrophobic regions; Eisenberg alpha and beta amphipathic regions; Emini
surface-forming
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
153
regions; and Jameson-Wolf high antigenic index regions, as predicted using the
default
parameters of these computer programs. Polynucleotides encoding these
polypeptides are also
encompassed by the invention.
As mentioned above, even if deletion of one or more amino acids from the N-
terminus
of a protein results in modification of loss of one or more biological
functions of the protein,
other functional activities (e.g., biological activities, ability to
multimerize, ability to bind TR14
ligand) may still be retained. For example, the ability of shortened TR14
muteins to induce
and/or bind to antibodies which recognize the complete or mature forms of the
polypeptides
generally will be retained when less than the majority of the residues of the
complete or mature
to polypeptide are removed from the N-terminus. Whether a particular
polypeptide lacking N
terminal residues of a complete polypeptide retains such immunologic
activities can readily be
determined by routine methods described herein and otherwise known in the art.
It is not
unlikely that an TR14 mutein with a large number of deleted N-terminal amino
acid residues
may retain some biological or immunogenic activities. In fact, peptides
composed of as few as
six TR14 amino acid residues may often evoke an immune response.
Accordingly, the present invention further provides polypeptides having one or
more
residues deleted from the amino terminus of the TR14 amino acid sequence shown
depicted
preferably Figures l0A-H (SEQ ID N0:61) or, alternatively, in Figures 4A-D
(SEQ ID NO:S),
up to the methionine residue at position number 231 of SEQ ID N0:61 (or,
number 226 of
2o SEQ ID NO:S) and polynucleotides encoding such polypeptides. In particular
preferred
embodiments for TR14, the present invention provides polypeptides comprising,
or
alternatively consisting of, the amino acid sequence of residues n'-231 of
Figures l0A-H,
where n' is an integer from 1 to 231 corresponding to the position of the
amino acid residue in
Figures l0A-H. In alternative embodiments, the present invention provides
polypeptides
comprising, or alternatively consisting of, the amino acid sequence of
residues n'-226 of
Figures 4A-D, where n' is an integer from 1 to 226 corresponding to the
position of the amino
acid residue in Figures 4A-D.
1n specific embodiments, N-terminal deletions of the TR14 polypeptides of the
invention
can be described by the general formula n2-231, where n2 is a number from 2 to
226,
corresponding to the position of amino acid identified in Figures l0A-H (SEQ
ID N0:61 ). N-
terminal deletions of the TR14 polypeptide of the invention shown as SEQ ID
N0:61 include
polypeptides comprising, or alternatively consisting of, the amino acid
sequence of residues: D-
2 to M-231; C-3 to M-231; Q-4 to M-231; E-5 to M-231; N-6 to M-231; E-7 to M-
231; Y-8 to
M-231; W-9 to M-231; D-10 to M-231; Q- I 1 to M-231; W-12 to M-231; G-13 to M-
231; R-14
to M-231; C-15 to M-231; V -16 to M-231; T-17 to M-231; C-18 to M-231; Q-19 to
M-231; R-
20 to M-231; C-21 to M-231; G-22 to M-231; P-23 to M-231; G-24 to M-231; Q-25
to M-231;
E-26 to M-23 I ; L-27 to M-231; S-28 to M-231; K-29 to M-231; D-30 to M-231; C-
31 to 1VI-
231; G-32 to M-231; Y-33 to M-231; G-34 to M-231; E-35 to M-231; G-36 to M-23
I ; G-37 to
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
154
M-231; D-38 to M-231; A-39 to M-231; Y-40 to M-231; W-41 to M-231; H-42 to M-
231; S-43
to M-231; L-44 to M-231; P-45 to M-231; S-46 to M-231; S-47 to M-231; Q-48 to
M-231; Y-
49 to M-23 I ; K-50 to M-231; S-51 to M-231; S-52 to M-231; W-53 to M-231; G-
54 to M-23 I ;
H-55 to M-231; H-56 to M-231; K-57 to M-231; C-5 8 to M-231; Q-59 to M-231; S-
60 to M-
231; C-61 to M-231; I-62 to M-231; T-63 to M-231; C-64 to M-231; A-65 to M-
231; V -66 to
M-231; I-67 to M-231; N-68 to M-231; R-69 to M-231; V -70 to M-231; Q-71 to M-
231; K-72
to M-231; V-73 to M-231; N-74 to M-231; C-75 to M-231; T-76 to M-231; P-77 to
M-231; T-
78 to M-231; S-79 to M-231; N-80 to M-231; A-81 to M-231; V -82 to M-231; C-83
to M-231;
G-84 to M-231; D-85 to M-231; C-86 to M-231; L-87 to M-231; P-8 8 to M-231; R-
89 to M-
231; F-90 to M-231; Y-91 to M-231; R-92 to M-231; K-93 to M-231; T-94 to M-
231; R-95 to
M-231; I-96 to M-231; G-97 to M-231; G-98 to M-231; L-99 to M-231; Q-100 to M-
231; D-
1 O l to M-231; Q-102 to M-231; E-103 to M-231; C-104 to M-231; I-105 to M-
231; P-106 to
M-231; C-107 to M-231; T-108 to M-231; K-109 to M-231; Q-110 to M-231; T-111
to M-231;
P-112 to M-231; T-113 to M-231; S-114 to M-231; E-115 to M-231; V-116 to M-
231; Q-117
to M-231; C-118 to M-231; A-119 to M-231; F-120 to M-231; Q-121 to M-231; L-
122 to M-
231; S-123 to M-23 I ; L-124 to M-231; V -125 to M-231; E-126 to M-231; A-127
to M-231; D-
128 to M-231; A-129 to M-231; P-130 to M-231; T-131 to M-231; V-132 to M-231;
P-133 to
M-231; P-134 to M-231; Q-135 to M-231; E-136 to M-231; A-137 to M-231; T-138
to M-231;
L-139 to M-231; V-140 to M-231; A-141 to M-231; L-142 to M-231; V-143 to M-
231; S-144
2o to M-231; S-145 to M-23 I ; L-146 to M-231; L-147 to M-231; V -148 to M-
231; V -149 to M-
231; F-150 to M-231; T-151 to M-231; L-152 to M-231; A-153 to M-231; F-154 to
M-231; L-
155 to M-231; G-156 to M-231; L-157 to M-231; F-158 to M-231; F-159 to M-231;
L-160 to
M-231; Y - I 61 to M-231; C-162 to M-231; K-163 to M-231; Q-164 to M-231; F-
165 to M-231;
F- I 66 to M-231; N-167 to M-23 I ; R-168 to M-231; H-169 to M-231; C-170 to M-
231; Q-171
to M-231; R-172 to M-231; G-173 to M-231; G-174 to M-231; L-175 to M-231; L-
176 to M-
231; Q-177 to M-231; F-178 to M-231; E-179 to M-231; A-180 to M-231; D-181 to
M-231; K-
182 to M-231; T-183 to M-231; A-184 to M-231; K-185 to M-231; E-186 to M-231;
E-187 to
M-231; S-188 to M-231; L-189 to M-231; F-190 to M-231; P-191 to M-231; V-192
to M-231;
P-193 to M-231; P-194 to M-231; S-195 to M-231; K-196 to M-231; E-197 to M-
231; T-198
3o to M-231; S-199 to M-231; A-200 to M-231; E-201 to M-231; S-202 to M-231; Q-
203 to M-
231; V-204 to M-231; S-205 to M-231; W-206 to M-231; A-207 to M-231; P-208 to
M-231;
G-209 to M-231; S-210 to M-231; L-211 to M-231; A-212 to M-231; Q-213 to M-
231; L-214
to M-231; F-215 to M-231; S-216 to M-231; L-217 to M-231; D-218 to M-231; S-
219 to M-
231; V-220 to M-231; P-221 to M-23 I ; I-222 to M-23 I ; P-223 to M-231; Q-224
to M-231; Q-
225 to M-231 and Q-226 to M-231 of SEQ ID N0:61.
In additional embodiments, N-terminal deletions of the TR14 polypeptides of
the
invention can be described by the general formula n2-226, where n2 is a number
from 2 to 221,
corresponding to the position of amino acid identified in Figures 4A-D (SEQ ID
NO:S). N-
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
155
terminal deletions of the TR14 polypeptide of the invention shown as SEQ ID
NO:S include
polypeptides comprising, or alternatively consisting of, the amino acid
sequence of residues: S-
2 to M-226; T-3 to M-226; G-4 to M-226; T-5 to M-226; N-6 to M-226; G-7 to M-
226; D-8 to
M-226; G-9 to M-226; V-10 to M-226; S-11 to M-226; P-12 to M-226; A-13 to M-
226; N-14
to M-226; G-15 to M-226; V -16 to M-226; V -17 to M-226; L-18 to M-226; D-19
to M-226; R-
20 to M-226; S-21 to M-226; Y-22 to M-226; P-23 to M-226; R-24 to M-226; I-25
to M-226;
V-26 to M-226; V-27 to M-226; M-28 to M-226; E-29 to M-226; R-30 to M-226; V-
31 to M-
226; E-32 to M-226; M-33 to M-226; P-34 to M-226; T-35 to M-226; A-36 to M-
226; Q-37 to
M-226; P-38 to M-226; A-39 to M-226; L-40 to M-226; L-41 to M-226; A-42 to M-
226; V-43
to M-226; Q-44 to M-226; K-45 to M-226; Q-46 to M-226; L-47 to M-226; G-48 to
M-226; P-
49 to M-226; P-50 to M-226; Q-51 to M-226; M-52 to M-226; C-53 to M-226; R-54
to M-226;
V-55 to M-226; A-56 to M-226; C-57 to M-226; T-58 to M-226; C-59 to M-226; A-
60 to M-
226; V-61 to M-226; I-62 to M-226; N-63 to M-226; R-64 to M-226; V-65 to M-
226; Q-66 to
M-226; K-67 to M-226; V-68 to M-226; N-69 to M-226; C-70 to M-226; T-71 to M-
226; P-72
to M-226; T-73 to M-226; S-74 to M-226; N-75 to M-226; A-76 to M-226; V-77 to
M-226; C-
78 to M-226; G-79 to M-226; D-80 to M-226; C-81 to M-226; L-82 to M-226; P-83
to M-226;
R-84 to M-226; F-85 to M-226; Y-86 to M-226; R-87 to M-226; K-88 to M-226; T-
89 to M-
226; R-90 to M-226; I-91 to M-226; G-92 to M-226; G-93 to M-226; L-94 to M-
226; Q-95 to
M-226; D-96 to M-226; Q-97 to M-226; E-98 to M-226; C-99 to M-226; I-100 to M-
226; P-
2o 101 to M-226; C-102 to M-226; T-103 to M-226; K-104 to M-226; Q-105 to M-
226; T-106 to
M-226; P-107 to M-226; T-108 to M-226; S-109 to M-226; E-110 to M-226; V-11 I
to M-226;
Q-112 to M-226; C-113 to M-226; A-114 to M-226; F-115 to M-226; Q-116 to M-
226; L-117
to M-226; S-118 to M-226; L-119 to M-226; V -120 to M-226; E-121 to M-226; A-
122 to M-
226; D-123 to M-226; A-124 to M-226; P-125 to M-226; T-126 to M-226; V-127 to
M-226; P-
128 to M-226; P-129 to M-226; Q-130 to M-226; E-131 to M-226; A-132 to M-226;
T-133 to
M-226; L-134 to M-226; V-135 to M-226; A-136 to M-226; L-137 to M-226; V-138
to M-226;
S-139 to M-226; S-140 to M-226; L-141 to M-226; L-142 to M-226; V-143 to M-
226; V-144
to M-226; F-145 to M-226; T-146 to M-226; L-147 to M-226; A-148 to M-226; F-
149 to M-
226; L-150 to M-226; G-151 to M-226; L-152 to M-226; F-153 to M-226; F-154 to
M-226; L-
155 to M-226; Y-156 to M-226; C-157 to M-226; K-158 to M-226; Q-159 to M-226;
F-160 to
M-226; F-161 to M-226; N-162 to M-226; R-163 to M-226; H-164 to M-226; C-165
to M-226;
Q-166 to M-226; R-167 to M-226; G-168 to M-226; G-169 to M-226; L-170 to M-
226; L-171
to M-226; Q-172 to M-226; F-173 to M-226; E-174 to M-226; A-175 to M-226; D-
176 to M-
226; K-177 to M-226; T-178 to M-226; A-179 to M-226; K-180 to M-226; E-181 to
M-226; E-
182 to M-226; S-183 to M-226; L-184 to M-226; F-185 to M-226; P-186 to M-226;
V-187 to
M-226; P-188 to M-226; P-189 to M-226; S-190 to M-226; K-191 to M-226; E-192
to M-226;
T-193 to M-226; S-194 to M-226; A-195 to M-226; E-196 to M-226; S-197 to M-
226; Q-198
to M-226; V-199 to M-226; S-200 to M-226; W-201 to M-226; A-202 to M-226; P-
203 to M-
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
156
226; G-204 to M-226; S-205 to M-226; L-206 to M-226; A-207 to M-226; Q-208 to
M-226; L-
209 to M-226; F-210 to M-226; S-211 to M-226; L-212 to M-226; D-213 to M-226;
S-214 to
M-226; V-215 to M-226; P-216 to M-226; I-217 to M-226; P-218 to M-226; Q-219
to M-226;
Q-220 to M-226; Q-221 to M-226; of SEQ ID NO:S. Polynucleotides encoding these
polypeptides are also encompassed by the invention.
In another embodiment, N-terminal deletions of the extracellular domain of the
TR14
polypeptide can be described by the general formula n2-133, where n2 is a
number from 1 to
128, corresponding to the position of amino acids identified in Figures 4A-D.
N-terminal
deletions of the extracellular domain of the TR14 polypeptide of the invention
shown as SEQ
t0 ID N0:7 include polypeptides comprising, or alternatively consisting of,
the amino acid
sequence of residues: S-2 to T-133; T-3 to T-133; G-4 to T-133; T-5 to T-133;
N-6 to T-133;
G-7 to T-133; D-8 to T-133; G-9 to T-133; V-10 to T-133; S-11 to T-133; P-12
to T-133; A-13
to T-133; N-14 to T-133; G-15 to T-133; V-16 to T-133; V-17 to T-133; L-18 to
T-133; D-19
to T-133; R-20 to T-133; S-21 to T-133; Y-22 to T-133; P-23 to T-133; R-24 to
T-133; I-25 to
T-133; V-26 to T-133; V-27 to T-133; M-28 to T-133; E-29 to T-133; R-30 to T-
133; V-31 to
T-133; E-32 to T-133; M-33 to T-133; P-34 to T-133; T-35 to T-133; A-36 to T-
133; Q-37 to
T-133; P-38 to T-133; A-39 to T-133; L-40 to T-133; L-41 to T-133; A-42 to T-
133; V-43 to
T-133; Q-44 to T-133; K-45 to T-133; Q-46 to T-133; L-47 to T-133; G-48 to T-
133; P-49 to
T-133; P-SO to T-133; Q-51 to T-133; M-52 to T-133; C-53 to T-133; R-54 to T-
133; V-55 to
2o T-133; A-56 to T-133; C-57 to T-133; T-58 to T-133; C-59 to T-133; A-60 to
T-133; V-61 to
T-133; I-62 to T-133; N-63 to T-133; R-64 to T-133; V-65 to T-133; Q-66 to T-
133; K-67 to
T-133; V-68 to T-133; N-69 to T-133; C-70 to T-133; T-71 to T-133; P-72 to T-
133; T-73 to
T-133; S-74 to T-133; N-75 to T-133; A-76 to T-133; V-77 to T-133; C-78 to T-
133; G-79 to
T-133; D-80 to T-133; C-81 to T-133; L-82 to T-133; P-83 to T-133; R-84 to T-
133; F-85 to
T-133; Y-86 to T-133; R-87 to T-133; K-88 to T-133; T-89 to T-133; R-90 to T-
133; I-91 to T-
133; G-92 to T-133; G-93 to T-133; L-94 to T-133; Q-95 to T-133; D-96 to T-
133; Q-97 to T-
133; E-98 to T-133; C-99 to T-133; I-100 to T-133; P-101 to T-133; C-102 to T-
133; T-103 to
T-133; K-104 to T-133; Q-105 to T-133; T-106 to T-133; P-107 to T-133; T-108
to T-133; S-
109 to T-133; E-110 to T-133; V-111 to T-133; Q-112 to T-133; C-113 to T-133;
A-114 to T-
133; F-115 to T-133; Q-116 to T-133; L-117 to T-133; S-118 to T-133; L-119 to
T-133; V-120
to T-133; E-121 to T-133; A-122 to T-133; D-123 to T-133; A-124 to T-133; P-
125 to T-133;
T-126 to T-133; V-127 to T-133; P-128 to T-133; of SEQ ID N0:7 (or the
corresponding
amino acid sequences in SEQ ID N0:61, as the sequence of amino acid residues T-
78 to T-138
of SEQ ID N0:61 is identical to the sequence of amino acid residues T-73 to T-
133 of SEQ ID
N0:7). Polynucleotides encoding these polypeptides are also encompassed by the
invention.
Also as mentioned above, even if deletion of one or more amino acids from the
C-
terminus of a protein results in modification or loss of one or more
biological functions of the
protein, other functional activities (e.g., biological activities), ability to
multimerize, ability to
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
157
bind TR14 ligand) may still be retained. For example the ability of the
shortened TR14 mutein
to induce and/or bind to antibodies which recognize the complete or mature
forms of the
polypeptide generally will be retained when less than the majority of the
residues of the
complete or mature polypeptide are removed from the C-terminus. Whether a
particular
polypeptide lacking C-terminal residues of a complete polypeptide retains such
immunologic
activities can readily be determined by routine methods described herein and
otherwise known
in the art. It is not unlikely that an TR14 mutein with a large number of
deleted C-terminal
amino acid residues may retain some biological or immunogenic activities. In
fact, peptides
composed of as few as six TR14 amino acid residues may often evoke an immune
response.
1o Accordingly, the present invention further provides polypeptides having one
or more
residues deleted from the carboxy terminus of the amino acid sequence of the
TR14 polypeptide
shown in Figures IOA-H (SEQ ID N0:61), up to the glutamic acid residue at
position number
7, and polynucleotides encoding such polypeptides. In particular, the present
invention
provides polypeptides comprising, or alternatively consisting of, the amino
acid sequence of
residues 1-m' of Figures l0A-H, where m' is an integer from 7 to 231
corresponding to the
position of the amino acid residue in Figures l0A-H (which is identical to the
sequence shown
as SEQ ID N0:61).
Moreover, the invention provides polypeptides comprising, or alternatively
consisting
of, the amino acid residues: M-1 to E-230; M-1 to P-229; M-1 to G-228; M-1 to
Q-227; M-1 to
Q-226; M-1 to Q-225; M-1 to Q-224; M-I to P-223; M-1 to I-222; M-1 to P-221; M-
1 to V-
220; M-1 to S-219; M-1 to D-218; M-1 to L-217; M-1 to S-216; M-1 to F-215; M-1
to L-214;
M-1 to Q-213; M-1 to A-212; M-1 to L-211; M-1 to S-210; M-1 to G-209; M-1 to P-
208; M-1
to A-207; M-1 to W-206; M-1 to S-205; M-1 to V-204; M-1 to Q-203; M-1 to S-
202; M-1 to E-
201; M-1 to A-200; M-1 to S-199; M-1 to T-198; M-1 to E-197; M-1 to K-196; M-1
to S-195;
M-1 to P-194; M-1 to P-193; M-1 to V-192; M-1 to P-191; M-1 to F-190; M-1 to L-
189; M-1
to S-188; M-1 to E-187; M-1 to E-186; M-1 to K-185; M-1 to A-184; M-1 to T-
183; M-1 to K-
182; M-1 to D-181; M-1 to A-180; M-1 to E-179; M-1 to F-178; M-1 to Q-177; M-1
to L-176;
M-1 to L-175; M-I to G-174; M-I to G-173; M-1 to R-172; M-1 to Q-171; M-1 to C-
170; M-1
to H-169; M-1 to R-168; M-t to N-167; M-1 to F-166; M-1 to F-165; M-1 to Q-
164; M-1 to K-
163; M-1 to C-162; M-1 to Y-161; M-1 to L-160; M-1 to F-159; M-1 to F-158; M-1
to L-157;
M-1 to G-156; M-1 to L-155; M-1 to F-154; M-1 to A-153; M-1 to L-152; M-1 to T-
151; M-1
to F-150; M-1 to V-149; M-1 to V-148; M-1 to L-147; M-1 to L-146; M-1 to S-
145; M-1 to S-
144; M-1 to V-143; M-1 to L-142; M-1 to A-141; M-1 to V-140; M-1 to L-139; M-1
to T-138;
M-1 to A-137; M-1 to E-136; M-1 to Q-135; M-1 to P-134; M-1 to P-133; M-1 to V-
132; M-1
to T-131; M-1 to P-130; M-1 to A-129; M-1 to D-128; M-1 to A-127; M-1 to E-
126; M-1 to V-
125; M-1 to L-124; M-1 to S-123; M-1 to L-122; M-1 to Q-121; M-1 to F-120; M-1
to A-119;
M-1 to C-118; M-1 to Q-117; M-1 to V-116; M-1 to E-115; M-1 to S-114; M-1 to T-
113; M-1
to P-112; M-1 to T-111; M-1 to Q-110; M-1 to K-109; M-1 to T-108; M-1 to C-
107; M-1 to P-
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
158
106; M-1 to I-105; M-1 to C-104; M-1 to E-103; M-1 to Q-102; M-1 to D-101; M-1
to Q-100;
M-1 to L-99; M-1 to G-98; M-1 to G-97; M-1 to I-96; M-1 to R-95 ; M-1 to T-94;
M-1 to K-93 ;
M-1 to R-92; M-1 to Y-91; M-1 to F-90; M-1 to R-89; M-1 to P-88; M-1 to L-87;
M-1 to C-86;
M-1 to D-85; M-1 to G-84; M-1 to C-83; M-1 to V-82; M-1 to A-81; M-1 to N-80;
M-1 to S-
79; M-1 to T-78; M-1 to P-77; M-1 to T-76; M-1 to C-75; M-1 tv N-74; M-1 to V-
73; M-1 to
K-72; M-1 to Q-71; M-1 to V-70; M-1 to R-69; M-1 to N-68; M-1 to I-67; M-1 to
V-66; M-1 to
A-65 ; M-1 to C-64; M-1 to T-63 ; M-1 to I-62; M-1 to C-61; M-1 to S-60; M-1
to Q-59; M-1 to
C-58; M-1 to K-57; M-1 to H-56; M-1 to H-55; M-1 to G-54; M-1 to W-53; M-1 to
S-52; M-1
to S-51; M-1 to K-50; M-1 to Y-49; M-1 to Q-48; M-1 to S-47; M-1 to S-4b; M-1
to P-45; M-1
1o to L-44; M-1 to S-43; M-1 to H-42; M-1 to W-41; M-1 to Y-40; M-1 to A-39; M-
1 to D-38; M-
1 to G-37; M-1 to G-36; M-1 to E-35; M-1 to G-34; M-1 to Y-33; M-1 to G-32; M-
1 to C-31;
M-1 to D-30; M-1 to K-29; M-1 to S-28; M-1 to L-27; M-1 to E-26; M-1 to Q-25;
M-1 to G-24;
M-1 to P-23 ; M-1 to G-22; M-1 to C-21; M-1 to R-20; M-1 to Q-19; M-1 to C-18;
M-1 to T-17;
M-1 to V-16; M-1 to C-15; M-1 to R-14; M-1 to G-13; M-1 to W-12; M-1 to Q-11;
M-1 to D-
10; M-1 to W-9; M-1 to Y-8 and M-1 to E-7 of SEQ ID N0:61. Polynucleotides
encoding
these polypeptides are also encompassed by the invention.
Alternatively, the present invention further provides polypeptides having one
or more
residues deleted from the carboxy terminus of the amino acid sequence of the
TR14 polypeptide
shown in Figures 4A-D (SEQ ID NO:S), up to the asparagine residue at position
number 6, and
2o polynucleotides encoding such polypeptides. In particular, the present
invention provides
polypeptides comprising, or alternatively consisting of, the amino acid
sequence of residues 1-
m' of Figures 4A-D, where m' is an integer from 6 to 226 corresponding to the
position of the
amino acid residue in Figures 4A-D (which is identical to the sequence shown
as SEQ ID
NO:S).
Moreover, the invention provides polypeptides comprising, or alternatively
consisting
of, the amino acid residues: M-I to E-225; M-1 to P-224; M-1 to G-223; M-1 to
Q-222; M-1 to
Q-221; M-1 to Q-220; M-1 to Q-219; M-1 to P-218; M-1 to I-217; M-1 to P-216; M-
1 to V-
215 ; M-1 to S-214; M-1 to D-213 ; M-1 to L-212; M-1 to S-211; M-1 to F-210; M-
1 to L-209;
M-1 to Q-208 ; M-1 to A-207 ; M-1 to L-206; M-1 to S-205 ; M-1 to G-204; M-1
to P-203 ; M-1
to A-202; M-1 to W-201; M-1 to S-200; M-1 to V -199; M-1 to Q-198; M-1 to S-
197; M-1 to E-
196; M-1 to A-195; M-1 to S-194; M-1 to T-193; M-1 to E-192; M-1 to K-191; M-1
to S-190;
M-1 to P-189; M-1 to P-188; M-1 to V-187; M-1 to P-186; M-1 to F-185; M-1 to L-
184; M-1
to S-183; M-1 to E-182; M-1 to E-181; M-1 to K-180; M-1 to A-179; M-1 to T-
178; M-1 to K-
177; M-1 to D-176; M-1 to A-175; M-1 to E-174; M-1 to F-173; M-1 to Q-172; M-1
to L-171;
M-1 to L-170; M-1 to G-169; M-1 to G-168; M-1 to R-167; M-I to Q-166; M-1 to C-
165; M-1
to H-164; M-1 to R-163; M-1 to N-162; M-1 to F-161; M-1 to F-160; M-1 to Q-
159; M-1 to K-
158; M-1 to C-157; M-1 to Y-156; M-1 to L-155; M-1 to F-154; M-1 to F-153; M-1
to L-152;
M-1 to G-151; M-1 to L-150; M-1 to F-149; M-1 to A-148; M-1 to L-147; M-1 to T-
146; M-1
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
159
to F-145; M-I to V-144; M-1 to V-143; M-1 to L-142; M-I to L-141; M-1 to S-
140; M-1 to S-
139; M-1 to V-138; M-1 to L-137; M-1 to A-136; M-I to V-135; M-1 to L-134; M-1
to T-133;
M-1 to A-132; M-1 to E-131; M-1 to Q-130; M-1 to P-129; M-1 to P-128; M-1 to V-
127; M-1
to T-126; M-1 to P-125; M-1 to A-124; M-1 to D-123; M-1 to A-122; M-1 to E-
121; M-1 to V-
120; M-1 to L-119; M-1 to S-I 18; M-I to L-117; M-1 to Q-116; M-1 to F-115; M-
1 to A-114;
M-1 to C-113; M-1 to Q-112; M-1 to V-111; M-1 to E-110; M-1 to S-109; M-1 to T-
108; M-1
to P-107; M-1 to T-106; M-1 to Q-105; M-1 to K-104; M-1 to T-103; M-I to C-
102; M-1 to P-
101; M-1 to I-100; M-1 to C-99; M-1 to E-98; M-1 to Q-97; M-1 to D-96; M-1 to
Q-95; M-1 to
L-94; M-1 to G-93; M-1 to G-92; M-1 to I-91; M-1 to R-90; M-1 to T-89; M-I to
K-88; M-1 to
to R-87; M-1 to Y-86; M-1 to F-85; M-1 to R-84; M-1 to P-83; M-1 to L-82; M-1
to C-81; M-I to
D-80; M-1 to G-79; M-1 to C-78; M-1 to V-77; M-1 to A-76; M-1 to N-75; M-1 to
S-74; M-I
to T-73; M-1 to P-72; M-1 to T-71; M-1 to C-70; M-1 to N-69; M-1 to V-68; M-1
to K-67; M-1
to Q-66; M-1 to V-65; M-1 to R-64; M-1 to N-63; M-1 to I-62; M-1 to V-61; M-1
to A-60; M-1
to C-59; M-1 to T-58; M-1 to C-57; M-1 to A-56; M-1 to V-55; M-1 to R-54; M-1
to C-53; M-1
to M-52; M-1 to Q-51; M-1 to P-50; M-1 to P-49; M-1 to G-48; M-1 to L-47; M-1
to Q-46; M-
1 to K-45; M-1 to Q-44; M-1 to V-43; M-1 to A-42; M-1 to L-41; M-1 to L-40; M-
1 to A-39;
M-1 to P-38; M-1 to Q-37; M-1 to A-36; M-1 to T-35; M-1 to P-34; M-1 to M-33;
M-1 to E-32;
M-1 to V-31; M-1 to R-30; M-I to E-29; M-1 to M-28; M-1 to V-27; M-1 to V-26;
M-1 to I-25;
M-1 to R-24; M-1 to P-23 ; M- I to Y-22; M-1 to S-21; M-1 to R-20; M-1 to D-
19; M-1 to L-18 ;
M-1 to V-17; M-1 to V-16; M-1 to G-15; M-1 to N-14; M-1 to A-13; M-1 to P-12;
M-1 to S-
11; M-1 to V-10; M-1 to G-9; M-1 to D-8; M-1 to G-7; M-1 to N-6; of SEQ ID
NO:S.
Polynucleotides encoding these polypeptides are also encompassed by the
invention.
Also as mentioned above, even if deletion of one or more amino acids from the
C-
terminus of a protein results in modification or loss of one or more
biological functions of the
protein, other functional activities (e.g., biological activities), ability to
multimerize, ability to
bind TR14 ligand) may still be retained. For example the ability of the
shortened TR14 mutein
to induce and/or bind to antibodies which recognize the complete or mature
forms of the
polypeptide generally will be retained when less than the majority of the
residues of the
complete or mature polypeptide are removed from the C-terminus. Whether a
particular
3o polypeptide lacking C-terminal residues of a complete polypeptide retains
such immunologic
activities can readily be determined by routine methods described herein and
otherwise known
in the art. It is not unlikely that an TR14 mutein with a large number of
deleted C-terminal
amino acid residues may retain some biological or immunogenic activities. 1n
fact, peptides
composed of as few as six TR14 amino acid residues may often evoke an immune
response.
The present invention further provides polypeptides having one or more
residues
deleted from the carboxy terminus of the amino acid sequence of the TR14
polypeptide shown
in Figures 4A-D (SEQ ID NO:S) and polynucleotides encoding such polypeptides.
In
particular, the present invention provides polypeptides comprising, or
alternatively consisting
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
160
of, the amino acid sequence of residues 133-m' of Figures 4A-D, where m' is an
integer from
6 to 132 corresponding to the position of the amino acid residue in Figures 4A-
D (which is
identical to the sequence shown as SEQ ID NO:S).
Moreover, the invention provides TR14 polypeptides comprising, or
alternatively
consisting of, the amino acid sequence of residues: M-1 to A-132; M-1 to E-
131; M-1 to Q-
130; M-1 to P-129; M-1 to P-128; M-1 to V-127; M-1 to T-126; M-1 to P-125; M-1
to A-124;
M-1 to D-123; M-1 to A-122; M-1 to E-121; M-1 to V-120; M-1 to L-119; M-1 to S-
118; M-1
to L-117; M-1 to Q-116; M-1 to F-115; M-1 to A-114; M-1 to C-113; M-1 to Q-I
12; M-1 to V-
111; M-1 to E-110; M-1 to S-109; M-1 to T-108; M-1 to P-107; M-1 to T-106; M-1
to Q-105;
t0 M-1 to K-104; M-1 to T-103; M-1 to C-102; M-1 to P-101; M-1 to I-100; M-1
to C-99; M-1 to
E-98; M-1 to Q-97; M- I to D-96; M-1 to Q-95; M-1 to L-94; M-1 to G-93 ; M-1
to G-92; M-1
to I-91; M-1 to R-90; M-1 to T-89; M-1 to K-88; M-1 to R-87; M-1 to Y-86; M-1
to F-85; M-1
to R-84; M-1 to P-83; M-1 to L-82; M-1 to C-81; M-1 to D-80; M-1 to G-79; M-1
to C-78; M-1
to V-77; M-1 to A-76; M-1 to N-75; M-1 to S-74; M-1 to T-73; M-1 to P-72; M-1
to T-71; M-1
to C-70; M-1 to N-69; M-1 to V-68; M-1 to K-67; M-1 to Q-66; M-1 to V-65; M-1
to R-64; M-
1 to N-63; M-1 to I-62; M-1 to V-61; M-1 to A-60; M-1 to C-59; M-1 to T-58; M-
1 to C-57; M-
1 to A-56; M-1 to V-55; M-1 to R-54; M-1 to C-53; M-1 to M-52; M-1 to Q-51; M-
1 to P-50;
M-1 to P-49; M-1 to G-48; M-1 to L-47; M-1 to Q-46; M-1 to K-45; M-1 to Q~4; M-
1 to V-
43 ; M-1 to A-42; M-1 to L-41; M-1 to L-40; M-1 to A-39; M-1 to P-3 8 ; M-1 to
Q-37; M-1 to
2o A-36; M-1 to T-35; M-1 to P-34; M-1 to M-33; M-1 to E-32; M-1 to V-31; M-1
to R-30; M-1 to
E-29; M-1 to M-28; M-1 to V-27; M-1 to V-26; M-1 to I-25; M-1 to R-24; M-1 to
P-23; M-1 to
Y-22; M-1 to S-21; M-1 to R-20; M-1 to D-19; M-1 to L-18; M-1 to V-17; M-1 to
V-16; M-1 to
G-15; M-1 to N-14; M-1 to A-13; M-1 to P-12; M-1 to S-11; M-1 to V-10; M-1 to
G-9; M-1 to
D-8; M-1 to G-7; M-1 to N-6; of SEQ ID N0:7. Polynucleotides encoding these
polypeptides
are also encompassed by the invention.
The invention also provides polypeptides having one or more amino acids
deleted from
both the amino and the carboxyl termini, which may be described generally as
having residues
n'- m', n2- m', n'-mz and/or n2-mZ, where n', n2, m', and m2 are integers as
described above.
Thus, any of the above listed N- or C-terminal deletions can be combined to
produce an N- and
C-terminal deleted TR14 polypeptide.
It will be recognized in the art that some amino acid sequences of TR14
polypeptides
can be varied without significant effect on the structure or function of the
protein. If such
differences in sequence are contemplated, it should be remembered that there
will be critical
areas on the protein which determine activity. Thus, the invention further
includes variations of
the TR14 polypeptide, which show substantial TR14 receptor activity or which
include regions
of TR14 polypeptides, such as the polypeptide portions discussed herein. Such
mutants
include deletions, insertions, inversions, repeats, and type substitutions. As
indicated above,
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
161
guidance concerning which amino acid changes are likely to be phenotypically
silent can be
found in J.U. Bowie et al., Science 247:1306-1310 (1990).
Thus, the fragment, derivative, or analog of the polypeptide encoded by the
cDNA
deposited as ATCC Deposit No. PTA-348 or shown, preferably, in SEQ ID N0:61
or,
alternatively, in SEQ ID N0:5, 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
to substituent group, or (iii) one in which the mature polypeptide is fused
with another
compound, such as a compound to increase the half-life of the polypeptide (for
example,
polyethylene glycol), or (iv) one in which the additional amino acids are
fused to the mature
polypeptide, such as an IgG Fc fusion region peptide or leader or secretory
sequence or a
sequence which is employed for purification of the mature polypeptide or a
proprotein
sequence. Such fragments, derivatives and analogs are deemed to be within the
scope of those
skilled in the art from the teachings herein.
Of particular interest are substitutions of charged amino acids with another
charged
amino acid and with neutral or negatively charged amino acids. The latter
results in proteins
with reduced positive charge to improve the characteristics of the TR14
polypeptide. The
prevention of aggregation is highly desirable. Aggregation of proteins not
only results in a loss
of activity but can also be problematic when preparing pharmaceutical
formulations, because
they can be immunogenic. (Pinckard et al., Clin Exp. Immunol. 2:331-340
(1967); Robbins et
al., Diabetes 36:838-845 (1987); Cleland et al. Crit. Rev. Therapeutic Drug
Carrier Systems
70:307-377 (1993)).
The replacement of amino acids can also change the selectivity of binding to
cell surface
receptors. Ostade et al., Nature 361:266-268 (1993), describes certain
mutations resulting in
selective binding of TNF-a to only one of the two known types of TNF
receptors. Thus, the
TR14 receptor of the present invention may include one or more amino acid
substitutions,
deletions, or additions, either from natural mutations or human manipulation.
3o 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 IV).
In specific embodiments, the number of substitutions, additions or deletions
in the
amino acid sequence of Figures l0A-H (SEQ ID N0:61) and/or any of the
polypeptide
fragments described herein (e.g., the cysteine-rich domain, 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.
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
162
In additional embodiments, the number of substitutions, additions or deletions
in the
amino acid sequence of Figures 4A-D (SEQ ID NO:S) and/or any of the
polypeptide fragments
described herein (e.g., the cysteine-rich domain, 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-S, 1-3 or 1-2.
Amino acids in the TR14 polypeptides 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
to resulting mutant molecules are then tested for biological activity such as
receptor binding or in
vitro proliferative activity. Sites that are critical for ligand-receptor
binding can also be
determined by structural analysis such as crystallization, nuclear magnetic
resonance or
photoaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904 (1992) and de
Vos et al. Science
255:306-312 (1992)).
To improve or alter the characteristics of TR14 polypeptides, protein
engineering may
be employed. Recombinant DNA technology known to those skilled in the art can
be used to
create novel mutant proteins or "muteins including single or multiple amino
acid substitutions,
deletions, additions or fusion proteins. Such modified polypeptides can show,
e.g., enhanced
activity or increased stability. In addition, they may be purified in higher
yields and show
2o better solubility than the corresponding natural polypeptide, at least
under certain purification
and storage conditions.
Non-naturally occurring variants may be produced using art-known mutagenesis
techniques, which include, but are not limited to oligonucleotide mediated
mutagenesis, alanine
scanning, PCR mutagenesis, site directed mutagenesis (see e.g., Carter et al.,
Nucl. Acids
Res. 13:4331 (1986); and Zoller et al., Nucl. Acids Res. 10:6487 (1982)),
cassette
mutagenesis (see e.g., Wells et al., Gene 34:315 (1985)), restriction
selection mutagenesis
(see e.g., Wells et al., Philos. Trans. R. Soc. London SerA 317:415 (1986)).
Thus, the invention also encompasses TR14 derivatives and analogs that have
one or
more amino acid residues deleted, added, or substituted to generate TR14
polypeptides that are
3o 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 TR14 polypeptides of
the invention,
and/or an amino acid deletion at the second position of any one or more such
recognition
sequences will prevent glycosylation of the TR14 at the modified tripeptide
sequence (see, e.g.,
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
163
Miyajimo et al., EMBO J 5(6):1193-1197). Additionally, one or more of the
amino acid
residues of the polypeptides of the invention (e.g., arginine and lysine
residues) may be deleted
or substituted with another residue to eliminate undesired processing by
proteases such as, for
example, furins or kexins.
The polypeptides of the present invention include a polypeptide comprising, or
alternatively, consisting of the polypeptide encoded by the cDNA deposited as
ATCC Deposit
No. PTA-348; a polypeptide comprising, or alternatively, consisting of amino
acids from 1 to
about 231 of SEQ ID N0:61 or from 1 to about 226 of SEQ ID NO:S; a polypeptide
comprising, or alternatively, consisting of amino acids from about from 2 to
about 231 of SEQ
1o ID N0:61 or 2 to about 226 of SEQ ID NO:S; a polypeptide comprising, or
alternatively,
consisting of amino acids from from 1 to about 138 of SEQ ID N0:61 or from 1
to about 133
of SEQ ID NO:S; a polypeptide comprising, or alternatively, consisting of the
extracellular
domain of the polypeptide encoded by the cDNA deposited as ATCC Deposit No.
PTA-348; a
polypeptide comprising, or alternatively, consisting of the cysteine rich
domain of the
polypeptide encoded by the cDNA deposited as ATCC Deposit No. PTA-348, or as
shown in
amino acids about 31 to about 104 of SEQ ID N0:61, or shown in amino acids
from about 65
to about 85 of SEQ ID NO:S; a polypeptide comprising, or alternatively,
consisting of the
transmembrane domain of the polypeptide encoded by the cDNA deposited as ATCC
Deposit
No. PTA-348 (predicted to constitute amino acids from about 139 to about 155
of SEQ ID
2o N0:61 or from 134 to about 150 of SEQ ID NO:S); a polypeptide comprising,
or alternatively,
consisting of the intracellular domain (predicted to constitute amino acids
from about 155 to
about 231 of SEQ ID N0:61 or from about 151 to about 226 of SEQ ID NO:S); a
polypeptide
comprising, or alternatively, consisting of the extracellular and
intracellular domains with all or
part of the transmembrane domain deleted; as well as polypeptides which are at
least 80%
identical, more preferably at least 90% or 95% identical, still more
preferably at least 96%,
97%, 98%, or 99% identical to the polypeptides described above (e.g., the
polypeptide
encoded by the cDNA in ATCC Deposit No. PTA-348, the polypeptide of Figures
l0A-H
(SEQ ID N0:61); or the polypeptide of Figures 4A-D (SEQ ID NO:S)) or
polypeptide
fragments thereof, such as those disclosed herein), and also include portions
of such
3o polypeptides with at least 30 amino acids and more preferably at least 50
amino acids. 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. Polynucleotides
encoding these
polypeptides are also encompassed by the invention.
By a polypeptide (protein) comprising, or alternatively consisting of, an
amino acid
sequence at least, for example, 95% "identical" to a reference amino acid
sequence of a TR14
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 TR14
polypeptide. In
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
164
other words, to obtain a polypeptide having an amino acid sequence at least
95% identical to a
reference amino acid sequence, up to 5% of the amino acid residues in the
reference sequence
may be deleted or substituted with another amino acid, or a number of amino
acids up to 5% of
the total amino acid residues in the reference sequence may be inserted into
the reference
sequence. These alterations of the reference sequence may occur at the amino
or carboxy
terminal positions of the reference amino acid sequence or anywhere between
those terminal
positions, interspersed either individually among residues in the reference
sequence or in one or
more contiguous groups within the reference sequence.
As a practical matter, whether any particular polypeptide is at least 90%,
95%, 96%,
97%, 98%, or 99% identical to, for instance, the amino acid sequence shown in
SEQ ID NO:S,
or to the amino acid sequence encoded by the cDNA deposited as ATCC Deposit
No. PTA
348, 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.
2o 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
3o 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
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
165
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
t5 calculated by FASTDB is not manually corrected. Once again, only residue
positions outside
the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB
alignment,
which are not matched/aligned with the query sequence are manually corrected
for. No other
manual corrections are made for the purposes of this embodiment.
The present application is also directed to proteins cotaining polypeptides at
least 90%,
95%, 96%, 97%, 98% or 99% identical to the TR14 polypeptide sequence set forth
as n'-m',
n2- m', n'- m2, and/or n2-m2 described herein. In preferred embodiments, the
application is
directed to proteins comprising, or alternatively consisting of, polypeptide
sequence at least
90%, 95%, 96%, 97%, 98% or 99% identical to polypeptides having the amino acid
sequence
of the specific TR14 N- and C-terminal deletions recited herein.
Polynucleotides encoding
these polypeptides are also encompassed by the invention.
In certain preferred embodiments, TR14 proteins of the invention comprise
fusion
proteins as described above wherein the TR14 polypeptides are those described
as n'-m',
and/or n2- m' herein. In preferred embodiments, the application is directed to
nucleic acid
molecules at least 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic
acid sequences
3o encoding polypeptides having the amino acid sequence of the specific N- and
C-terminal
deletions recited herein. Polynucleotides encoding these polypeptides are also
encompassed by
the invention.
In another aspect, the invention provides a TR14 polypeptide comprising an
epitope-
bearing portion of a polypeptide of the invention.. The epitope of this
polypeptide portion is an
immunogenic or antigenic epitope of a polypeptide described herein. An
"immunogenic
epitope" is defined as a part of a protein that elicits an antibody response
when the whole
protein is the immunogen. On the other hand, a region of a protein molecule to
which an
antibody can bind is defined as an "antigenic epitope." The number of
immunogenic epitopes
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
166
of a protein generally is less than the number of antigenic epitopes. See, for
instance, Geysen
et al., Proc. Natl. Acad. Sci. USA 87:3998- 4002 (1983).
As to the selection of peptides or polypeptides bearing an antigenic epitope
(i.e., that
contain a region of a protein molecule to which an antibody can bind), it is
well known in that
art that relatively short synthetic peptides that mimic part of a protein
sequence are routinely
capable of eliciting an antiserum that reacts with the partially mimicked
protein. See, for
instance, J.G. Sutcliffe et al., "Antibodies That React With Predetermined
Sites on Proteins,"
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.
Antigenic epitope-bearing peptides and polypeptides of the invention are
therefore
useful to raise antibodies, including monoclonal antibodies, that bind
specifically to a
polypeptide of the invention. See, for instance, Wilson et al., Cell 37:767-
778 ( 1984) at 777.
Antigenic epitope-bearing peptides and polypeptides of the invention
preferably contain a
sequence of at least seven, more preferably at least nine, at least 20, at
least 25, at least 30, at
least 40, at least 50 and most preferably between at least about SS to about
100 amino acids
contained within the amino acid sequence of a polypeptide of the invention. In
this context
"about" includes the particularly recited ranges, larger or smaller by several
(5, 4, 3, 2, or 1)
2o amino acids, at either extreme or at both extremes.
Non-limiting examples of predicted antigenic polypeptides that can be used to
generate
TR14-specific antibodies include polypeptides comprising about: Asp-2 to Asp-
10, Thr-17 to
Asp-38, Pro-45 to Ser-52, Pro-88 to Arg-95, Thr-108 to Glu-115, Thr-131 to Glu-
136, Phe-
166 to Gly-174, Ala-180 to Ala-200, and Gln-224 to Met-231 of SEQ ID N0:61.
Fragments
and/or variants of these polypeptides, such as, for example, fragments and/or
variants as
described herein, are encompassed by the invention. Polynucleotides encoding
these
polypeptides (including fragments and/or variants) are also encompassed by the
invention, as
are antibodies that bind these polypeptides.
Additional non-limiting examples of predicted antigenic polypeptides that can
be used to
generate TR14-specific antibodies include: a polypeptide comprising, or
alternatively consisting
of amino acid residues from about 2 to about 24 in Figures 4A-D (corresponding
to about
amino acid 2 to about 24 in SEQ ID NO:S); a polypeptide comprising amino acid
residues from
about 42 to about 52 in Figures 4A-D (corresponding to about amino acid 42 to
about 52 in
SEQ ID NO:S); a polypeptide comprising amino acid residues from about 80 to
about 115 in
Figures 4A-D (corresponding to about amino acid 80 to about 115 in SEQ ID
NO:S); and a
polypeptide comprising amino acid residues from about 155 to about 226 in
Figures 4A-D
(corresponding to about amino acid 155 to about 226 in SEQ ID NO:S), and the
corresponding
amino acid sequences of SEQ ID N0:61, as the sequence of amino acid residues T-
78 to M-
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
167
231 of SEQ ID N0:61 is identical to the sequence of amino acid residues T-73
to M-226 of
SEQ ID NO:S. As indicated above, the inventors have determined that the above
polypeptide
fragments are antigenic regions of the TR14 receptor protein. 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. Polynucleotides encoding these
polypeptides are also
encompassed by the invention.
Additional non-limiting examples of predicted antigenic polypeptides that can
be used to
generate TR14-specific antibodies include: a polypeptide comprising, or
alternatively consisting
of, amino acid residues from about T3 to about S11, from about V 16 to about
R24, from about
to Q44 to about M52, from about F85 to about G93, from about T103 to about V 1
I 1, from about
F161 to about 6169, from about V 187 to about A 195, from about P218 to about
M226 of SEQ
ID NO:S (Figures 4A-D, and the corresponding amino acid sequences of SEQ ID
N0:61, as
the sequence of amino acid residues T-78 to M-231 of SEQ ID N0:61 is identical
to the
sequence of amino acid residues T-73 to M-226 of SEQ ID NO:S) correspond to
the highly
antigenic regions of the TR14 protein, predicted using the Jameson-Wolf
antigenic index (See
Figure 6 and Table II). These highly antigenic fragments correspond to the
amino acid residues
illustrated in Figure 4A-D and in SEQ ID NO:S. 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. Polynucleotides encoding these polypeptides are also
encompassed by the
invention.
The epitope-bearing peptides and polypeptides of the invention may be produced
by any
conventional means. R.A. Houghten, "General Method for the Rapid Solid-phase
Synthesis of
Large Numbers of Peptides: Specificity of Antigen-Antibody Interaction at the
Level of
Individual Amino Acids," Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985). This
"Simultaneous Multiple Peptide Synthesis (SMPS)" process is further described
in U.S. Patent
No. 4,631,211 to Houghten et al. (1986).
As one of skill in the art will appreciate, TR14 receptor polypeptides of the
present
invention 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 about 149,
from about 2 to about 24, from about 42 to about 52, from about 80 to about
115, and/or from
about 155 to about 226 of SEQ ID N0:5), can be combined with heterologous
polypeptide
sequences, for example, the polypeptides of the present invention may be fused
with the
constant domain of immunoglobulins (IgA, IgE, IgG, IgM) or portions thereof
(CH1, CH2,
CH3, and any combination thereof, including both entire domains and portions
thereof),
resulting in chimeric polypeptides. These fusion proteins facilitate
purification and show an
increased half-life 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.,
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
168
Nature 337:84-86 (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 TR14 protein or protein fragment alone (Fountoulakis et al., J.
Biochem.
270:3958-3964 (1995)). 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.
Preferred TR14 Fc fusions of the present invention include, but are not
limited to
constructs comprising, or alternatively consisting of, amino acid residues: 1
to 138, 50 to 138,
70 to 90, 1 to 231, 10 to 231, 20 to 231, 30 to 231, 40 to 231, 1 to 221, 1 to
211, 1 to 201, 1
to 191, 10 to 221, 10 to 201, and/or 10 to 191 of SEQ ID N0:61.
Polynucleotides encoding
these TR14 fusions are also encompassed by the invention.
Additonal TR14 Fc fusions of the present invention include, but are not
limited to
constructs comprising, or alternatively consisting of, amino acid residues: 1
to 133, 50 to 133,
65 to 85, 1 to 226, 10 to 226, 20 to 226, 30 to 226, 40 to 226, 1 to 216, 1 to
206, 1 to 196, 1
to 186, 10 to 216, 10 to 206, and/or 10 to 196 of SEQ ID NO:S. Polynucleotides
encoding
t5 these TR14 fusions are also encompassed by the invention.
The polypeptides of the present invention have uses which include, but are not
limited
to, as sources for generating antibodies that bind the polypeptides of the
invention, and as
molecular weight markers on SDS-PAGE gels or on molecular sieve gel filtration
columns
using methods well known to those of skill in the art.
Diagnostic Assays
The compounds of the present invention are useful for diagnosis or treatment
of various
immune system-related disorders in mammals, preferably humans. Such disorders
include but
are not limited to tumors. (e.g., T cell, B cell and monocytic cell leukemias
and lymphomas)
and tumor metastasis, infections by bacteria, viruses and other parasites,
immunodeficiencies,
inflammatory diseases, lymphadenopathy, autoimmune diseases, and graft versus
host disease.
TR13 and TR14 are expressed in immune cells and tissue. For a number of immune
system-related disorders, substantially altered (increased or decreased)
levels TR13 and/or
TR14 gene expression can be detected in immune system tissue or other cells or
bodily fluids
(e.g., sera, plasma, urine, synovial fluid or spinal fluid) taken from an
individual having such a
disorder, relative to a "standard" TR13 and/or TR14 gene expression level,
that is, the TR13
and/or TR14 expression level in immune system tissues or bodily fluids from an
individual not
having the immune system disorder. Thus, the invention provides a diagnostic
method useful
during diagnosis of an immune system disorder, which involves measuring the
expression
level of the gene encoding the TR13 and/or TR14 polypeptide in immune system
tissue or other
cells or body fluid from an individual and comparing the measured gene
expression level with a
standard TR13 and/or TR14 gene expression level, respectively, whereby an
increase or
decrease in the gene expression level compared to the standard is indicative
of an immune
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
169
system disorder or normal activation, proliferation, differentiation, and/or
death.
In particular, it is believed that certain tissues in mammals with cancer
(such as, for
example, cancer of cells or tissue of the immune system) express significantly
enhanced or
reduced levels of normal or altered TR13 and/or TR14 polypeptide and mRNA
encoding the
TR13 and/or TR14 polypeptide when compared to a corresponding "standard"
level. Further,
it is believed that enhanced or depressed levels of the TR13 and/or TR14
polypeptide can be
detected in certain body fluids (e.g., sera, plasma, urine, and spinal fluid)
or cells or tissue
from mammals with such a cancer when compared to sera from mammals of the same
species
not having the cancer.
to For example, polynucleotides of the invention (e.g., polynucleotide
sequences
complementary to all or a portion of TR13 and/or TR14 mRNA) and antibodies
(and antibody
fragments) directed against the polypeptides of the invention may be used to
quantitate or
qualitate concentrations of cells of T cell lineage and/or B cell lineage
(e.g., B cell leukemia
cells) expressing TR13 and/or TR14 on their cell surfaces. These antibodies
additionally have
diagnostic applications in detecting abnormalities in the level of TR13 and/or
TR14 gene
expression, or abnormalities in the structure and/or temporal, tissue,
cellular, or subcellular
location of TR13 and/or TR14. These diagnostic assays may be performed in vivo
or in vitro,
such as, for example, on blood samples, biopsy tissue or autopsy tissue.
For example, as disclosed herein, TR13 or TR14 is expressed in T cells.
Accordingly,
2o polynucleotides of the invention (e.g., polynucleotide sequences
complementary to all or a
portion of TR13 or TR14 mRNA) and antibodies (and antibody fragments) directed
against the
polypeptides of the invention may be used to quantitate or qualitate
concentrations of cells of T
cell lineage (e.g., T cell leukemia cells) expressing TR13 or TR14 on their
cell surfaces. These
polypeptides and antibodies additionally have diagnostic applications in
detecting abnormalities
in the level of TR13 or TR14 gene expression, or abnormalities in the
structure and/or
temporal, tissue, cellular, or subcellular location of TR13 or TR14. These
diagnostic assays
may be performed in vivo or in vitro, such as, for example, on blood samples,
biopsy tissue or
autopsy tissue.
Thus, the invention provides a diagnostic method useful during diagnosis of a
immune
system disorder (including cancers of this system) and/or cell proliferation
disorder (e.g.,
cancer, such as a cancer disclosed herein) which involves measuring the
expression level of the
gene encoding the TR13 and/or TR14 polypeptide in immune system tissue or
other cells or
body fluid from an individual and comparing the measured gene expression level
with a
standard TR13 and/or TR14 gene expression level, whereby an increase or
decrease in the gene
expression level compared to the standard is indicative of an immune system
disorder and/or
cell proliferation disorder.
Where a diagnosis of a disorder in the immune system (including diagnosis of a
tumor)
and/or diagnosis of a cell proliferation disorder has already been made
according to
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
170
conventional methods, the present invention is useful as a prognostic
indicator, whereby
patients exhibiting enhanced or depressed TR13 and/or TR14 gene expression
will experience a
worse clinical outcome relative to patients expressing the gene at a level
nearer the standard
level.
By "assaying the expression level of the gene encoding the TR13 and/or TR14
polypeptide" is intended qualitatively or quantitatively measuring or
estimating the level of the
TR13 and/or TR14 polypeptide or the level of the mRNA encoding the TR13 and/or
TR14
polypeptide in a first biological sample either directly (e.g., by determining
or estimating
absolute protein level or mRNA level) or relatively (e.g., by comparing to the
TR13 and/or
to TR14 polypeptide level or mRNA level in a second biological sample).
Preferably, the TR13
and/or TR14 polypeptide level or mRNA level in the first biological sample is
measured or
estimated and compared to a standard TR13 and/or TR14 polypeptide level or
mRNA level, the
standard being taken from a second biological sample obtained from an
individual not having
the disorder or being determined by averaging levels from a population of
individuals not
having a disorder of the immune system. As will be appreciated in the art,
once a standard
TR13 and/or TR14 polypeptide level or mRNA level is known, it can be used
repeatedly as a
standard for comparison.
By "biological sample" is intended any biological sample obtained from an
individual,
cell line, tissue culture, or other source containing TR13 and/or TR14
receptor protein
(including portions thereof) or mRNA. As indicated, biological samples include
body fluids
(such as sera, plasma, urine, synovial fluid and spinal fluid) which contain
free extracellular
domains of the TR13 and/or TR14 polypeptide, immune system tissue, and other
tissue
sources found to express complete or free extracellular domain of the TR13
and/or TR14
receptor. 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.
Total cellular RNA can be isolated from a biological sample using any suitable
technique such as, for example, the single-step guanidinium-thiocyanate-phenol-
chloroform
method described in Chomczynski and Sacchi, Anal. Biochem. 162:156-159 (1987).
Levels
of mRNA encoding the TR13 and/or TR14 polypeptide are then assayed using any
appropriate
method. These include Northern blot analysis, S1 nuclease mapping, the
polymerase chain
reaction (PCR), reverse transcription in combination with the polymerase chain
reaction
(RT-PCR), and reverse transcription in combination with the ligase chain
reaction (RT-LCR).
The present invention also relates to diagnostic assays such as quantitative
and
diagnostic assays for detecting levels of TR13 polypeptide, or the soluble
form thereof, in cells
and tissues, including determination of normal and abnormal levels. Thus, for
instance, a
diagnostic assay in accordance with the invention for detecting over-
expression of TR13, or
soluble form thereof, compared to normal control tissue samples may be used to
detect the
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
171
presence of tumors, for example. Assay techniques that can be used to
determine levels of a
protein, such as a TR13 polypeptide of the present invention, or a soluble
form thereof, in a
biological sample derived from a host are well-known to those of skill in the
art. Such assay
methods include radioimmunoassays, competitive-binding assays, Western Blot
analysis and
ELISA assays. Preferred for assaying TR13 polypeptide levels in a biological
sample are
antibody-based techniques. For example, TR13 polypeptide expression in tissues
can be
studied with classical immunohistological methods. (M. Jalkanen et al., J.
Cell. Biol.
101:976-985 (1985); M. Jalkanen et al., J. Cell. Biol. 705:3087-3096 (1987)).
Other
antibody-based methods useful for detecting TR13 gene expression include
immunoassays,
such as the enzyme linked immunosorbent assay (EL1SA) and the radioimmunoassay
(RIA).
The present invention also relates to diagnostic assays such as quantitative
and
diagnostic assays for detecting levels of TR14 polypeptide, or the soluble
form thereof, in cells
and tissues, including determination of normal and abnormal levels. Thus, for
instance, a
diagnostic assay in accordance with the invention for detecting over-
expression of TR14, or
~5 soluble form thereof, compared to normal control tissue samples may be used
to detect the
presence of tumors, for example. Assay techniques that can be used to
determine levels of a
protein, such as a TR14 polypeptide of the present invention, or a soluble
form thereof, in a
biological sample derived from a host are well-known to those of skill in the
art. Such assay
methods include radioimmunoassays, competitive-binding assays, Western Blot
analysis and
2o ELISA assays. Preferred for assaying TR14 polypeptide levels in a
biological sample are
antibody-based techniques. For example, TR14 polypeptide expression in tissues
can be
studied with classical immunohistological methods. (M. Jalkanen et al., J.
Cell. Biol.
101:976-985 (1985); M. Jalkanen et al., J. Cell. Biol. 705:3087-3096 (1987)).
Other
antibody-based methods useful for detecting TR14 gene expression include
immunoassays,
25 such as the enzyme linked immunosorbent assay (ELISA) and the
radioimmunoassay (RIA).
Suitable antibody assay labels are known in the art and include enzyme labels,
such as
glucose oxidase, and radioisotopes, such as iodine ('3'I~ 125I~ 123I~ lz~I)~
carbon ('4C), sulfur
(3s5), tritium (3H), indium ("smln, "3'"In, "zln, "'In), and technetium (99Tc,
99mTC), thallium
(zo~Ti), gallium (6gGa, 6'Ga), palladium ('°3Pd), molybdenum (99Mo),
xenon ('33Xe), fluorine
30 (~sF)> >s3sm~ »~Lu~ 159Gd' 149Pm' ~aoLa~ ms~,b~ 166Ho~ 9oY~ a~Sc~ is6Re~
~ggRe~ iazPr~ iosRh~
9'Ru; luminescent labels, such as luminol; and fluorescent labels, such as
fluorescein and
rhodamine, and biotin.
The tissue or cell type to be analyzed will generally include those which are
known, or
suspected, to express the TR13 and/or TR14 gene (such as, for example, cells
of T cell
35 lineage) or cells or tissue which are known, or suspected, to express the
TR13 ligand and/or
TR14 ligand gene (such as, for example, cells of monocytic lineage and the
spleen). The
protein isolation methods employed herein may, for example, be such as those
described in
Harlow and Lane (Harlow, E. and Lane, D., 1988, "Antibodies: A Laboratory
Manual", Cold
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
172
Spring Harbor Laboratory Press, Cold Spring Harbor, New York), which is
incorporated
herein by reference in its entirety. The isolated cells can be derived from
cell culture or from a
patient. The analysis of cells taken from culture may be a necessary step in
the assessment of
cells that could be used as part of a cell-based gene therapy technique or,
alternatively, to test
the effect of compounds on the expression of the TR13 and/or TR14 gene or TR13
ligand
and/or TR14 ligand gene.
For example, antibodies, or fragments of antibodies, such as those described
herein, may
be used to quantitatively or qualitatively detect the presence of TR13 and/or
TR14 gene
products or conserved variants or peptide fragments thereof. This can be
accomplished, for
to example, by immunofluorescence techniques employing a fluorescently labeled
antibody
coupled with light microscopic, flow cytometric, or fluorimetric detection.
The antibodies (or fragments thereof) or TR13 and/or TR14 polypeptides or TR13
ligand
and/or TR14 ligand polypeptides of the present invention may, additionally, be
employed
histologically, as in immunofluorescence, immunoelectron microscopy or non-
immunological
assays, for in situ detection of TR13 and/or TR14 gene products or conserved
variants or
polypeptide fragments thereof, or for TR13 and/or TR14 binding to TR13 and/or
TR14 ligand,
respectively. In situ detection may be accomplished by removing a histological
specimen from
a patient, and applying thereto a labeled antibody, TR13 polypeptide, or TR14
polypeptide of
the present invention. The antibody (or fragment) or TR13 and/or TR14
polypeptide is
2o preferably applied by overlaying the labeled antibody (or fragment) onto a
biological sample.
Through the use of such a procedure, it is possible to determine not only the
presence of the
TR13 and/or TR14 gene product, or conserved variants or peptide fragments, or
TR13 and/or
TR14 polypeptide binding, but also its distribution in the examined tissue.
Using the present
invention, those of ordinary skill will readily perceive that any of a wide
variety of histological
methods (such as staining procedures) can be modified in order to achieve such
in situ
detection.
Immunoassays and non-immunoassays for TR13 and/or TR14 gene products or
conserved variants or peptide fragments thereof will typically comprise
incubating a sample,
such as a biological fluid, a tissue extract, freshly harvested cells, or
lysates of cells which have
been incubated in cell culture, in the presence of a detectably labeled
antibody capable of TR13
and/or TR14 gene products or conserved variants or peptide fragments thereof,
and detecting
the bound antibody by any of a number of techniques well-known in the art.
Immunoassays and non-immunoassays for TR13 ligand and/or TR14 ligand gene
products or conserved variants or peptide fragments thereof will typically
comprise incubating a
sample, such as a biological fluid, a tissue extract, freshly harvested cells,
or lysates of cells
which have been incubated in cell culture, in the presence of a detectable or
labeled TR13
and/or TR14 polypeptide capable of identifying TR13 ligand and/or TR14 ligand
gene products
or conserved variants or polypeptide fragments thereof, and detecting the
bound TR13 and/or
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
173
TR14 polypeptide by any of a number of techniques well-known in the art.
The biological sample may be brought in contact with and immobilized onto a
solid phase
support or carrier such as nitrocellulose, or other solid support which is
capable of
immobilizing cells, cell particles or soluble proteins. The support may then
be washed with
suitable buffers followed by treatment with the detectably labeled anti-TR13
and/or TR14
antibody or detectable TR13 and/or TR14 polypeptide. The solid phase support
may then be
washed with the buffer a second time to remove unbound antibody or
polypeptide. Optionally
the antibody is subsequently labeled. The amount of bound label on solid
support may then be
detected by conventional means.
1o By "solid phase support or carrier" is intended any support capable of
binding an antigen
or an antibody. Well-known supports or carriers include glass, polystyrene,
polypropylene,
polyethylene, dextran, nylon, amylases, natural and modified celluloses,
polyacrylamides,
gabbros, and magnetite. The nature of the carrier can be either soluble to
some extent or
insoluble for the purposes of the present invention. The support material may
have virtually
any possible structural configuration so long as the coupled molecule is
capable of binding to
an antigen or antibody. Thus, the support configuration may be spherical, as
in a bead, or
cylindrical, as in the inside surFace of a test tube, or the external surface
of a rod. Alternatively,
the surface may be flat such as a sheet, test strip, etc. Preferred supports
include polystyrene
beads. Those skilled in the art will know many other suitable carriers for
binding antibody or
antigen, or will be able to ascertain the same by use of routine
experimentation.
Assaying TR13 or TR14 protein levels in a biological sample can occur using
any art-known
method.
The binding activity of a given lot of anti-TR13 and/or anti-TR14 antibody or
TR13
and/or TR14 polypeptide may be determined according to well known methods.
Those
skilled in the art will be able to determine operative and optimal assay
conditions for each
determination by employing routine experimentation.
In addition to assaying TR13 and/or TR14 polypeptide levels or polynucleotide
levels in a
biological sample obtained from an individual, TR13 and/or TR14 polypeptide or
polynucleotide can also be detected in vivo by imaging. For example, in one
embodiment of
3o the invention, TR13 and/or TR14 polypeptide is used to image monocytic
leukemias or
lymphomas. In another embodiment, TR13 and/or TR14 polynucleotides of the
invention
(e.g., polynucleotides complementary to all or a portion of TR13 and/or TR14
mRNA) is used
to image T cell leukemias or lymphomas.
Antibody labels or markers for in vivo imaging of TR13 and/or TR14 polypeptide
include those detectable by X-radiography, NMR, MRI, CAT-scans or ESR. For
X-radiography, suitable labels include radioisotopes such as barium or cesium,
which emit
detectable radiation but are not overtly harmful to the subject. Suitable
markers for NMR and
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
174
ESR include those with a detectable characteristic spin, such as deuterium,
which may be
incorporated into the antibody by labeling of nutrients for the relevant
hybridoma. Where in
vivo imaging is used to detect enhanced levels of TR13 and/or TR14 polypeptide
for diagnosis
in humans, it may be preferable to use human antibodies or "humanized"
chimeric monoclonal
antibodies. Such antibodies can be produced using techniques described herein
or otherwise
known in the art. For example methods for producing chimeric antibodies are
known in the
art. See, for review, Morrison, Science 229:1202 (1985); Oi et al.,
BioTechnigues 4:214
(1986); Cabilly et al., U.S. Patent No. 4,816,567; Taniguchi et al., EP
171496; Morrison et
al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671;
Boulianne et
al., Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985).
Additionally, any TR13 and/or TR14 polypeptide whose presence can be detected,
can be
administered. For example, TR13 and/or TR14 polypeptides labeled with a radio-
opaque or
other appropriate compound can be administered and visualized in vivo, as
discussed, above
for labeled antibodies. Further such TR13 and/or TR14 polypeptides can be
utilized for in vitro
diagnostic procedures.
A TR13 and/or TR14 polypeptide-specific antibody or antibody fragment which
has
been labeled with an appropriate detectable imaging moiety, such as a
radioisotope (for
example,'3'I, "zln, ~'mTc), a radio-opaque substance, or a material detectable
by nuclear
magnetic resonance, is introduced (for example, parenterally, subcutaneously
or
2o intraperitoneally) into the mammal to be examined for an immune system
disorder and/or cell
proliferation disorder. 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
~'"'Tc. The labeled
antibody or antibody fragment will then preferentially accumulate at the
location of cells which
contain TR13 and/or TR14 protein. In vivo tumor imaging is described in S.W.
Burchiel et
al., "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments"
(Chapter 13
in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B.
A. Rhodes,
eds., Masson Publishing Inc. ( 1982)).
3o With respect to antibodies, one of the ways in which the anti-TR13 and/or
anti-TR14
antibody can be detectably labeled is by linking the same to an enzyme and
using the linked
product in an enzyme immunoassay (EIA) (Volley, A., "The Enzyme Linked
Immunosorbent
Assay (ELISA)", 1978, Diagnostic Horizons 2:1-7, Microbiological Associates
Quarterly
Publication, Walkersville, MD); Volley et al., J. Clin. Pathol. 31:507-520
(1978); Butler, J.E.,
Meth. Enzymol. 73:482-523 (1981); Maggio, E. (ed.), 1980, Enzyme Immunoassay,
CRC
Press, Boca Raton, FL,; Ishikawa, E. et al., (eds.), 1981, Enzyme Immunoassay,
Kgaku
Shoin, Tokyo). The enzyme which is bound to the antibody will react with an
appropriate
substrate, preferably a chromogenic substrate, in such a manner as to produce
a chemical
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
175
moiety which can be detected, for example, by spectrophotometric, fluorimetric
or by visual
means. Enzymes which can be used to detectably label the antibody include, but
are not limited
to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase,
yeast alcohol
dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphate
isomerase,
horseradish peroxidase,alkaline phosphatase,asparaginase, glucose oxidase,beta-
galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate
dehydrogenase,
glucoamylase and acetylcholinesterase. Additionally, the detection can be
accomplished by
colorimetric methods which employ a chromogenic substrate for the enzyme.
Detection may
also be accomplished by visual comparison of the extent of enzymatic reaction
of a substrate in
l0 comparison with similarly prepared standards.
Detection may also be accomplished using any of a variety of other
immunoassays. For
example, by radioactively labeling the antibodies or antibody fragments, it is
possible to detect
TR13 and/or TR14 through the use of a radioimmunoassay (RIA) (see, for
example,
Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on
Radioligand
Assay Techniques, The Endocrine Society, March, 1986, which is incorporated by
reference
herein). The radioactive isotope can be detected by means including, but not
limited to, a
gamma counter, a scintillation counter, or autoradiography.
It is also possible to label the antibody with a fluorescent compound. When
the
fluorescently labeled antibody is exposed to light of the proper wave length,
its presence can
2o then be detected due to fluorescence. Among the most commonly used
fluorescent labeling
compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,
phycocyanin,
allophycocyanin, ophthaldehyde and fluorescamine.
The antibody can also be detectably labeled using fluorescence emitting metals
such as
~szEu, or others of the lanthanide series. These metals can be attached to the
antibody using
such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or
ethylenediaminetetraacetic acid (EDTA).
The antibody also can be detectably labeled by coupling it to a
chemiluminescent
compound. The presence of the chemiluminescent-tagged antibody is then
determined by
detecting the presence of luminescence that arises during the course of a
chemical reaction.
3o Examples of particularly useful chemiluminescent labeling compounds are
luminol, isoluminol,
theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
Likewise, a bioluminescent compound may be used to label the antibody of the
present
invention. Bioluminescence is a type of chemiluminescence found in biological
systems in,
which a catalytic protein increases the efficiency of the chemiluminescent
reaction. The
presence of a bioluminescent protein is determined by detecting the presence
of luminescence.
Important bioluminescent compounds for purposes of labeling are luciferin,
luciferase and
aequorin.
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
176
TR13 and TR14 Binding Peptides and Other Molecules
The invention also encompasses screening methods for identifying polypeptides
and
nonpolypeptides that bind TR13 or TR14, and the TR13 or TR14 binding molecules
identified
thereby. These binding molecules are useful, for example, as agonists and
antagonists of the
TR13 or TR14 receptor proteins. Such agonists and antagonists can be used, in
accordance
with the invention, in the therapeutic embodiments described in detail, below.
This method comprises the steps of:
a. contacting a TR13 or TR14 protein or TR13 or TR14-like protein with a
plurality of
molecules; and
l0 b. identifying a molecule that binds the TR13 or TR14 protein or TR13 or
TR14-like
protein.
The step of contacting the TR13 or TR14 protein or TR13 or TR14-like protein
with
the plurality of molecules may be effected in a number of ways. For example,
one may
contemplate immobilizing the TR13 or TR14 protein or TR13 or TR14-like protein
on a solid
15 support and bringing a solution of the plurality of molecules in contact
with the immobilized
TR13 or TR14 protein or TR13 or TR14-like protein. Such a procedure would be
akin to an
affinity chromatographic process, with the affinity matrix being comprised of
the immobilized
TR13 or TR14 protein or TR13 or TR14-like protein. The molecules having a
selective
affinity for the TR13 or TR14 protein or TR13 or TR14-like protein can then be
purified by
20 affinity selection. The nature of the solid support, process for attachment
of the TR13 or
TR14 protein or TR13 or TR14-like protein to the solid support, solvent, and
conditions of
the affinity isolation or selection are largely conventional and well known to
those of ordinary
skill in the art.
Alternatively, one may also separate a plurality of polypeptides into
substantially
25 separate fractions comprising a subset of or individual polypeptides. For
instance, one can
separate the plurality of polypeptides by gel electrophoresis, column
chromatography, or like
method known to those of ordinary skill for the separation of polypeptides.
The individual
polypeptides can also be produced by a transformed host cell in such a way as
to be expressed
on or about its outer surface (e.g., a recombinant phage). Individual isolates
can then be
30 "probed" by the TR13 or TR14 protein or TR13 or TR14-like protein,
optionally in the
presence of an inducer should one be required for expression, to determine if
any selective
affinity interaction takes place between the TR13 or TR14 protein or TR13 or
TR14-like protein
and the individual clone. Prior to contacting the TR13 or TR14 protein or TR13
or TR14-like
protein with each fraction comprising individual polypeptides, the
polypeptides could first be
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
177
transferred to a solid support for additional convenience. Such a solid
support may simply be a
piece of filter membrane, such as one made of nitrocellulose or nylon. In this
manner, positive
clones could be identified from a collection of transformed host cells of an
expression library,
which harbor a DNA construct encoding a polypeptide having a selective
affinity for TR13 or
TR14 protein or TR13 or TR14-like protein. Furthermore, the amino acid
sequence of the
polypeptide having a selective affinity for the TR13 or TR14 protein or TR13
or TR14-like
protein can be determined directly by conventional means or the coding
sequence of the DNA
encoding the polypeptide can frequently be determined more conveniently. The
primary
sequence can then be deduced from the corresponding DNA sequence. If the amino
acid
to sequence is to be determined from the polypeptide itself, one may use
microsequencing
techniques. The sequencing technique may include mass spectroscopy.
In certain situations, it may be desirable to wash away any unbound TR13 or
TR14 protein or TR13 or TR14-like protein, or alterntatively, unbound
polypeptides, from a
mixture of the TR13 or TR14 protein or TR13 or TR14-like protein and the
plurality of
polypeptides prior to attempting to determine or to detect the presence of a
selective affinity
interaction. Such a wash step may be particularly desirable when the TR13 or
TR14 protein
or TR13 or TR14-like protein or the plurality of polypeptides is bound to a
solid support.
The plurality of molecules provided according to this method may be provided
by way
of diversity libraries, such as random or combinatorial peptide or nonpeptide
libraries which
can be screened for molecules that specifically bind to TR13 or TR14. Many
libraries are
known in the art that can be used, e.g., chemically synthesized libraries,
recombinant (e.g.,
phage display libraries), and in vitro translation-based libraries. Examples
of chemically
synthesized libraries are described in Fodor et al., 1991, Science 251:767-
773; Houghten et
al., 1991, Nature 354:84-86; Lam et al., 1991, Nature 354:82-84; Medynski,
1994,
Bio/Technology 12:709-710;Gallop et al., 1994, J. Medicinal Chemistry
37(9):1233-1251;
Ohlmeyer et al., 1993, Proc. Natl. Acad. Sci. USA 90:10922-10926; Erb et al.,
1994, Proc.
Natl. Acad. Sci. USA 91:11422-11426; Houghten et al., 1992, Biotechniques
13:412;
Jayawickreme et al., 1994, Proc. Natl. Acad. Sci. USA 91:1614-1618; Salmon et
al., 1993,
Proc. Natl. Acad. Sci. USA 90:11708-11712; PCT Publication No. WO 93/20242;
and
3o Brenner and Lerner, 1992, Proc. Natl. Acad. Sci. USA 89:5381-5383.
Examples of phage display libraries are described in Scott and Smith, 1990,
Science
249:386-390; Devlin et al., 1990, Science, 249:404-406; Christian, R. B., et
al., 1992, J.
Mol. Biol. 227:711-718); Lenstra, 1992, J. Immunol. Meth. 152:149-157; Kay et
al., 1993,
Gene 128:59-65; and PCT Publication No. WO 94/18318 dated Aug. 18, 1994.
In vitro translation-based libraries include but are not limited to those
described in PCT
Publication No. WO 91/05058 dated Apr. 18, 1991; and Mattheakis et al., 1994,
Proc. Natl.
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
178
Acad. Sci. USA 91:9022-9026.
By way of examples of nonpeptide libraries, a benzodiazepine library (see
e.g., Bunin
et al., 1994, Proc. Natl. Acad. Sci. USA 91:4708-4712) can be adapted for use.
Peptoid
libraries (Simon et al., 1992, Proc. Natl. Acad. Sci. USA 89:9367-9371) can
also be used.
Another example of a library that can be used, in which the amide
functionalities in peptides
have been permethylated to generate a chemically transformed combinatorial
library, is
described by Ostresh et al. (1994, Proc. Natl. Acad. Sci. USA 91:11138-11142).
The variety of non-peptide libraries that are useful in the present invention
is great. For
example, Ecker and Crooke, 1995, Bio/Technology 13:351-360 list
benzodiazepines,
1o hydantoins, piperazinediones, biphenyls, sugar analogs, beta-
mercaptoketones, arylacetic
acids, acylpiperidines, benzopyrans, cubanes, xanthines, aminimides, and
oxazolones as
among the chemical species that form the basis of various libraries.
Non-peptide libraries can be classified broadly into two types: decorated
monomers and
oligomers. Decorated monomer libraries employ a relatively simple scaffold
structure upon
which a variety functional groups is added. Often the scaffold will be a
molecule with a known
useful pharmacological activity. For example, the scaffold might be the
benzodiazepine
structure.
Non-peptide oligomer libraries utilize a large number of monomers that are
assembled
together in ways that create new shapes that depend on the order of the
monomers. Among the
2o monomer units that have been used are carbamates, pyrrolinones, and
morpholinos. Peptoids,
peptide-like oligomers in which the side chain is attached to the alpha amino
group rather than
the alpha carbon, form the basis of another version of non-peptide oligomer
libraries. The first
non-peptide oligomer libraries utilized a single type of monomer and thus
contained a repeating
backbone. Recent libraries have utilized more than one monomer, giving the
libraries added
flexibility.
Screening the libraries can be accomplished by any of a variety of commonly
known
methods. See, e.g., the following references, which disclose screening of
peptide libraries:
Parmley and Smith, 1989, Adv. Exp. Med. Biol. 251:215-218; Scott and Smith,
1990,
Science 249:386-390; Fowlkes et al., 1992; BioTechniques 13:422-427; Oldenburg
et al.,
1992, Proc. Natl. Acad. Sci. USA 89:5393-5397; Yu et al., 1994, Cell 76:933-
945; Staudt et
al., 1988, Science 241:577-580; Bock et al., 1992, Nature 355:564-566; Tuerk
et al., 1992,
Proc. Natl. Acad. Sci. USA 89:6988-6992; Ellington et al., 1992, Nature
355:850-852; U.S.
Pat. No. 5,096,815, U.S. Pat. No. 5,223,409, and U.S. Pat. No. 5,198,346, all
to Ladner et
al.; Rebar and Pabo, 1993, Science 263:671-673; and CT Publication No. WO
94/18318.
In a specific embodiment, screening to identify a molecule that binds TR13 or
TR14 can
be carried out by contacting the library members with a TR13 or TR14 protein
or TR13 or
TR14-like protein immobilized on a solid phase and harvesting those library
members that bind
to the TR13 or TR14 protein or TR13 or TR14-like protein. Examples of such
screening
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
179
methods, termed "panning" techniques are described by way of example in
Parmley and Smith,
1988, Gene 73:305-318; Fowlkes et al., 1992, BioTechniques 13:422-427; PCT
Publication
No. WO 94/18318; and in references cited herein.
In another embodiment, the two-hybrid system for selecting interacting
proteins in yeast
(Fields and Song, 1989, Nature 340:245-246; Chien et al., 1991, Proc. Natl.
Acad. Sci. USA
88:9578-9582) can be used to identify molecules that specifically bind to TR13
or TR14 or
TR13 or TR14-like proteins.
Where the TR13 or TR14 binding molecule is a polypeptide, the polypeptide can
be
conveniently selected from any peptide library, including random peptide
libraries,
to combinatorial peptide libraries, or biased peptide libraries. The term
"biased" is used herein to
mean that the method of generating the library is manipulated so as to
restrict one or more
parameters that govern the diversity of the resulting collection of molecules,
in this case
peptides.
Thus, a truly random peptide library would generate a collection of peptides
in which
15 the probability of finding a particular amino acid at a given position of
the peptide is the same
for all 20 amino acids. A bias can be introduced into the library, however, by
specifying, for
example, that a lysine occur every fifth amino acid or that positions 4, 8,
and 9 of a decapeptide
library be fixed to include only arginine. Clearly, many types of biases can
be contemplated,
and the present invention is not restricted to any particular bias.
Furthermore, the present
2o invention contemplates specific types of peptide libraries, such as phage
displayed peptide
libraries and those that utilize a DNA construct comprising a lambda phage
vector with a DNA
insert.
As mentioned above, in the case of a TR13 or TR14 binding molecule that is a
polypeptide, the polypeptide may have about 6 to less than about 60 amino acid
residues,
25 preferably about 6 to about 10 amino acid residues, and most preferably,
about 6 to about 22
amino acids. In another embodiment, a TR13 or TR14 binding polypeptide has in
the range of
15-100 amino acids, or 20-SO amino acids.
The selected TR13 or TR14 binding polypeptide can be obtained by chemical
synthesis
or recombinant expression.
Enitopes
The present invention encompasses polypeptides comprising, or alternatively
consisting of, an epitope of the TR13 and TR14 polypeptides described in
detail above
or encoded by a polynucleotide that hybridizes to the complement of the
sequence of
TR13 and TR14 coding sequences described in detail above, under stringent
hybridization conditions or lower stringency hybridization conditions as
defined supra.
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
180
The present invention further encompasses polynucleotide sequences encoding an
epitope of a polypeptide sequence of the, polynucleotide sequences of the
complementary strand of a polynucleotide sequence encoding an epitope of the
invention, and polynucleotide sequences which hybridize to the complementary
strand
under stringent hybridization conditions or lower stringency hybridization
conditions
defined supra.
The term "epitopes," as used herein, refers to portions of a polypeptide
having
antigenic or immunogenic activity in an animal, preferably a mammal, and most
preferably in a human. In a preferred embodiment, the present invention
encompasses
1o a polypeptide comprising an epitope, as well as the polynucleotide encoding
this
polypeptide. An "immunogenic epitope," as used herein, is defined as a portion
of a
protein that elicits an antibody response in an animal, as determined by any
method
known in the art, for example, by the methods for generating antibodies
described
infra. (See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-
4002
(1983)). The term "antigenic epitope," as used herein, is defined as a portion
of a
protein to which an antibody can immunospecifically bind its antigen as
determined by
any method well known in the art, for example, by the immunoassays described
herein. Immunospecific binding excludes non-specific binding but does not
necessarily exclude cross-reactivity with other antigens. Antigenic epitopes
need not
2o necessarily be immunogenic.
Fragments that 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. 4,631,211).
In the present invention, antigenic epitopes preferably contain a sequence of
at
least 4, at least 5, at least 6, at least 7, more preferably at least 8, at
least 9, at least 10,
at least 15, at least 20, at least 25, and, most preferably, between about 15
to about 30
amino acids. Preferred polypeptides comprising immunogenic or antigenic
epitopes
are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,
90, 95, or
100 amino acid residues in length. Antigenic epitopes are useful, for example,
to raise
antibodies, including monoclonal antibodies, that specifically bind the
epitope.
Antigenic epitopes can be used as the target molecules in immunoassays. (See,
for
instance, Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al., Science
219:660-666
( 1983)).
Similarly, immunogenic epitopes can be used, for example, to induce
antibodies according to methods well known in the art. (See, for instance,
Sutcliffe et
al., supra; Wilson et al., supra; Chow et al., Proc. Natl. Acad. Sci. USA
82:910-914;
and Bittle et al., J. Gen. Virol. 66:2347-2354 (1985). The polypeptides
comprising
one or more immunogenic epitopes may be presented for eliciting an antibody
response
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
181
together with a carrier protein, such as an albumin, to an animal system (such
as, for
example, rabbit or mouse), or, if the polypeptide is of sufficient length (at
least about
25 amino acids), the polypeptide may be presented without a carrier. However,
immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown
to
be sufficient to raise antibodies capable of binding to, at the very least,
linear epitopes
in a denatured polypeptide (e.g., in Western blotting).
Epitope-bearing polypeptides of the present invention may be used to induce
antibodies according to methods well known in the art including, but not
limited to, in
vivo immunization, in vitro immunization, and phage display methods. See,
e.g.,
1o Sutcliffe et al., supra; Wilson et al., supra, and Bittle et al., J. Gen.
Virol., 66:2347-
2354 (1985). If in vivo immunization is used, animals may be immunized with
free
peptide; however, anti-peptide antibody titer may be boosted by coupling the
peptide to
a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus
toxoid.
For instance, peptides containing cysteine residues may be coupled to a
carrier using a
15 linker such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while
other
peptides may be coupled to carriers using a more general linking agent such as
glutaraldehyde. Animals such as, for example, rabbits, rats, and mice are
immunized
with either free or carrier-coupled peptides, for instance, by intraperitoneal
and/or
intradermal injection of emulsions containing about 100 micrograms of peptide
or
20 carrier protein and Freund's adjuvant or any other adjuvant known for
stimulating an
immune response. Several booster injections may be needed, for instance, at
intervals
of about two weeks, to provide a useful titer of anti-peptide antibody that
can be
detected, for example, by ELISA assay using free peptide adsorbed to a solid
surface.
The titer of anti-peptide antibodies in serum from an immunized animal may be
25 increased by selection of anti-peptide antibodies, for instance, by
adsorption to the
peptide on a solid support and elution of the selected antibodies according to
methods
well known in the art.
As one of skill in the art will appreciate, and as discussed above, the
polypeptides of the present invention comprising an immunogenic or antigenic
epitope
3o can be fused to other polypeptide sequences. For example, the polypeptides
of the
present invention may be fused with the constant domain of immunoglobulins
(IgA,
IgE, IgG, IgM), or portions thereof (CH1, CH2, CH3, or any combination thereof
and portions thereof) resulting in chimeric polypeptides. Such fusion proteins
may
facilitate purification and may increase half-life in vivo. This has been
shown for
35 chimeric proteins consisting of the first two domains of the human CD4-
polypeptide
and various domains of the constant regions of the heavy or light chains of
mammalian
immunoglobulins. See, e.g., EP 394,827; Traunecker et al., Nature, 331:84-86
(1988). IgG Fusion proteins that have a disulfide-linked dimeric structure due
to the
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
182
IgG portion desulfide bonds have also been found to be more efficient in
binding and
neutralizing other molecules than monomeric polypeptides or fragments thereof
alone.
See, e.g., Fountoulakis et al., J. Biochem., 270:3958-3964 (1995). Nucleic
acids
encoding the above epitopes can also be recombined with a gene of interest as
an
epitope tag (e.g., the hemagglutinin ("HA") tag or flag tag) to aid in
detection and
purification of the expressed polypeptide. For example, a system described by
Janknecht et al. allows for the ready purification of non-denatured fusion
proteins
expressed in human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci.
USA
88:8972- 897). In this system, the gene of interest is subcloned into a
vaccinia
recombination plasmid such that the open reading frame of the gene is
translationally
fused to an amino-terminal tag consisting of six histidine residues. The tag
serves as a
matrix-binding domain for the fusion protein. Extracts from cells infected
with the
recombinant vaccinia virus are loaded onto Niz+ nitriloacetic acid-agarose
column and
histidine-tagged proteins can be selectively eluted with imidazole-containing
buffers.
Additional fusion proteins of the invention may be generated through the
techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-
shuffling
(collectively referred to as "DNA shuffling"). DNA shuffling may be employed
to
modulate the activities of polypeptides of the invention, such methods can be
used to
generate polypeptides with altered activity, as well as agonists and
antagonists of the
2o polypeptides. See, generally, U.S. Patent Nos. 5,605,793; 5,811,238;
5,830,721;
5,834,252; and 5,837,458, and Patten et al., Curr. Opinion Biotechnol. 8:724-
33
(1997); Harayama, Trends Biotechnol. 16(2):76-82 (1998); Hansson, et al., J.
Mol.
Biol. 287:265-76 (1999); and Lorenzo and Blasco, Biotechniques 24(2):308- 13
(1998) (each of these patents and publications are hereby incorporated by
reference in
its entirety). In one embodiment, alteration of polynucleotides corresponding
to SEQ
ID NO:l or 60 and the polypeptides encoded by these polynucleotides may be
achieved
by DNA shuffling. DNA shuffling involves the assembly of two or more DNA
segments by homologous or site-specific recombination to generate variation in
the
polynucleotide sequence. In another embodiment, polynucleotides of the
invention, or
the encoded polypeptides, may be altered by being subjected to random
mutagenesis
by error-prone PCR, random nucleotide insertion or other methods prior to
recombination. In another embodiment, one or more components, motifs,
sections,
parts, domains, fragments, etc., of a polynucleotide coding a polypeptide of
the
invention may be recombined with one or more components, motifs, sections,
parts,
domains, fragments, etc. of one or more heterologous molecules. (each of these
patents and publications are hereby incorporated by reference). In one
embodiment,
alteration of TR13 and/or TR14 polynucleotides and corresponding polypeptides
may
be achieved by DNA shuffling. DNA shuffling involves the assembly of two or
more
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
183
DNA segments into a desired TR13 and/or TR14 molecule by homologous, or site-
specific, recombination. In another embodiment, TR13 and/or TR14
polynucleotides
and corresponding polypeptides may be altered by being subjected to random
mutagenesis by error-prone PCR, random nucleotide insertion or other methods
prior
to recombination. In another embodiment, one or more components, motifs,
sections,
parts, domains, fragments, etc., of TR13 and/or TR14 may be recombined with
one or
more components, motifs, sections, parts, domains, fragments, etc. of one or
more
heterologous molecules. In preferred embodiments, the heterologous molecules
are
receptors for TNF-alpha, TNF-beta, lymphotoxin-alpha, lymphotoxin-beta, FAS
ligand, and APRIL. In further preferred embodiments, the heterologous
molecules are
any member of the TNF family.
Antibodies
The present invention further relates to antibodies and T-cell antigen
receptors
(TCR) which immunospecifically bind a polypeptide, preferably an epitope, of
the
present invention (as determined by immunoassays well known in the art for
assaying
specific antibody-antigen binding). Antibodies of the invention include, but
are not
limited to, polyclonal, monoclonal, multispecific, human, humanized or
chimeric
antibodies, single chain antibodies, Fab fragments, F(ab') fragments,
fragments
2o produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies
(including,
e.g., anti-Id antibodies to antibodies of the invention), and epitope-binding
fragments
of any of the above. The term "antibody," as used herein, refers to
immunoglobulin
molecules and immunologically active portions of immunoglobulin molecules,
i.e.,
molecules that contain an antigen binding site that immunospecifically binds
an
antigen. The immunoglobulin molecules of the invention can be of any type
(e.g.,
IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGI, IgG2, IgG3, IgG4, IgAl
and
IgA2) or subclass of immunoglobulin molecule.
Most preferably the antibodies are human antigen-binding antibody fragments
of the present invention and include, but are not limited to, Fab, Fab' and
F(ab')2, Fd,
3o single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs
(sdFv) and
fragments comprising either a VL or VH domain. Antigen-binding antibody
fragments, including single-chain antibodies, may comprise the variable
regions)
alone or in combination with the entirety or a portion of the following: hinge
region,
CH1, CH2, and CH3 domains. Also included in the invention are antigen-binding
fragments also comprising any combination of variable regions) with a hinge
region,
CH1, CH2, and CH3 domains. The antibodies of the invention may be from any
animal origin including birds and mammals. Preferably, the antibodies are
human,
murine, donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken. As
used
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
184
herein, "human" antibodies include antibodies having the amino acid sequence
of a
human immunoglobulin and include antibodies isolated from human immunoglobulin
libraries or from animals transgenic for one or more human immunoglobulin and
that
do not express endogenous immunoglobulins, as described infra and, for example
in,
U.S. Patent No. 5,939,598 by Kucherlapati et al.
The antibodies of the present invention may be monospecific, bispecific,
trispecific or of greater multispecificity. Multispecific antibodies may be
specific for
different epitopes of a polypeptide of the present invention or may be
specific for both
a polypeptide of the present invention as well as for a heterologous epitope,
such as a
heterologous polypeptide or solid support material. See, e.g., PCT'
publications WO
93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol.
147:60-69 (1991); U.S. Patent Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920;
5,601,819; Kostelny et al., J. Immunol. 148:1547-1553 (1992).
Antibodies of the present invention may be described or specified in terms of
~5 the epitope(s) or portions) of a polypeptide of the present invention that
they recognize
or specifically bind. The epitope(s) or polypeptide portions) may be specified
as
described herein, e.g., by N-terminal and C-terminal positions, by size in
contiguous
amino acid residues, or listed in the Tables and Figures. Antibodies that
specifically
bind any epitope or polypeptide of the present invention may also be excluded.
2o Therefore, the present invention includes antibodies that specifically bind
polypeptides
of the present invention, and allows for the exclusion of the same.
Antibodies of the present invention may also be described or specified in
terms
of their cross-reactivity. Antibodies that do not bind any other analog,
ortholog, or
homolog of a polypeptide of the present invention are included. Antibodies
that bind
25 polypeptides with at least 95%, at least 90%, at least 85%, at least 80%,
at least 75%,
at least 70%, at least 65%, at least 60%, at least 55%, and at least 50%
identity (as
calculated using methods known in the art and described herein) to a
polypeptide of the
present invention are also included in the present invention. Antibodies that
do not
bind polypeptides with less than 95%, less than 90%, less than 85%, less than
80%,
30 less than 75%, less than 70%, less than 65%, less than 60%, less than 55%,
and less
than 50% identity (as calculated using methods known in the art and described
herein)
to a polypeptide of the present invention are also included in the present
invention.
Further included in the present invention are antibodies that bind
polypeptides encoded
by polynucleotides which hybridize to a polynucleotide of the present
invention under
35 stringent hybridization conditions (as described herein). Antibodies of the
present
invention may also be described or specified in terms of their binding
affinity to a
polypeptide of the invention. Preferred binding affinities include those with
a
dissociation constant or Kd less than SX10-ZM, 10-2M, 5X10-3M, 10-3M, SX10-4M,
10-
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
185
4M, 5X10~5M, 10-5M, SX10-6M, 10-6M, SX10-'M, 10-'M, SX10-$M, 10-8M, 5X10-9M,
10-9M, SX10-'°M, 10-'°M, SX10-"M, 10-"M, 5X10-'ZM, 10-'2M, 5X10-
'3M, 10-'3M,
SX10-'4M, 10''°M, 5X10-'SM, and 10-'5M.
The invention also provides antibodies that competitively inhibit binding of
an
antibody to an epitope of the invention as determined by any method known in
the art
for determining competitive binding, for example, the immunoassays described
herein.
In preferred embodiments, the antibody competitively inhibits binding to the
epitope by
at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.
Antibodies of the present invention may act as agonists or antagonists of the
1o polypeptides of the present invention. For example, the present invention
includes
antibodies which disrupt the receptor/ligand interactions with the
polypeptides of the
invention either partially or fully. The invention features both receptor-
specific
antibodies and ligand-specific antibodies. The invention also features
receptor-specific
antibodies which do not prevent ligand binding but prevent receptor
activation.
15 Receptor activation (i.e., signaling) may be determined by techniques
described herein
or otherwise known in the art. For example, receptor activation can be
determined by
detecting the phosphorylation (e.g., tyrosine or serine/threonine) of the
receptor or its
substrate by immunoprecipitation followed by western blot analysis (for
example, as
described supra). In specific embodiments, antibodies are provided that
inhibit ligand
20 or receptor activity by at least 90%, at least 80%, at least 70%, at least
60%, or at least
50% of the activity in absence of the antibody.
The invention also features receptor-specific antibodies which both prevent
ligand binding and receptor activation as well as antibodies that recognize
the receptor-
ligand complex, and, preferably, do not specifically recognize the unbound
receptor or
25 the unbound ligand. Likewise, included in the invention are neutralizing
antibodies
which bind the ligand and prevent binding of the ligand to the receptor, as
well as
antibodies which bind the ligand, thereby preventing receptor activation, but
do not
prevent the ligand from binding the receptor. Further included in the
invention are
antibodies which activate the receptor. These antibodies may act as receptor
agonists,
30 i.e., potentiate or activate either all or a subset of the biological
activities of the ligand-
mediated receptor activation. The antibodies may be specified as agonists,
antagonists
or inverse agonists for biological activities comprising the specific
biological activities
of the peptides of the invention disclosed herein. The above antibody agonists
can be
made using methods known in the art. See, e.g., PCT publication WO 96/40281;
35 U.S. Patent No. 5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chen,
et al.,
Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol. 161(4):1786-
1794
(1998); Zhu et al., Cancer Res. 58(15):3209-3214 (1998); Yoon, et al., J.
Immunol.
160(7):3170-3179 ( 1998); Prat et al ., J. Cell . Sci . 11 I (Pt2):237-247 (
1998); Pitard et
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
186
al., J. Immunol. Methods 205(2):177-190 (1997); Liautard et al., Cytokine
9(4):233-
241 (1997); Carlson et al., J. Biol. Chem. 272(17):11295-11301 (1997); Taryman
et
al., Neuron 14(4):755-762 (1995); Muller et al., Structure 6(9):1153-1167
(1998);
Bartunek et al., Cytokine 8(1):14-20 (1996) (which are all incorporated by
reference
herein in their entireties).
Antibodies of the present invention may be used, for example, but not limited
to, to purify, detect, and target the polypeptides of the present invention,
including
both in vitro and in vivo diagnostic and therapeutic methods. For example, the
antibodies have use in immunoassays for qualitatively and quantitatively
measuring
to levels of the polypeptides of the present invention in biological samples.
See, e.g.,
Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory
Press, 2nd ed. 1988) (incorporated by reference herein in its entirety).
As discussed in more detail below, the antibodies of the present invention may
be used either alone or in combination with other compositions. The antibodies
may
further be recombinantly fused to a heterologous polypeptide at the N- or C-
terminus
or chemically conjugated (including covalently and non-covalently
conjugations) to
polypeptides or other compositions. For example, antibodies of the present
invention
may be recombinantly fused or conjugated to molecules useful as labels in
detection
assays and effector molecules such as heterologous polypeptides, drugs, or
toxins.
See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Patent
No. 5,314,995; and EP 396,387.
The antibodies of the invention include derivatives that are modified, i.e, by
the
covalent attachment of any type of molecule to the antibody such that covalent
attachment does not prevent the antibody from generating an anti-idiotypic
response.
For example, but not by way of limitation, the antibody derivatives include
antibodies
that have been modified, e.g., by glycosylation, acetylation, pegylation,
phosphylation, amidation, derivatization by known protecting/blocking groups,
proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any
of
numerous chemical modifications may be carried out by known techniques,
including,
but not limited to specific chemical cleavage, acetylation, formylation,
metabolic
synthesis of tunicamycin, etc. Additionally, the derivative may contain one or
more
non-classical amino acids.
The antibodies of the present invention may be generated by any suitable
method known in the art. Polyclonal antibodies to an antigen-of- interest can
be
produced by various procedures well known in the art. For example, a
polypeptide of
the invention can be administered to various host animals including, but not
limited to,
rabbits, mice, rats, etc. to induce the production of sera containing
polyclonal
antibodies specific for the antigen. Various adjuvants may be used to increase
the
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
187
immunological response, depending on the host species, and include but are not
limited to, Freund's (complete and incomplete), mineral gels such as aluminum
hydroxide, surface active substances such as lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,
dinitrophenol, and
potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and
corynebacterium parvum. Such adjuvants are also well known in the art.
Monoclonal antibodies can be prepared using a wide variety of techniques
known in the art including the use of hybridoma, recombinant, and phage
display
technologies, or a combination thereof. For example, monoclonal antibodies can
be
1o produced using hybridoma techniques including those known in the art and
taught, for
example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring
Harbor
Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies
and
T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporated
by
reference in their entireties). The term "monoclonal antibody" as used herein
is not
15 limited to antibodies produced through hybridoma technology. The term
"monoclonal
antibody" refers to an antibody that is derived from a single clone, including
any
eukaryotic, prokaryotic, or phage clone, and not the method by which it is
produced.
Methods for producing and screening for specific antibodies using hybridoma
technology are routine and well-known in the art and are discussed in detail
in Example
20 5, below. Briefly, mice can be immunized with a polypeptide of the
invention or a cell
expressing such peptide. Once an immune response is detected, e.g., antibodies
specific for the antigen are detected in the mouse serum, the mouse spleen is
harvested
and splenocytes isolated. The splenocytes are then fused by well-known
techniques to
any suitable myeloma cells, for example cells from cell line SP20 available
from the
25 ATCC. Hybridomas are selected and cloned by limited dilution. The hybridoma
clones are then assayed by methods known in the art for cells that secrete
antibodies
capable of binding a polypeptide of the invention. Ascites fluid, which
generally
contains high levels of antibodies, can be generated by immunizing mice with
positive
hybridoma clones.
3o Accordingly, the present invention provides methods of generating
monoclonal
antibodies as well as antibodies produced by the method comprising culturing a
hybridoma cell secreting an antibody of the invention wherein, preferably, the
hybridoma is generated by fusing splenocytes isolated from a mouse immunized
with
an antigen of the invention with myeloma cells and then screening the
hybridomas
35 resulting from the fusion for hybridoma clones that secrete an antibody
able to bind a
polypeptide of the invention.
Antibody fragments that recognize specific epitopes may be generated by
known techniques. For example, Fab and F(ab')2 fragments of the invention may
be
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
188
produced by proteolytic cleavage of immunoglobulin molecules, using enzymes
such
as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
F(ab')2 fragments contain the variable region, the light chain constant region
and the
CH1 domain of the heavy chain.
For example, the antibodies of the present invention can also be generated
using various phage display methods known in the art. In phage display
methods,
functional antibody domains are displayed on the surface of phage particles
which
carry the polynucleotide sequences encoding them. In a particular, such phage
can be
utilized to display antigen-binding domains expressed from a repertoire or
to combinatorial antibody library (e.g., human or murine). Phage expressing an
antigen
binding domain that binds the antigen of interest can be selected or
identified with
antigen, e.g., using labeled antigen or antigen bound or captured to a solid
surface or
bead. Phage used in these methods are typically filamentous phage including fd
and
M13 binding domains expressed from phage with Fab, Fv or disulfide stabilized
Fv
t5 antibody domains recombinantly fused to either the phage gene III or gene
VIII
protein. Examples of phage display methods that can be used to make the
antibodies
of the present invention include those disclosed in Brinkman et al., J.
Immunol.
Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186 (1995);
Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al., Gene
187 9-
20 18 (1997); Burton et al., Advances in Immunology 57:191-280 (1994); PCT
application No. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737;
WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and
U.S. Patent Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908;
5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727;
25 5,733,743 and 5,969,108; each of which is incorporated herein by reference
in its
entirety.
As described in the above references, after phage selection, the antibody
coding
regions from the phage can be isolated and used to generate whole antibodies,
including human antibodies, or any other desired antigen binding fragment, and
3o expressed in any desired host, including mammalian cells, insect cells,
plant cells,
yeast, and bacteria, e.g., as described in detail below. For example,
techniques to
~recombinantly produce Fab, Fab' and F(ab')2 fragments can also be employed
using
methods known in the art such as those disclosed in PCT publication WO
92/22324;
Mullinax et al., BioTechniques 12(6):864-869 ( 1992); and Sawai et al., AJRI
34:26-
35 34 (1995); and Better et al., Science 240:1041-1043 (1988) (said references
incorporated by reference in their entireties).
Examples of techniques which can be used to produce single-chain Fvs and
antibodies include those described in U.S. Patents 4,946,778 and 5,258,498;
Huston
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
189
et al., Methods in Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999
(1993); and Skerra et al., Science 240:1038-1040 (1988). For some uses,
including
in vivo use of antibodies in humans and in vitro detection assays, it may be
preferable
to use chimeric, humanized, or human antibodies. A chimeric antibody is a
molecule
in which different portions of the antibody are derived from different animal
species,
such as antibodies having a variable region derived from a murine monoclonal
antibody and a human immunoglobulin constant region. Methods for producing
chimeric antibodies are known in the art. See e.g., Morrison, Science 229:1202
(1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J.
Immunol.
Methods 125:191-202; U.S. Patent Nos. 5,807,715; 4,816,567; and 4,816397,
which
are incorporated herein by reference in their entireties. Humanized antibodies
are
antibody molecules from non-human species antibody that binds the desired
antigen
having one or more complementarity determining regions (CDRs) from the non-
human
species and framework regions from a human immunoglobulin molecule. Often,
framework residues in the human framework regions will be substituted with the
corresponding residue from the CDR donor antibody to alter, preferably
improve,
antigen binding. These framework substitutions are identified by methods well
known
in the art, e.g., by modeling 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; Riechmann et al., Nature 332:323 (1988), which are
incorporated herein by reference in their entireties.) Antibodies can be
humanized
using a variety of techniques known in the art including, for example, CDR-
grafting
(EP 239,400; PCT publication WO 91/09967; U.S. Patent Nos. 5,225,539;
5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596;
Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein
Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994)), and
chain shuffling (U.S. Patent No. 5,565,332).
Completely human antibodies are particularly desirable for therapeutic
treatment
of human patients. Human antibodies can be made by a variety of methods known
in
the art including phage display methods described above using antibody
libraries
derived from human immunoglobulin sequences. See also, U.S. Patent Nos.
4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO
98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of
which is incorporated herein by reference in its entirety.
Human antibodies can also be produced using transgenic mice which are
incapable of expressing functional endogenous immunoglobulins, but which can
express human immunoglobulin genes. For example, the human heavy and light
chain
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
190
immunoglobulin gene complexes may be introduced randomly or by homologous
recombination into mouse embryonic stem cells. Alternatively, the human
variable
region, constant region, and diversity region may be introduced into mouse
embryonic
stem cells in addition to the human heavy and light chain genes. The mouse
heavy and
light chain immunoglobulin genes may be rendered non-functional separately or
simultaneously with the introduction of human immunoglobulin loci by
homologous
recombination. In particular, homozygous deletion of the JH region prevents
endogenous antibody production. The modified embryonic stem cells are expanded
and microinjected into blastocysts to produce chimeric mice. The chimeric mice
are
then bred to produce homozygous offspring that express human antibodies. The
transgenic mice are immunized in the normal fashion with a selected antigen,
e.g., all
or a portion of a polypeptide of the invention. Monoclonal antibodies directed
against
the antigen can be obtained from the immunized, transgenic mice using
conventional
hybridoma technology. The human immunoglobulin transgenes harbored by the
~5 transgenic mice rearrange during B cell differentiation, and subsequently
undergo class
switching and somatic mutation. Thus, using such a technique, it is possible
to
produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an
overview of
this technology for producing human antibodies, see Lonberg and Huszar (1995,
Int.
Rev. Immunol. 13:65-93). For a detailed discussion of this technology for
producing
human antibodies and human monoclonal antibodies and protocols for producing
such
antibodies, see, e.g., PCT publications WO 98/24893; WO 96/34096; WO 96/33735;
U.S. Patent Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016;
5,545,806; 5,814,318; and 5,939,598, which are incorporated by reference
herein in
their entirety. In addition, companies such as Abgenix, Inc. (Freemont, CA)
and
Genpharm (San Jose, CA) can be engaged to provide human antibodies directed
against a selected antigen using technology similar to that described above.
Completely human antibodies which recognize a selected epitope can be
generated using a technique referred to as "guided selection." In this
approach a
selected non-human monoclonal antibody, e.g., a mouse antibody, is used to
guide the
selection of a completely human antibody recognizing the same epitope.
(Jespers et
al., Biotechnology 12:899-903 (1988)).
Further, antibodies~to the polypeptides of the invention can, in turn, be
utilized
to generate anti-idiotype antibodies that "mimic" polypeptides of the
invention using
techniques well known to those skilled in the art. (See, e.g., Greenspan &
Bona,
FASEB J. 7(5):437-444; (1989) and Nissinoff, J. Immunol. 147(8):2429-2438
(1991)). For example, antibodies which bind to and competitively inhibit
polypeptide
multimerization and/or binding of a polypeptide of the invention to a ligand
can be used
to generate anti-idiotypes that "mimic" the polypeptide multimerization and/or
binding
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
191
domain and, as a consequence, bind to and neutralize polypeptide and/or its
ligand.
Such neutralizing anti-idiotypes or Fab fragments of such anti-idiotypes can
be used in
therapeutic regimens to neutralize polypeptide ligand. For example, such anti-
idiotypic
antibodies can be used to bind a polypeptide of the invention and/or to bind
its
ligands/receptors, and thereby block its biological activity.
Polynucleotides Encoding Antibodies
The invention further provides polynucleotides comprising a nucleotide
sequence encoding an antibody of the invention and fragments thereof. The
invention
also encompasses polynucleotides that hybridize under stringent or lower
stringency
hybridization conditions, e.g., as defined supra, to polynucleotides that
encode an
antibody, preferably, that specifically binds to a polypeptide of the
invention,
preferably, an antibody that binds to TR13 or TR14 polypeptide of the
invention, as
described above.
~ 5 The polynucleotides may be obtained, and the nucleotide sequence of the
po(ynucleotides determined, by any method known in the art. For example, if
the
nucleotide sequence of the antibody is known, a polynucleotide encoding the
antibody
may be assembled from chemically synthesized oligonuc(eotides (e.g., as
described in
Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly, involves the
synthesis
2o of overlapping oligonucleotides containing portions of the sequence
encoding the
antibody, annealing and ligation of those oligonucleotides, and then
amplification of
the ligated oligonucleotides by PCR.
Alternatively, a polynucleotide encoding an antibody may be generated
from nucleic acid from a suitable source. If a clone containing a nucleic acid
encoding
25 a particular antibody is not available, but the sequence of the antibody
molecule is
known, a nucleic acid encoding the immunoglobulin may be obtained from a
suitable
source (e.g., an antibody cDNA library, or a cDNA library generated from, or
nucleic
acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing
the
antibody, such as hybridoma cells selected to express an antibody of the
invention) by
3o 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.
35 Once the nucleotide sequence and corresponding amino acid sequence of
the antibody is determined, the nucleotide sequence of the antibody may be
manipulated using methods well known in the art for the manipulation of
nucleotide
sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR,
etc.
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
192
(see, for example, the techniques described in Sambrook et al., 1990,
Molecular
Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold
Spring
Harbor, NY and Ausubel et al., eds., 1998, Current Protocols in Molecular
Biology,
John Wiley & Sons, NY, which are both incorporated by reference herein in
their
entireties ), to generate antibodies having a different amino acid sequence,
for example
to create amino acid substitutions, deletions, and/or insertions.
In a specific embodiment, the amino acid sequence of the heavy and/or light
chain variable domains may be inspected to identify the sequences of the
complementarity determining regions (CDRs) by methods that are well know in
the art,
e.g., by comparison to known amino acid sequences of other heavy and light
chain
variable regions to determine the regions of sequence hypervariability. Using
routine
recombinant DNA techniques, one or more of the CDRs may be inserted within
framework regions, e.g., into human framework regions to humanize a non-human
antibody, as described supra. The framework regions may be naturally occurring
or
is consensus framework regions, and preferably human framework regions (see,
e.g.,
Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for a listing of human
framework
regions). Preferably, the polynucleotide generated by the combination of the
framework regions and CDRs encodes an antibody that specifically binds a
polypeptide of the invention. Preferably, as discussed supra, one or more
amino acid
2o substitutions may be made within the framework regions, and, preferably,
the amino
acid substitutions improve binding of the antibody to its antigen.
Additionally, such
methods may be used to make amino acid substitutions or deletions of one or
more
variable region cysteine residues participating in an intrachain disulfide
bond to
generate antibody molecules lacking one or more intrachain disulfide bonds.
Other
25 alterations to the polynucleotide are encompassed by the present invention
and within
the skill of the art.
In addition, techniques developed for the production of "chimeric
antibodies" (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855;
Neuberger et
al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454) by
splicing
3o genes from a mouse antibody molecule of appropriate antigen specificity
together with
genes from a human antibody molecule of appropriate biological activity can be
used.
As described supra, a chimeric antibody is a molecule in which different
portions are
derived from different animal species, such as those having a variable region
derived
from a murine mAb and a human immunoglobulin constant region, e.g., humanized
35 antibodies.
Alternatively, techniques described for the production of single chain
antibodies (U.S. Patent No. 4,694,778; Bird, 1988, Science 242:423- 42; Huston
et
al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989,
Nature
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
193
334:544-54) can be adapted to produce single chain antibodies. Single chain
antibodies are formed by linking the heavy and light chain fragments of the Fv
region
via an amino acid bridge, resulting in a single chain polypeptide. Techniques
for the
assembly of functional Fv fragments in E. coli may also be used (Skerra et
al., 1988,
Science 242:1038- 1041).
Methods of Producing Antibodies
The antibodies of the invention can be produced by any method known in
the art for the synthesis of antibodies, in particular, by chemical synthesis
or
preferably, by recombinant expression techniques.
Recombinant expression of an antibody of the invention, or fragment,
derivative or analog thereof, e.g., a heavy or light chain of an antibody of
the
invention, requires construction of an expression vector containing a
polynucleotide
that encodes the antibody. Once a polynucleotide encoding an antibody molecule
or a
~5 heavy or light chain of an antibody, or portion thereof (preferably
containing the heavy
or light chain variable domain), of the invention has been obtained, the
vector for the
production of the antibody molecule may be produced by recombinant DNA
technology using techniques well known in the art. Thus, methods for preparing
a
protein by expressing a polynucleotide containing an antibody encoding
nucleotide
2o sequence are described herein. Methods which are well known to those
skilled in the
art can be used to construct expression vectors containing antibody coding
sequences
and appropriate transcriptional and translational control signals. These
methods
include, for example, in vitro recombinant DNA techniques, synthetic
techniques, and
in vivo genetic recombination. The invention, thus, provides replicable
vectors
25 comprising a nucleotide sequence encoding an antibody molecule of the
invention, or
a heavy or light chain thereof, or a heavy or light chain variable domain,
operably
linked to a promoter. Such vectors may include the nucleotide sequence
encoding the
constant region of the antibody molecule (see, e.g., PCT Publication WO
86/05807;
PCT Publication WO 89/01036; and U.S. Patent No. 5,122,464.) and the variable
3o domain of the antibody may be cloned into such a vector for expression of
the entire
heavy or light chain.
The expression vector is transferred to a host cell by conventional
techniques and the transfected cells are then cultured by conventional
techniques to
produce an antibody of the invention. Thus, the invention includes host cells
35 containing a polynucleotide encoding an antibody of the invention, or a
heavy or light
chain thereof, operably linked to a heterologous promoter. In preferred
embodiments
for the expression of double-chained antibodies, vectors encoding both the
heavy and
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
194
light chains may be co-expressed in the host cell for expression of the entire
immunoglobulin molecule, as detailed below.
A variety of host-expression vector systems may be utilized to express the
antibody molecules of the invention. Such host-expression systems represent
vehicles
by which the coding sequences of interest may be produced and subsequently
purified,
but also represent cells which may, when transformed or transfected with the
appropriate nucleotide coding sequences, express an antibody molecule of the
invention in situ. These include but are not limited to microorganisms such as
bacteria
(e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA,
plasmid
DNA or cosmid DNA expression vectors containing antibody coding sequences;
yeast
(e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression
vectors
containing antibody coding sequences; insect cell systems infected with
recombinant
virus expression vectors (e.g., baculovirus) containing antibody coding
sequences;
plant cell systems infected with recombinant virus expression vectors (e.g.,
cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with
recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody
coding
sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 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., 1986, Gene
45:101;
Cockett et al., 1990, Bio/Technology 8:2).
In bacterial systems, a number of expression vectors may be
advantageously selected depending upon the use intended for the antibody
molecule
being expressed. For example, when a large quantity of such a protein is to be
produced, for the generation of pharmaceutical compositions of an antibody
molecule,
vectors which direct the expression of high levels of fusion protein products
that are
readily purified may be desirable. Such vectors include, but are not limited,
to the E.
coli expression vector pUR278 (Ruther et al., 1983, EMBO J. 2:1791 ), in which
the
antibody coding sequence may be ligated individually into the vector in frame
with the
lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye
&
Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J.
Biol. Chem. 24:5503-5509); and the like. pGEX vectors may also be used to
express
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
195
foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
In
general, such fusion proteins are soluble and can easily be purified from
lysed cells by
adsorption and binding to a matrix glutathione-agarose beads followed by
elution in the
presence of free glutathione. The pGEX vectors are designed to include
thrombin or
factor Xa protease cleavage sites so that the cloned target gene product can
be released
from the GST moiety.
In an insect system, Autographa californica nuclear polyhedrosis virus
(AcNPV) is used as a vector to express foreign genes. The virus grows in
Spodoptera frugiperda cells. The antibody coding sequence may be cloned
1o individually into non-essential regions (for example the polyhedrin gene)
of the virus
and placed under control of an AcNPV promoter (for example the polyhedrin
promoter).
In mammalian host cells, a number of viral-based expression systems may
be utilized. In cases where an adenovirus is used as an expression vector, the
~5 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 E1 or E3) will result in a recombinant virus that is viable and
capable of
2o expressing the antibody molecule in infected hosts. (e.g., see Logan &
Shenk, 1984,
Proc. Natl. Acad. Sci. USA 81:355-359). Specific initiation signals may also
be
required for efficient translation of inserted antibody coding sequences.
These signals
include the ATG initiation codon and adjacent sequences. Furthermore, the
initiation
codon must be in phase with the reading frame of the desired coding sequence
to
25 ensure translation of the entire insert. These exogenous translational
control signals
and initiation codons can be of a variety of origins, both natural and
synthetic. The
efficiency of expression may be enhanced by the inclusion of appropriate
transcription
enhancer elements, transcription terminators, etc. (see Bittner et al., 1987,
Methods in
Enzymol. 153:51-544).
3o 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-
35 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,
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
196
glycosylation, and phosphorylation of the gene product may be used. Such
mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS,
MDCK, 293, 3T3, WI38, and in particular, breast cancer cell lines such as, for
example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell
line such as, for example, CRL7030 and Hs578Bst.
For long-term, high-yield production of recombinant proteins, stable
expression is preferred. For example, cell lines which stably express the
antibody
molecule may be engineered. Rather than using expression vectors which contain
viral
origins of replication, host cells can be transformed with DNA controlled by
appropriate expression control elements (e.g., promoter, enhancer, sequences,
transcription terminators, polyadenylation sites, etc.), and a selectable
marker.
Following the introduction of the foreign DNA, engineered cells may be allowed
to
grow for 1-2 days in an enriched media, and then are switched to a selective
media.
The selectable marker in the recombinant plasmid confers resistance to the
selection
and allows cells to stably integrate the plasmid into their chromosomes and
grow to
form foci which in turn can be cloned and expanded into cell lines. This
method may
advantageously be used to engineer cell lines which express the antibody
molecule.
Such engineered cell lines may be particularly useful in screening and
evaluation of
compounds that interact directly or indirectly with the antibody molecule.
2o A number of selection systems may be used, including but not limited to
the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223),
hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 192,
Proc.
Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et
al.,
1980, Cell 22:817) genes can be employed in tk-, hgprt- or aprt- cells,
respectively.
Also, antimetabolite resistance can be used as the basis of selection for the
following
genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980,
Natl.
Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA
78:1527);
gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981,
Proc.
Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the
aminoglycoside
3o G-418 Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95;
Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993,
Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem.
62:191-217; May, 1993, TIB TECH 11(5):155-215); and hygro, which confers
resistance to hygromycin (Santerre et al., 1984, Gene 30:147). Methods
commonly
known in the art of recombinant DNA technology which can be used are described
in
Ausubel et al. (eds.), 1993, Current Protocols in Molecular Biology, John
Wiley &
Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual,
Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al. (eds), 1994,
Current
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
197
Protocols in Human Genetics, John Wiley & Sons, NY.; Colberre-Garapin et al.,
1981, J. Mol. Biol. 150:1, which are incorporated by reference herein in their
entireties.
The expression levels of an antibody molecule can be increased by vector
amplification (for a review, see Bebbington and Hentschel, The use of vectors
based
on gene amplification for the expression of cloned genes in mammalian cells in
DNA
cloning, Vol.3. (Academic Press, New York, 1987)). When a marker in the vector
system expressing antibody is amplifiable, increase in the level of inhibitor
present in
culture of host cell will increase the number of copies of the marker gene.
Since the
amplified region is associated with the antibody gene, production of the
antibody will
also increase (Grouse et al., 1983, Mol. Cell. Biol. 3:257).
The host cell may be co-transfected with two expression vectors of the
invention, the first vector encoding a heavy chain derived polypeptide and the
second
vector encoding a light chain derived polypeptide. The two vectors may contain
identical selectable markers which enable equal expression of heavy and light
chain
polypeptides. Alternatively, a single vector may be used which encodes both
heavy
and light chain polypeptides. In such situations, the light chain should be
placed
before the heavy chain to avoid an excess of toxic free heavy chain
(Proudfoot, 1986,
Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197). The coding
sequences for the heavy and light chains may comprise cDNA or genomic DNA.
Once an antibody molecule of the invention has been recombinantly
expressed, it may be purified by any method known in the art for purification
of an
immunoglobulin molecule, for example, by chromatography (e.g., ion exchange,
affinity, particularly by affinity for the specific antigen after Protein A,
and sizing
column chromatography), centrifugation, differential solubility, or by any
other
standard technique for the purification of proteins.
Antibody Conjugates
The present invention encompasses antibodies recombinantly fused or
3o chemically conjugated (including both covalently and non-covalently
conjugations) to
a polypeptide (or portion thereof, preferably at least 10, 20 or 50 amino
acids of the
polypeptide) of the present invention to generate fusion proteins. The fusion
does not
necessarily need to be direct, but may occur through linker sequences. The
antibodies
may be specific for antigens other than polypeptides (or portion thereof,
preferably at
least 10, 20 or 50 amino acids of the polypeptide) of the present invention.
For
example, antibodies may be used to target the polypeptides of the present
invention to
particular cell types, either in vitro or in vivo, by fusing or conjugating
the
polypeptides of the present invention to antibodies specific for particular
cell surface
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
198
receptors. Antibodies fused or conjugated to the polypeptides of the present
invention
may also be used in in vitro immunoassays and purification methods using
methods
known in the art. See e.g., Harbor et al., supra, and PCT publication WO
93/21232;
EP 439,095; Naramura et al., Immunol. Lett. 39:91-99 (1994); U.S. Patent
5,474,981; Gillies et al., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol.
146:2446-2452(1991), which are incorporated by reference in their entireties.
The present invention further includes compositions comprising the
polypeptides of the present invention fused or conjugated to antibody domains
other
than the variable regions. For example, the polypeptides of the present
invention may
be fused or conjugated to an antibody Fc region, or portion thereof. The
antibody
portion fused to a polypeptide of the present invention may comprise the
constant
region, hinge region, CH 1 domain, CH2 domain, and CH3 domain or any
combination of whole domains or portions thereof. The polypeptides may also be
fused or conjugated to the above antibody portions to form multimers. For
example,
Fc portions fused to the polypeptides of the present invention can form dimers
through
disulfide bonding between the Fc portions. Higher multimeric forms can be made
by
fusing the polypeptides to portions of IgA and IgM. Methods for fusing or
conjugating the polypeptides of the present invention to antibody portions are
known
in the art. See, e.g., U.S. Patent Nos. 5,336,603; 5,622,929; 5,359,046;
5,349,053;
5,447,851; 5,112,946; EP 307,434; EP 367,166; PCT publications WO 96/04388;
WO 91/06570; Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88:10535-10539
(1991);
Zheng et al., J. Immunol. 154:5590-5600 (1995); and Vil et al., Proc. Natl.
Acad.
Sci. USA 89:11337- 11341(1992) (said references incorporated by reference in
their
entireties).
As discussed, supra, the polypeptides of the present invention may be
fused or conjugated to the above antibody portions to increase the in vivo
half life of
the polypeptides or for use in immunoassays using methods known in the art.
Further, the polypeptides of the present invention may be fused or conjugated
to the
above antibody portions to facilitate purification. One reported example
describes
chimeric proteins consisting of the first two domains of the human CD4-
polypeptide
and various domains of the constant regions of the heavy or light chains of
mammalian
immunoglobulins. (EP 394,827; Traunecker et al., Nature 331:84-86 (1988). The
polypeptides of the present invention fused or conjugated to an antibody
having
disulfide- linked dimeric structures (due to the IgG) may also be more
efficient in
binding and neutralizing other molecules, than the monomeric secreted protein
or
protein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964
(1995)). In
many cases, the Fc part in a fusion protein is beneficial in therapy and
diagnosis, and
thus can result in, for example, improved pharmacokinetic properties. (EP A
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
199
232,262). Alternatively, deleting the Fc part after the fusion protein has
been
expressed, detected, and purified, would be desired. For example, the Fc
portion may
hinder therapy and diagnosis if the fusion protein is used as an antigen for
immunizations. In drug discovery, for example, human proteins, such as hIL-5,
have
been fused with Fc portions for the purpose of high-throughput screening
assays to
identify antagonists of hIL-5. (See, D. Bennett et al., J. Molecular
Recognition 8:52-
58 (1995); K. Johanson et al., J. Biol. Chem. 270:9459-9471 (1995)0.
Moreover, the antibodies or fragments thereof of the present invention can
be fused to marker sequences, such as a peptide to facilitates their
purification. In
1o preferred embodiments, the marker amino acid sequence is a hexa-histidine
peptide,
such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue,
Chatsworth, CA, 91311), among others, many of which are commercially
available.
As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989),
for
instance, hexa-histidine provides for convenient purification of the fusion
protein.
Other peptide tags useful for purification include, but are not limited to,
the "HA" tag,
which corresponds to an epitope derived from the influenza hemagglutinin
protein
(Wilson et al., Cell 37:767 (1984)) and the "flag" tag.
The present invention further encompasses antibodies or fragments thereof
conjugated to a diagnostic or therapeutic agent. The antibodies can be used
diagnostically to, for example, monitor the development or progression of a
tumor as
part of a clinical testing procedure to, e.g., determine the efficacy of a
given treatment
regimen. Detection can be facilitated by coupling the antibody to a detectable
substance. Examples of detectable substances include various enzymes,
prosthetic
groups, fluorescent materials, luminescent materials, bioluminescent
materials,
radioactive materials, positron emitting metals using various positron
emission
tomographies, and nonradioactive paramagnetic metal ions. See, for example,
U.S.
Patent No. 4,741,900 for metal ions which can be conjugated to antibodies for
use as
diagnostics according to the present invention. Examples of suitable enzymes
include
horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or
3o acetylcholinesterase; examples of suitable prosthetic group complexes
include
streptavidin/biotin and avidin/biotin; examples of suitable fluorescent
materials include
umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an
example of a
luminescent material includes luminol; examples of bioluminescent materials
include
luciferase, luciferin, and aequorin; and examples of suitable radioactive
material
include iodine ('3'I,'zsl,'z3I,'z'1), carbon ('4C), sulfur (35S), tritium
(3H), indium
("smln, "3mln, "zln, "'In), and technetium (~''Tc, ~''"Tc), thallium
(z°'Ti), gallium
(6~Ga, 6'Ga), palladium ('°3Pd), molybdenum (~''Mo), xenon ('33Xe),
fluorine ('8F),
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
200
153Sm' 177Lu' 159Gd' 149Pm' l4oLa~ l7sYb' 166HO' 90Y' 47SC' 186Re' 188Re'
142Pr' lOSRh,
97Ru .
Further, an antibody or fragment thereof may be conjugated to a therapeutic
moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a
therapeutic agent or
a radioactive metal ion. A cytotoxin or cytotoxic agent includes any agent
that is
detrimental to cells. Examples include paclitaxol, cytochalasin B, gramicidin
D,
ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine,
vinblastine,
colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,
mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,
tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs
thereof.
Therapeutic agents include, but are not limited to, antimetabolites (e.g.,
methotrexate,
6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine),
alkylating
agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine
(BSNU)
and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,
streptozotocin, mitomycin C, and cis- dichlorodiamine platinum (II) (DDP)
cisplatin),
anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin),
antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin,
mithramycin, and
anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and
vinblastine).
The conjugates of the invention can be used for modifying a given
2o biological response, the therapeutic agent or drug moiety is not to be
construed as
limited to classical chemical therapeutic agents. For example, the drug moiety
may be
a protein or polypeptide possessing a desired biological activity. Such
proteins may
include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or
diphtheria toxin; a protein such as tumor necrosis factor, a-interferon,13-
interferon,
nerve growth factor, platelet derived growth factor, tissue plasminogen
activator, a
thrombotic agent or an anti- angiogenic agent, e.g., angiostatin or
endostatin; or,
biological response modifiers such as, for example, lymphokines, interleukin-I
("IL-
1 "), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophase
colony
stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-
CSF"), or
other growth factors.
Antibodies may also be attached to solid supports, which are particularly
useful for immunoassays or purification of the target antigen. Such solid
supports
include, but are not limited to, glass, cellulose, polyacrylamide, nylon,
polystyrene,
polyvinyl chloride or polypropylene.
Techniques for conjugating such therapeutic moiety to antibodies are well
known, see, e.g., Arnon 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
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
201
Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.),
pp.
623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic
Agents
In Cancer Therapy: A Review", in Monoclonal Antibodies'84: Biological And
Clinical
Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results,
And
Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer
Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin
et
al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., "The
Preparation
And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev. 62:119-
58
( 1982).
to Alternatively, an antibody can be conjugated to a second antibody to form
an antibody heteroconjugate as described by Segal in U.S. Patent No.
4,676,980,
which is incorporated herein by reference in its entirety.
An antibody, with or without a therapeutic moiety conjugated to it,
administered alone or in combination with cytotoxic factors) and/or
cytokine(s) can
be used as a therapeutic.
Assays For Antibody Binding
The antibodies of the invention may be assayed for immunospecific binding
by any method known in the art. The immunoassays which can be used include but
2o are not limited to competitive and non-competitive assay systems using
techniques
such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent
assay), "sandwich" immunoassays, immunoprecipitation assays, precipitin
reactions,
gel diffusion precipitin reactions, immunodiffusion assays, agglutination
assays,
complement-fixation assays, immunoradiometric assays, fluorescent
immunoassays,
protein A immunoassays, to name but a few. Such assays are routine and well
known
in the art (see, e.g., Ausubel et al, eds, 1994, Current Protocols in
Molecular
Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by
reference herein in its entirety). Exemplary immunoassays are described
briefly below
(but are not intended by way of limitation).
3o Immunoprecipitation protocols generally comprise lysing a population of
cells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X- 100, 1%
sodium
deoxycholate, 0.1% SDS, 0.15 M NaCI, 0.01 M sodium phosphate at pH 7.2, 1%
Trasylol) supplemented with protein phosphatase and/or protease inhibitors
(e.g.,
EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to
the cell
lysate, incubating for a period of time (e.g., 1-4 hours) at 4° C,
adding protein A
and/or protein G sepharose beads to the cell lysate, incubating for about an
hour or
more at 4° C, washing the beads in lysis buffer and resuspending the
beads in
SDS/sample buffer. The ability of the antibody of interest to
immunoprecipitate a
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
202
particular antigen can be assessed by, e.g., western blot analysis. One of
skill in the
art would be knowledgeable as to the parameters that can be modified to
increase the
binding of the antibody to an antigen and decrease the background (e.g., pre-
clearing
the cell lysate with sepharose beads). For further discussion regarding
immunoprecipitation protocols see, e.g., Ausubel et al, eds, 1994, Current
Protocols
in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.
Western blot analysis generally comprises preparing protein samples,
electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%- 20%
SDS-
PAGE depending on the molecular weight of the antigen), transferring the
protein
sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF
or
nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or
non-
fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20),
blocking
the membrane with primary antibody (the antibody of interest) diluted in
blocking
buffer, washing the membrane in washing buffer, blocking the membrane with a
secondary antibody (which recognizes the primary antibody, e.g., an anti-human
antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase
or
alkaline phosphatase) or radioactive molecule (e.g., 32P or 125I) 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
2o be modified to increase the signal detected and to reduce the background
noise. For
further discussion regarding western blot protocols see, e.g., Ausubel et al,
eds,
1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,
New
York at 10.8.1.
ELISAs comprise preparing antigen, coating the well of a 96 well microtiter
plate with the antigen, adding the antibody of interest conjugated to a
detectable
compound such as an enzymatic substrate (e.g., horseradish peroxidase or
alkaline
phosphatase) to the well and incubating for a period of time, and detecting
the
presence of the antigen. In ELISAs the antibody of interest does not have to
be
conjugated to a detectable compound; instead, a second antibody (which
recognizes
3o the antibody of interest) conjugated to a detectable compound may be added
to the
well. Further, instead of coating the well with the antigen, the antibody may
be coated
to the well. In this case, a second antibody conjugated to a detectable
compound may
be added following the addition of the antigen of interest to the coated well.
One of
skill in the art would be knowledgeable as to the parameters that can be
modified to
increase the signal detected as well as other variations of ELISAs known in
the art.
For further discussion regarding ELISAs see, e.g., Ausubel et al, eds, 1994,
Current
Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at
11.2.1.
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
203
The binding affinity of an antibody to an antigen and the off-rate of an
antibody-antigen interaction can be determined by competitive binding assays.
One
example of a competitive binding assay is a radioimmunoassay comprising the
incubation of labeled antigen (e.g., 3H or 125I) with the antibody of interest
in the
presence of increasing amounts of unlabeled antigen, and the detection of the
antibody
bound to the labeled antigen. The affinity of the antibody of interest for a
particular
antigen and the binding off-rates can be determined from the data by scatchard
plot
analysis. Competition with a second antibody can also be determined using
radioimmunoassays. In this case, the antigen is incubated with antibody of
interest is
1o conjugated to a labeled compound (e.g., 3H or 125I) in the presence of
increasing
amounts of an unlabeled second antibody.
Therapeutic Uses
The present invention is further directed to antibody-based therapies which
involve administering antibodies of the invention to an animal, preferably a
mammal,
and most preferably a human, patient for treating one or more of the described
disorders. Therapeutic compounds of the invention include, but are not limited
to,
antibodies of the invention (including fragments, analogs and derivatives
thereof as
described herein) and nucleic acids encoding antibodies of the invention
(including
fragments, analogs and derivatives thereof as described herein). The
antibodies of the
invention can be used to treat, inhibit or prevent diseases and disorders
associated
with aberrant expression and/or activity of a polypeptides of the invention,
including,
but not limited to, immune disorders. The treatment and/or prevention of
diseases
and disorders associated with aberrant expression and/or activity of a
polypeptide of
the invention includes, but is not limited to, alleviating symptoms associated
with those
diseases and disorders. Antibodies of the invention may be provided in
pharmaceutically acceptable compositions as known in the art or as described
herein.
Antibodies that agonize the TR14 receptor can be used to ameliorate or treat
biological
activities associated with epithelial cell proliferation, tooth development,
growth of
3o mucosal layers, and the growth of epithelial surfaces, including hair
folicles, sweat
glands, basal cells, and dermis. Accordingly, TR14 agonistic antibodies may be
used
in the treatment of diseases and/or disorders relating to the epithelium
(e.g., anhidrotic
ectodermal dysplasia, hidrotic ectodermal dysplasia, sweat gland disorders,
venous
ulcers, psoriasis, prickly heat disorder, wounds healing, cancers of
epithelial origins,
male pattern baldness, and/or as described under "Epithelial Cell
Proliferation and
Wound Healing" below).
A summary of the ways in which the antibodies of the present invention
may be used therapeutically includes binding polynucleotides or polypeptides
of the
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
204
present invention locally or systemically in the body or by direct
cytotoxicity of the
antibody, e.g. as mediated by complement (CDC) or by effector cells (ADCC).
Some
of these approaches are described in more detail below. Armed with the
teachings
provided herein, one of ordinary skill in the art will know how to use the
antibodies of
the present invention for diagnostic, monitoring or therapeutic purposes
without undue
experimentation.
The antibodies of this invention may be advantageously utilized in
combination with other monoclonal or chimeric antibodies, or with lymphokines
or
hematopoietic growth factors (such as, e.g., IL-2, IL-3 and IL-7), for
example, which
serve to increase the number or activity of effector cells which interact with
the
antibodies.
The antibodies of the invention may be administered alone or in
combination with other types of treatments (e.g., radiation therapy,
chemotherapy,
hormonal therapy, immunotherapy and anti-tumor agents). Generally,
administration
of products of a species origin or species reactivity (in the case of
antibodies) that is
the same species as that of the patient is preferred. Thus, in a preferred
embodiment,
human antibodies, fragments derivatives, analogs, or nucleic acids, are
administered
to a human patient for therapy or prophylaxis.
It is preferred to use high affinity and/or potent in vivo inhibiting and/or
2o neutralizing antibodies against polypeptides or polynucleotides of the
present
invention, fragments or regions thereof, for both immunoassays directed to and
therapy of disorders related to polynucleotides or polypeptides, including
fragments
thereof, of the present invention. Such antibodies, fragments, or regions,
will
preferably have an affinity for polynucleotides or polypeptides, including
fragments
thereof. Preferred binding affinities include those with a dissociation
constant or Kd
less than 5 X 10-6 M, 10-6 M, 5 X 10-7 M, 10-7 M, 5 X 10-8 M, 10-8 M, 5 X 10-9
M, 10-9 M, 5 X 10-10 M, 10-10 M, 5 X 10-11 M, 10-11 M, 5 X 10-12 M, 10-12 M,
5 X 10-13 M, 10- 13 M, 5 X 10-14 M, 10-14 M, 5 X 10-15 M, and 10-15 M.
3o Gene Therapy
In a specific embodiment, nucleic acids comprising sequences encoding
antibodies or functional derivatives thereof, are administered to treat,
inhibit or prevent
a disease or disorder associated with aberrant expression and/or activity of a
polypeptide of the invention, by way of gene therapy. Gene therapy refers to
therapy
performed by the administration to a subject of an expressed or expressible
nucleic
acid. In this embodiment of the invention, the nucleic acids produce their
encoded
protein that mediates a therapeutic effect.
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
205
Any of the methods for gene therapy available in the art can be used
according to the present invention. Exemplary methods are described below.
For general reviews of the methods of gene therapy, see Goldspiel et al.,
1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95;
Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993,
Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem.
62:191-217; May, 1993, TIBTECH 11(5):155-215). Methods commonly known in
the art of recombinant DNA technology which can be used are described in
Ausubel et
al. (eds.), 1993, Current Protocols in Molecular Biology, John Wiley & Sons,
NY;
and Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual,
Stockton
Press, NY.
In a preferred aspect, the compound comprises nucleic acid sequences
encoding an antibody, said nucleic acid sequences being part of expression
vectors
that express the antibody or fragments or chimeric proteins or heavy or light
chains
thereof in a suitable host. In particular, such nucleic acid sequences have
promoters
operably linked to the antibody coding region, said promoter being inducible
or
constitutive, and, optionally, tissue- specific. In another particular
embodiment,
nucleic acid molecules are used in which the antibody coding sequences and any
other
desired sequences are flanked by regions that promote homologous recombination
at a
desired site in the genome, thus providing for intrachromosomal expression of
the
antibody nucleic acids (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA
86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438). In specific
embodiments,
the expressed antibody molecule is a single chain antibody; alternatively, the
nucleic
acid sequences include sequences encoding both the heavy and light chains, or
fragments thereof, of the antibody.
Delivery of the nucleic acids into a patient may be either direct, in which
case the patient is directly exposed to the nucleic acid or nucleic acid-
carrying vectors,
or indirect, in which case, cells are first transformed with the nucleic acids
in vitro,
then transplanted into the patient. These two approaches are known,
respectively, as
3o in vivo or ex vivo gene therapy.
In a specific embodiment, the nucleic acid sequences are directly
administered in vivo, where it is expressed to produce the encoded product.
This can
be accomplished by any of numerous methods known in the art, e.g., by
constructing
them as part of an appropriate nucleic acid expression vector and
administering it so
that they become intracellular, e.g., by infection using defective or
attenuated
retrovirals or other viral vectors (see U.S. Patent No. 4,980,286), or by
direct
injection of naked DNA, or by use of microparticle bombardment (e.g., a gene
gun;
Biolistic, Dupont), or coating with lipids or cell-surface receptors or
transfecting
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
206
agents, encapsulation in liposomes, microparticles, or microcapsules, or by
administering them in linkage to a peptide which is known to enter the
nucleus, by
administering it in linkage to a ligand subject to receptor-mediated
endocytosis (see,
e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432) (which can be used to
target
cell types specifically expressing the receptors), etc. In another embodiment,
nucleic
acid-ligand complexes can be formed in which the ligand comprises a fusogenic
viral
peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal
degradation. In yet another embodiment, the nucleic acid can be targeted in
vivo for
cell specific uptake and expression, by targeting a specific receptor (see,
e.g., PCT
to Publications WO 92/06180 dated April 16, 1992 (Wu et al.); WO 92/22635
dated
December 23, 1992 (Wilson et al.); W092/20316 dated November 26, 1992 (Findeis
et al.); W093/14188 dated July 22, 1993 (Clarke et al.), WO 93/20221 dated
October
14, 1993 (Young)). Alternatively, the nucleic acid can be introduced
intracellularly
and incorporated within host cell DNA for expression, by homologous
recombination
~5 (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935;
Zijlstra et al.,
1989, Nature 342:435-438).
In a specific embodiment, viral vectors that contains nucleic acid sequences
encoding an antibody of the invention are used. For example, a retroviral
vector can
be used (see Miller et al., 1993, Meth. Enzymol. 217:581-599). These
retroviral
20 vectors have been to delete retroviral sequences that are not necessary for
packaging of
the viral genome and integration into host cell DNA. The nucleic acid
sequences
encoding the antibody to be used in gene therapy are cloned into one or more
vectors,
which facilitates delivery of the gene into a patient. More detail about
retroviral vectors
can be found in Boesen et al., 1994, Biotherapy 6:291-302, which describes the
use
25 of a retroviral vector to deliver the mdrl gene to hematopoietic stem cells
in order to
make the stem cells more resistant to chemotherapy. Other references
illustrating the
use of retroviral vectors in gene therapy are: Clowes et al., 1994, J. Clin.
Invest.
93:644-651; Kiem et al., 1994, Blood 83:1467-1473; Salmons and Gunzberg, 1993,
Human Gene Therapy 4:129-141; and Grossman and Wilson, 1993, Curr. Opin. in
3o Genetics and Devel. 3:110-114.
Adenoviruses are other viral vectors that can be used in gene therapy.
Adenoviruses are especially attractive vehicles for delivering genes to
respiratory
epithelia. Adenoviruses naturally infect respiratory epithelia where they
cause a mild
disease. Other targets for adenovirus-based delivery systems are liver, the
central
35 nervous system, endothelial cells, and muscle. Adenoviruses have the
advantage of
being capable of infecting non-dividing cells. Kozarsky and Wilson, 1993,
Current
Opinion in Genetics and Development 3:499-503 present a review of adenovirus-
based
gene therapy. Bout et al., 1994, Human Gene Therapy 5:3-10 demonstrated the
use
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
207
of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus
monkeys.
Other instances of the use of adenoviruses in gene therapy can be found in
Rosenfeld
et al., 1991, Science 252:431-434; Rosenfeld et al., 1992, Cell 68:143- 155;
Mastrangeli et al., 1993, J. Clin. Invest. 91:225-234; PCT Publication
W094/12649;
and Wang, et al., 1995, Gene Therapy 2:775-783. In a preferred embodiment,
adenovirus vectors are used.
Adeno-associated virus (AAV) has also been proposed for use in gene
therapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med. 204:289-300; U.S.
Patent
No. 5,436,146).
to Another approach to gene therapy involves transferring a gene to cells in
tissue culture by such methods as electroporation, lipofection, calcium
phosphate
mediated transfection, or viral infection. Usually, the method of transfer
includes the
transfer of a selectable marker to the cells. The cells are then placed under
selection to
isolate those cells that have taken up and are expressing the transferred
gene. Those
is cells are then delivered to a patient.
In this embodiment, the nucleic acid is introduced into a cell prior to
administration in vivo of the resulting recombinant cell. Such introduction
can be
carried out by any method known in the art, including but not limited to
transfection,
electroporation, microinjection, infection with a viral or bacteriophage
vector
2o containing the nucleic acid sequences, cell fusion, chromosome-mediated
gene
transfer, microcell-mediated gene transfer, spheroplast fusion, etc. Numerous
techniques are known in the art for the introduction of foreign genes into
cells (see,
e.g., Loeffler and Behr, 1993, Meth. Enzymol. 217:599-618; Cohen et al., 1993,
Meth. Enzymol. 217:618-644; Cline, 1985, Pharmac. Ther. 29:69-92) and may be
25 used in accordance with the present invention, provided that the necessary
developmental and physiological functions of the recipient cells are not
disrupted. The
technique should provide for the stable transfer of the nucleic acid to the
cell, so that
the nucleic acid is expressible by the cell and preferably heritable and
expressible by
its cell progeny.
3o The resulting recombinant cells can be delivered to a patient by various
methods known in the art. Recombinant blood cells (e.g., hematopoietic stem or
progenitor cells) are preferably administered intravenously. The amount of
cells
envisioned for use depends on the desired effect, patient state, etc., and can
be
determined by one skilled in the art.
35 Cells into which a nucleic acid can be introduced for purposes of gene
therapy encompass any desired, available cell type, and include but are not
limited to
epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells,
hepatocytes;
blood cells such as Tlymphocytes, Blymphocytes, monocytes, macrophages,
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
208
neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or
progenitor
cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained
from bone
marrow, umbilical cord blood, peripheral blood, fetal liver, etc.
In a preferred embodiment, the cell used for gene therapy is autologous to
the patient.
In an embodiment in which recombinant cells are used in gene therapy,
nucleic acid sequences encoding an antibody are introduced into the cells such
that
they are expressible by the cells or their progeny, and the recombinant cells
are then
administered in vivo for therapeutic effect. In a specific embodiment, stem or
to progenitor cells are used. Any stem and/or progenitor cells which can be
isolated and
maintained in vitro can potentially be used in accordance with this embodiment
of the
present invention (see e.g. PCT Publication WO 94/08598, dated April 28, 1994;
Stemple and Anderson, 1992, Cell 71:973-985; Rheinwald, 1980, Meth. Cell Bio.
21A:229; and Pittelkow and Scott, 1986, Mayo Clinic Proc. 61:771).
1n a specific embodiment, the nucleic acid to be introduced for purposes of
gene therapy comprises an inducible promoter operably linked to the coding
region,
such that expression of the nucleic acid is controllable by controlling the
presence or
absence of the appropriate inducer of transcription.
2o Demonstration of Therapeutic or Prophylactic Activity
The compounds or pharmaceutical compositions of the invention are
preferably tested in vitro, and then in vivo for the desired therapeutic or
prophylactic
activity, prior to use in humans. For example, in vitro assays to demonstrate
the
therapeutic or prophylactic utility of a compound or pharmaceutical
composition
include, the effect of a compound on a cell line or a patient tissue sample.
The effect of
the compound or composition on the cell line and/or tissue sample can be
determined
utilizing techniques known to those of skill in the art including, but not
limited to,
rosette formation assays and cell lysis assays. In accordance with the
invention, in
vitro assays which can be used to determine whether administration of a
specific
3o compound is indicated, include in vitro cell culture assays in which a
patient tissue
sample is grown in culture, and exposed to or otherwise administered a
compound,
and the effect of such compound upon the tissue sample is observed.
Therapeutic/Prophylactic Administration and Composition
The invention provides methods of treatment, inhibition and prophylaxis by
administration to a subject of an effective amount of a compound or
pharmaceutical
composition of the invention, preferably an antibody of the invention. In a
preferred
aspect, the compound is substantially purified (e.g., substantially free from
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
209
substances that limit its effect or produce undesired side-effects). The
subject is
preferably an animal, including but not limited to animals such as cows, pigs,
horses,
chickens, cats, dogs, etc., and is preferably a mammal, and most preferably
human.
Formulations and methods of administration that can be employed when the
compound comprises a nucleic acid or an immunoglobulin are described above;
additional appropriate formulations and routes of administration can be
selected from
among those described herein below.
Various delivery systems are known and can be used to administer a
compound of the invention, e.g., encapsulation in liposomes, microparticles,
to microcapsules, recombinant cells capable of expressing the compound,
receptor-
mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-
4432),
construction of a nucleic acid as part of a retroviral or other vector, etc.
Methods of
introduction include but are not limited to intradermal, intramuscular,
intraperitoneal,
intravenous, subcutaneous, intranasal, epidural, and oral routes. The
compounds or
~5 compositions may be administered by any convenient route, for example by
infusion
or bolus injection, by absorption through epithelial or mucocutaneous linings
(e.g.,
oral mucosa, rectal and intestinal mucosa, etc.) and may be administered
together with
other biologically active agents. Administration can be systemic or local. In
addition,
it may be desirable to introduce the pharmaceutical compounds or compositions
of the
2o 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.
25 In a specific embodiment, it may be desirable to administer the
pharmaceutical compounds or compositions of the invention locally to the area
in need
of treatment; this may be achieved by, for example, and not by way of
limitation, local
infusion during surgery, topical application, e.g., in conjunction with a
wound
dressing after surgery, by injection, by means of a catheter, by means of a
3o suppository, or by means of an implant, said implant being of a porous, non-
porous,
or gelatinous material, including membranes, such as sialastic membranes, or
fibers.
Preferably, when administering a protein, including an antibody, of the
invention,
care must be taken to use materials to which the protein does not absorb.
In another embodiment, the compound or composition can be delivered in a
35 vesicle, in particular a liposome (see Langer, 1990, Science 249:1527-1533;
Treat et
al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-
Berestein
and Fidler (eds.), Liss, New York, pp. 353- 365 (1989); Lopez-Berestein,
ibid., pp.
317-327; see generally ibid.)
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
210
In yet another embodiment, the compound or composition can be delivered
in a controlled release system. In one embodiment, a pump may be used (see
Langer,
supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al.,
1980,
Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In another
embodiment, polymeric materials can be used (see Medical Applications of
Controlled
Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974);
Controlled
Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball
(eds.),
Wiley, New York (1984); Ranger and Peppas, J., 1983, Macromol. Sci. Rev.
Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228:190; During et
al.,
1989, Ann. Neurol. 25:351; Howard et al., 1989, J.Neurosurg. 71:105). In yet
another embodiment, a controlled release system can be placed in proximity of
the
therapeutic target, i.e., the brain, thus requiring only a fraction of the
systemic dose
(see, e.g., Goodson, in Medical Applications of Controlled Release, supra,
vol. 2,
pp. 115-138 (1984)).
Other controlled release systems are discussed in the review by Langer
(1990, Science 249:1527-1533).
In a specific embodiment where the compound of the invention is a nucleic
acid encoding a protein, the nucleic acid can be administered in vivo to
promote
expression of its encoded protein, by constructing it as part of an
appropriate nucleic
2o 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., 1991, Proc. Natl. Acad. Sci. USA 88:1864-1868), etc.
Alternatively, a
nucleic acid can be introduced intracellularly and incorporated within host
cell DNA
for expression, by homologous recombination.
The present invention also provides pharmaceutical compositions. Such
compositions comprise a therapeutically effective amount of a compound, and a
3o 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
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
211
solutions and aqueous dextrose and glycerol solutions can also be employed as
liquid
carriers, particularly for injectable solutions. Suitable pharmaceutical
excipients
include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,
silica gel,
sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim
milk,
glycerol, propylene, glycol, water, ethanol and the like. The composition, if
desired,
,can also contain minor amounts of wetting or emulsifying agents, or pH
buffering
agents. These compositions can take the form of solutions, suspensions,
emulsion,
tablets, pills, capsules, powders, sustained-release formulations and the
like. The
composition can be formulated as a suppository, with traditional binders and
carriers
such as triglycerides. Oral formulation can include standard carriers such as
pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium
saccharine, cellulose, magnesium carbonate, etc. Examples of suitable
pharmaceutical
carriers are described in "Remington's Pharmaceutical Sciences" by E.W.
Martin.
Such compositions will contain a therapeutically effective amount of the
compound,
preferably in purified form, together with a suitable amount of carrier so as
to provide
the form for proper administration to the patient. The formulation should suit
the mode
of administration.
In a preferred embodiment, the composition is formulated in accordance
with routine procedures as a pharmaceutical composition adapted for
intravenous
administration to human beings. Typically, compositions for intravenous
administration are solutions in sterile isotonic aqueous buffer. Where
necessary, the
composition may also include a solubilizing agent and a local anesthetic such
as
lignocaine to ease pain at the site of the injection. Generally, the
ingredients are
supplied either separately or mixed together in unit dosage form, for example,
as a dry
lyophilized powder or water free concentrate in a hermetically sealed
container such as
an ampoule or sachette indicating the quantity of active agent. Where the
composition
is to be administered by infusion, it can be dispensed with an infusion bottle
containing sterile pharmaceutical grade water or saline. Where the composition
is
administered by injection, an ampoule of sterile water for injection or saline
can be
provided so that the ingredients may be mixed prior to administration.
The compounds of the invention can be formulated as neutral or salt forms.
Pharmaceutically acceptable salts include those formed with anions such as
those
derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc.,
and those
formed with cations such as those derived from sodium, potassium, ammonium,
calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino
ethanol,
histidine, procaine, etc.
The amount of the compound of the invention which will be effective in the
treatment, inhibition and prevention of a disease or disorder associated with
aberrant
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
212
expression and/or activity of a polypeptide of the invention can be determined
by
standard clinical techniques. In addition, in vitro assays may optionally be
employed
to help identify optimal dosage ranges. The precise dose to be employed in the
formulation will also depend on the route of administration, and the
seriousness of the
disease or disorder, and should be decided according to the judgment of the
practitioner and each patient's circumstances. Effective doses may be
extrapolated
from dose-response curves derived from in vitro or animal model test systems.
For antibodies, the dosage administered to a patient is typically 0.1 mg/kg
to 100 mg/kg of the patient's body weight. Preferably, the dosage administered
to a
1o patient is between 0.1 mg/kg and 20 mg/kg of the patient's body weight,
more
preferably 1 mg/kg to 10 mg/kg of the patient's body weight. Generally, human
antibodies have a longer half-life within the human body than antibodies from
other
species due to the immune response to the foreign polypeptides. Thus, lower
dosages
of human antibodies and less frequent administration is often possible.
Further, the
~5 dosage and frequency of administration of antibodies of the invention may
be reduced
by enhancing uptake and tissue penetration (e.g., into the brain) of the
antibodies by
modifications such as, for example, lipidation.
The invention also provides a pharmaceutical pack or kit comprising one or
more containers filled with one or more of the ingredients of the
pharmaceutical
20 compositions of the invention. Optionally associated with such containers)
can be a
notice in the form prescribed by a governmental agency regulating the
manufacture,
use or sale of pharmaceuticals or biological products, which notice reflects
approval by
the agency of manufacture, use or sale for human administration.
25 Diagnosis and Imaging
Labeled antibodies, and derivatives and analogs thereof, which specifically
bind to a polypeptide of interest can be used for diagnostic purposes to
detect,
diagnose, or monitor diseases and/or disorders associated with the aberrant
expression
and/or activity of a polypeptide of the invention. The invention provides for
the
30 detection of aberrant expression of a polypeptide of interest, comprising
(a) assaying
the expression of the polypeptide of interest in cells or body fluid of an
individual
using one or more antibodies specific to the polypeptide interest and (b)
comparing the
level of gene expression with a standard gene expression level, whereby an
increase
or decrease in the assayed polypeptide gene expression level compared to the
standard
35 expression level is indicative of aberrant expression.
The invention provides a diagnostic assay for diagnosising a disorder,
comprising (a) assaying the expression of the polypeptide of interest in cells
or body
fluid of an individual using one or more antibodies specific to the
polypeptide interest
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
213
and (b) comparing the level of gene expression with a standard gene expression
level,
whereby an increase or decrease in the assayed polypeptide gene expression
level
compared to the standard expression level is indicative of a particular
disorder. With
respect to cancer, the presence of a relatively high amount of transcript in
biopsied
tissue from an individual may indicate a predisposition for the development of
the
disease, or may provide a means for detecting the disease prior to the
appearance of
actual clinical symptoms. A more definitive diagnosis of this type may allow
health
professionals to employ preventative measures or aggressive treatment earlier
thereby
preventing the development or further progression of the cancer.
Antibodies of the invention can be used to assay protein levels in a
biological sample using classical immunohistological methods known to~ those
of skill
in the art (e.g., see Jalkanen, M., et al., J. Cell. Biol. 101:976-985 (1985);
Jalkanen,
M., et al., J. Cell . Biol. 105:3087-3096 (1987)). Other antibody-based
methods
useful for detecting protein gene expression include immunoassays, such as the
enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
Suitable antibody assay labels are known in the art and include enzyme labels,
such as,
glucose oxidase; radioisotopes, such as iodine ('3'I,'ZSI, "~I,'z'I), carbon
('4C),
sulfur (35S), tritium (3H), indium ("5'"In, "3"'In, "ZIn, "'In), and
technetium (9~'Cc,
99"'Tc), thallium (Z°'Ti), gallium (~Ga, 6'Ga), palladium
('°3Pd), molybdenum (~'Mo),
2o xenon (~ssXe)~ Buorine (~sF)~ ls~sm~ »~Lu~ ~s9Gd~ ~a9Pm~ ~a°La~
mshb~ ~66Ho~ 9oY~
4'Sc,'g6Re,'~Re,'42Pr,'osRh, 9'Ru; luminescent labels, such as luminol; and
fluorescent labels, such as fluorescein and rhodamine, and biotin.
One aspect of the invention is the detection and diagnosis of a disease or
disorder associated with aberrant expression of a polypeptide of the interest
in an
animal, preferably a mammal and most preferably a human. In one embodiment,
diagnosis comprises: a) administering (for example, parenterally,
subcutaneously, or
intraperitoneally) to a subject an effective amount of a labeled molecule
which
specifically binds to the polypeptide of interest; b) waiting for a time
interval following
the administering for permitting the labeled molecule to preferentially
concentrate at
3o sites in the subject where the polypeptide is expressed (and for unbound
labeled
molecule to be cleared to background level); c) determining background level;
and d)
detecting the labeled molecule in the subject, such that detection of labeled
molecule
above the background level indicates that the subject has a particular disease
or
disorder associated with aberrant expression of the polypeptide of interest.
Background level can be determined by various methods including, comparing the
amount of labeled molecule detected to a standard value previously determined
for a
particular system.
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
214
It will be understood in the art that the size of the subject and the imaging
system used will determine the quantity of imaging moiety needed to produce
diagnostic images. In the case of a radioisotope moiety, for a human subject,
the
quantity of radioactivity injected will normally range from about 5 to 20
millicuries of
99mTc. The labeled antibody or antibody fragment will then preferentially
accumulate
at the location of cells which contain the specific protein. In vivo tumor
imaging is
described in S.W. Burchiel et al., "Immunopharmacokinetics of Radiolabeled
Antibodies and Their Fragments." (Chapter 13 in Tumor Imaging: The
Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds.,
Masson
to Publishing Inc. (1982).
Depending on several variables, including the type of label used and the
mode of administration, the time interval following the administration for
permitting
the labeled molecule to preferentially concentrate at sites in the subject and
for unbound
labeled molecule to be cleared to background level is 6 to 48 hours or 6 to 24
hours or
t5 6 to 12 hours. In another embodiment the time interval following
administration is 5 to
20 days or 5 to 10 days.
In an embodiment, monitoring of the disease or disorder is carried out by
repeating the method for diagnosing the disease or disease, for example, one
month
after initial diagnosis, six months after initial diagnosis, one year after
initial diagnosis,
20 etc.
Presence of the labeled molecule can be detected in the patient using
methods known in the art for in vivo scanning. These methods depend upon the
type
of label used. Skilled artisans will be able to determine the appropriate
method for
detecting a particular label. Methods and devices that may be used in the
diagnostic
25 methods of the invention include, but are not limited to, computed
tomography (CT),
whole body scan such as position emission tomography (PET), magnetic resonance
imaging (MRI), and sonography.
In a specific embodiment, the molecule is labeled with a radioisotope and is
detected in the patient using a radiation responsive surgical instrument
(Thurston et
3o al., U.S. Patent No. 5,441,050). In another embodiment, the molecule is
labeled
with a fluorescent compound and is detected in the patient using a
fluorescence
responsive scanning instrument. In another embodiment, the molecule is labeled
with
a positron emitting metal and is detected in the patent using positron
emission-
tomography. In yet another embodiment, the molecule is labeled with a
paramagnetic
35 label and is detected in a patient using magnetic resonance imaging (MRI).
Kits
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
215
The present invention provides kits that can be used in the above methods.
In one embodiment, a kit comprises an antibody of the invention, preferably a
purified
antibody, in one or more containers. In a specific embodiment, the kits of the
present
invention contain a substantially isolated polypeptide comprising an epitope
which is
specifically immunoreactive with an antibody included in the kit. Preferably,
the kits
of the present invention further comprise a control antibody which does not
react with
the polypeptide of interest. In another specific embodiment, the kits of the
present
invention contain a means for detecting the binding of an antibody to a
polypeptide of
interest (e.g., the antibody may be conjugated to a detectable substrate such
as a
1o fluorescent compound, an enzymatic substrate, a radioactive compound or a
luminescent compound, or a second antibody which recognizes the first antibody
may
be conjugated to a detectable substrate).
In another specific embodiment of the present invention, the kit is a
diagnostic kit for use in screening serum containing antibodies specific
against
proliferative and/or cancerous polynucleotides and polypeptides. Such a kit
may
include a control antibody that does not~react with the polypeptide of
interest. Such a
kit may include a substantially isolated polypeptide antigen comprising an
epitope
which is specifically immunoreactive with at least one anti-po(ypeptide
antigen
antibody. Further, such a kit includes means for detecting the binding of said
antibody
to the antigen (e.g., the antibody may be conjugated to a fluorescent compound
such
as fluorescein or rhodamine which can be detected by flow cytometry). In
specific
embodiments, the kit may include a recombinantly produced or chemically
synthesized
polypeptide antigen. The polypeptide antigen of the kit may also be attached
to a solid
support.
In a more specific embodiment the detecting means of the above-described
kit includes a solid support to which said polypeptide antigen is attached.
Such a kit
may also include a non-attached reporter-labeled anti-human antibody. In this
embodiment, binding of the antibody to the polypeptide antigen can be detected
by
binding of the said reporter-labeled antibody.
In an additional embodiment, the invention includes a diagnostic kit for use
in screening serum containing antigens of the polypeptide of the invention.
The
diagnostic kit includes a substantially isolated antibody specifically
immunoreactive
with polypeptide or polynucleotide antigens, and means for detecting the
binding of
the polynucleotide or polypeptide antigen to the antibody. In one embodiment,
the
antibody is attached to a solid support. In a specific embodiment, the
antibody may be
a monoclonal antibody. The detecting means of the kit may include a second,
labeled
monoclonal antibody. Alternatively, or in addition, the detecting means may
include a
labeled, competing antigen.
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
216
In one diagnostic configuration, test serum is reacted with a solid phase
reagent having a surface-bound antigen obtained by the methods of the present
invention. After binding with specific antigen antibody to the reagent and
removing
unbound serum components by washing, the reagent is reacted with reporter-
labeled
anti-human antibody to bind reporter to the reagent in proportion to the
amount of
bound anti-antigen antibody on the solid support. The reagent is again washed
to
remove unbound labeled antibody, and the amount of reporter associated with
the
reagent is determined. Typically, the reporter is an enzyme which is detected
by
incubating the solid phase in the presence of a suitable fluorometric,
luminescent or
1o colorimetric substrate (Sigma, St. Louis, MO).
The solid surface reagent in the above assay is prepared by known
techniques for attaching protein material to solid support material, such as
polymeric
beads, dip sticks, 96-well plate or filter material. These attachment methods
generally
include non-specific adsorption of the protein to the support or covalent
attachment of
the protein, typically through a free amine group, to a chemically reactive
group on the
solid support, such as an activated carboxyl, hydroxyl, or aldehyde group.
Alternatively, streptavidin coated plates can be used in conjunction with
biotinylated
antigen(s).
Thus, the invention provides an assay system or kit for carrying out this
diagnostic method. The kit generally includes a support with surface- bound
recombinant antigens, and a reporter-labeled anti-human antibody for detecting
surface-bound anti-antigen antibody.
Therapeutics
The Tumor Necrosis Factor (TNF) family ligands are known to be among the most
pleiotropic cytokines, inducing a large number of cellular responses,
including cytotoxicity,
anti-viral activity, immunoregulatory activities, and the transcriptional
regulation of several
genes (D.V. Goeddel et al., "Tumor Necrosis Factors: Gene Structure and
Biological
Activities," Symp. Quant. Biol. 51:597- 609 (1986), Cold Spring Harbor; B.
Beutler and A.
Cerami, Annu. Rev. Biochem. 57:505-518 (1988); L.J. Old, Sci. Am. 258:59-75
(1988); W.
Fiers, FEBS Lett. 285:199-224 (1991)). The TNF-family ligands induce such
various cellular
responses by binding to TNF-family receptors.
E~ithilial Disorder-Related Therapeutic Embodiments for TR14
TR14 polynucleotides or polypeptides, or agonists or antagonists of the
present
invention, can be used in assays to test for one or more biological
activities. If these
polynucleotides or polypeptides, or agonists or antagonists of the present
invention, do exhibit
activity in a particular assay, it is likely that these molecules may be
involved in the diseases
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
217
associated with the biological activity. Thus, the polynucleotides and
polypeptides, and
agonists or antagonists could be used to treat the associated disease.
TR14 polynucleotides and translation products are believed to be involved in
biological
activities associated with epithelial cell proliferation, tooth development,
growth of mucosal
layers, and the growth of epithelial surfaces, including hair folicles, sweat
glands, basal cells,
and dermis. Accordingly, compositions of the invention (including
polynucleotides,
polypeptides and antibodies of the invention, and fragments and variants
thereof) may be used
in the diagnosis, detection and/or treatment of diseases and/or disorders
associated with
aberrant TR14 activity. In preferred embodiments, compositions of the
invention (including
TR14 polynucleotides, polypeptides and TR14 agonists, including peptides and
antibodies of
the invention, and fragments and variants thereof) may be used in the
diagnosis, detection
and/or treatment of diseases and/or disorders relating to the epithelium
(e.g., anhidrotic
ectodermal dysplasia, hidrotic ectodermal dysplasia, sweat gland disorders,
venous ulcers,
psoriasis, prickly heat disorder, wounds healing, cancers of epithelial
origins, male pattern
baldness, and/or as described under "Epithelial Cell Proliferation and Wound
Healing" below).
Thus, polynucleotides, translation products and antibodies of the invention
are useful in the
diagnosis, detection and/or treatment of diseases and/or disorders associated
with activities that
include, but are not limited to, diseases and/or disorders of the epithelium
and epithelial cell
proliferation diseases and/or disorders.
2o More generally, polynucleotides, translation products and antibodies
corresponding to
this gene may be useful for the diagnosis, detection and/or treatment of
diseases and/or
disorders associated with the following systems.
Epithelial Cell Proliferation and Wound Healing
In accordance with yet a further aspect of the present invention, there is
provided a
process for utilizing polynucleotides or translation products, as well as
agonists or antagonists
of the present invention, for therapeutic purposes, for example, to stimulate
epithelial cell
proliferation and basal keratinocytes for the purpose of wound healing, and to
stimulate hair
follicle production and healing of dermal wounds. Polynucleotides or
polypeptides, as well as
3o agonists or antagonists of the present invention, may be clinically useful
in stimulating wound
healing including surgical wounds, excisional wounds, deep wounds involving
damage of the
dermis and epidermis, eye tissue wounds, dental tissue wounds, oral cavity
wounds, diabetic
ulcers, dermal ulcers, cubitus ulcers, arterial ulcers, venous stasis ulcers,
burns resulting from
heat exposure or chemicals, and other abnormal wound healing conditions such
as uremia,
malnutrition, vitamin deficiencies and complications associted with systemic
treatment with
steroids, radiation therapy and antineoplastic drugs and antimetabolites.
Polynucleotides or
polypeptides, as well as agonists or antagonists of the present invention,
could be used to
promote dermal reestablishment subsequent to dermal loss.
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
218
In specific, preferred embodiments, TR14 polynucleotides and polypeptides, as
well as
antibodies that agonize TR14 receptor (as described in the section on
Antibodies, above),
stimulate epithelial cell proliferation and/or development to ameliorate the
diseases and
disorders described in this section. Members of the TNF family of proteins are
known to
signal through the NF-xB singaling pathway. NF-xB is a transcription factor
activated by a
wide certain agents to stimulate cell activation and differentiation. It is
believed that the TR14
receptor of the instant invention signals through the NF-xB pathway to
activate proliferation
and development of cells. Thus, TR14 polynucleotides and polypeptides of the
invention as
well as antibodies and peptides that agonize TR14 may be used in accordance
with the
invention to stimulate NF-xB-mediated epithelial cell proliferation, and
thereby treat the
epithelial disorders described above.
It is believed that polynucleotides or polypeptides, as well as agonists or
antagonists of
the present invention, will also produce changes in hepatocyte proliferation,
and epithelial cell
proliferation in the lung, breast, pancreas, stomach, small intesting, and
large intestine.
Polynucleotides or polypeptides, as well as agonists or antagonists of the
present invention,
could promote proliferation of epithelial cells such as sebocytes, hair
follicles, hepatocytes,
type II pneumocytes, mucin-producing goblet cells, and other epithelial cells
and their
progenitors contained within the skin, lung, liver, and gastrointestinal
tract. Polynucleotides or
polypeptides, agonists or antagonists of the present invention, may promote
proliferation of
endothelial cells, keratinocytes, and basal keratinocytes.
Polynucleotides or polypeptides, as well as agonists or antagonists of the
present
invention, could be used to increase the adherence of skin grafts to a wound
bed and to
stimulate re-epithelialization from the wound bed. The following are types of
grafts that
polynucleotides or polypeptides, agonists or antagonists of the present
invention, could be used
to increase adherence to a wound bed: autografts, artificial skin, allografts,
autodermic graft,
autoepdermic grafts, avacular grafts, Blair-Brown grafts, bone graft,
brephoplastic grafts, cutis
graft, delayed graft, dermic graft, epidermic graft, fascia graft, full
thickness graft,
heterologous graft, xenograft, homologous graft, hyperplastic graft, lamellar
graft, mesh graft,
mucosal graft, Oilier-Thiersch graft, omenpal graft, patch graft, pedicle
graft, penetrating graft,
3o split skin graft, thick split graft. Polynucleotides or polypeptides, as
well as agonists or
antagonists of the present invention, can be used to promote skin strength and
to improve the
appearance of aged skin.
Polynucleotides or polypeptides, as well as agonists or antagonists of the
present
invention, could also be used to reduce the side effects of gut toxicity that
result from radiation,
chemotherapy treatments or viral infections. Polynucleotides or polypeptides,
as well as
agonists or antagonists of the present invention, may have a cytoprotective
effect on the small
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
219
intestine mucosa. Polynucleotides or polypeptides, as well as agonists or
antagonists of the
present invention, may also stimulate healing of mucositis (mouth ulcers) that
result from
chemotherapy and viral infections.
Polynucleotides or polypeptides, as well as agonists or antagonists of the
present
invention, could further be used in full regeneration of skin in full and
partial thickness skin
defects, including burns, (i.e., repopulation of hair follicles, sweat glands,
and sebaceous
glands), treatment of other skin defects such as psoriasis. Polynucleotides or
polypeptides, as
well as agonists or antagonists of the present invention, could be used to
treat epidermolysis
bullosa, a defect in adherence of the epidermis to the underlying dermis which
results in
to frequent, open and painful blisters by accelerating reepithelialization of
these lesions.
Polynucleotides or polypeptides, as well as agonists or antagonists of the
present invention,
could also be used to treat gastric and doudenal ulcers and help heal by scar
formation of the
mucosal lining and regeneration of glandular mucosa and duodenal mucosal
lining more
rapidly. Inflamamatory bowel diseases, such as Crohn's disease and ulcerative
colitis, are
diseases which result in destruction of the mucosal surface of the small or
large intestine,
respectively. Thus, polynucleotides or polypeptides, as well as agonists or
antagonists of the
present invention, could be used to promote the resurfacing of the mucosal
surface to aid more
rapid healing and to prevent progression of inflammatory bowel disease.
Treatment with
polynucleotides or polypeptides, agonists or antagonists of the present
invention, is expected to
2o have a significant effect on the production of mucus throughout the
gastrointestinal tract and
could be used to protect the intestinal mucosa from injurious substances that
are ingested or
following surgery. Polynucleotides or polypeptides, as well as agonists or
antagonists of the
present invention, could be used to treat diseases associate with the under
expression.
Moreover, polynucleotides or polypeptides, as well as agonists or antagonists
of the
present invention, could be used to prevent and heal damage to the lungs due
to various
pathological states. Polynucleotides or polypeptides, as well as agonists or
antagonists of the
present invention, which could stimulate proliferation and differentiation and
promote the repair
of alveoli and brochiolar epithelium to prevent or treat acute or chronic lung
damage. For
example, emphysema, which results in the progressive loss of aveoli, and
inhalation injuries,
i.e., resulting from smoke inhalation and burns, that cause necrosis of the
bronchiolar
epithelium and alveoli could be effectively treated using polynucleotides or
polypeptides,
agonists or antagonists of the present invention. Also, polynucleotides or
polypeptides, as well
as agonists or antagonists of the present invention, could be used to
stimulate the proliferation
of and differentiation of type II pneumocytes, which may help treat or prevent
disease such as
hyaline membrane diseases, such as infant respiratory distress syndrome and
bronchopulmonary displasia, in premature infants.
Polynucleotides or polypeptides, as well as agonists or antagonists of the
present
invention, could stimulate the proliferation and differentiation of
hepatocytes and, thus, could
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
220
be used to alleviate or treat liver diseases and pathologies such as fulminant
liver failure caused
by cirrhosis, liver damage caused by viral hepatitis and toxic substances
(i.e., acetaminophen,
carbon tetraholoride and other hepatotoxins known in the art).
In addition, polynucleotides or polypeptides, as well as agonists or
antagonists of the
present invention, could be used treat or prevent the onset of diabetes
mellitus. In patients with
newly diagnosed Types I and II diabetes, where some islet cell function
remains,
polynucleotides or polypeptides, as well as agonists or antagonists of the
present invention,
could be used to maintain the islet function so as to alleviate, delay or
prevent permanent
manifestation of the disease. Also, polynucleotides or polypeptides, as well
as agonists or
antagonists of the present invention, could be used as an auxiliary in islet
cell transplantation to
improve or promote islet cell function.
Additional Therapeutic Embodiments
TR13 nucleic acids, polypeptides (proteins), agonists and/or antagonists of
the
invention may be administered to a patient (e.g., mammal, preferably human)
afflicted with any
disease or disorder mediated (directly or indirectly) by defective, or
deficient levels of, TR13.
Alternatively, a gene therapy approach may be applied to treat such diseases
or disorders. In
one embodiment of the invention, TR13 polynucleotide sequences are used to
detect mutein
2o TR13 genes, including defective genes. Mutein genes may be identified in in
vitro diagnostic
assays using techniques known in the art, and by comparison of the TR13
nucleotide sequence
disclosed herein with that of a TR13 gene obtained from a patient suspected of
harboring a
defect in this gene. Defective genes may be replaced with normal TRI3-encoding
genes using
techniques known to one skilled in the art.
In another embodiment, the TR13 polypeptides, nucleic acids, agonists and/or
antagonists of the present invention are used as research tools for studying
the phenotypic
effects that result from inhibiting TR13/TR13 ligand interactions on various
cell types. TR13
polypeptides and antagonists (e.g. monoclonal antibodies to TR13) also may be
used in in vitro
assays for detecting TR13 or TR13 ligand(s) or the interactions thereof.
3o TR14 nucleic acids, polypeptides (proteins), agonists and/or antagonists of
the
invention may be administered to a patient (e.g., mammal, preferably human)
afflicted with any
disease or disorder mediated (directly or indirectly) by defective, or
deficient levels of, TR14.
Alternatively, a gene therapy approach may be applied to treat such diseases
or disorders. In
one embodiment of the invention, TR14 polynucleotide sequences are used to
detect mutein
TR14 genes, including defective genes. Mutein genes may be identified in in
vitro diagnostic
assays using techniques known in the art, and by comparison of the TR14
nucleotide sequence
disclosed herein with that of a TR14 gene obtained from a patient suspected of
harboring a
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
221
defect in this gene. Defective genes may be replaced with normal TR14-encoding
genes using
techniques known to one skilled in the art.
In another embodiment, the TR14 polypeptides, nucleic acids, agonists and/or
antagonists of the present invention are used as research tools for studying
the phenotypic
effects that result from inhibiting TR14/TR14 ligand interactions on various
cell types. TR14
polypeptides and antagonists (e.g. monoclonal antibodies to TR14) also may be
used in in vitro
assays for detecting TR14 or TR14 ligand(s) or the interactions thereof.
Cells which express the TR13 polypeptide and are believed to have a potent
cellular
response to TR13 ligands include pancrease tumor, endometrial tumor, adult
small intestine,
colon cancer, breast cancer cell line, resting T-cell, amygdala, rectum, T-
cell helper, pineal
gland, apoptotic T-cell, epididymus, greater omentum, prostate BPH,
osteoclastoma,
endometrial stromal cells, stromal cell, substantia nigra, activated T-cell,
tonsil, and testes
tissue. By "a cellular response to a TNF-family ligand" is intended any
genotypic, phenotypic,
and/or morphologic change to a cell, cell line, tissue, tissue culture or
patient that is induced by
IS a TNF-family ligand (such as, for example, a TNF-ligand disclosed herein).
As indicated,
such cellular responses include not only normal physiological responses to TNF-
family
ligands, but also diseases associated with increased apoptosis or the
inhibition of apoptosis.
Apoptosis-programmed cell death is a physiological mechanism involved in the
deletion of
peripheral T lymphocytes of the immune system, and its dysregulation can lead
to a number of
2o different pathogenic processes (J.C. Ameisen, AIDS 8:1197-1213 (1994); P.H.
Krammer et
al., Curr. Opin. Immunol. 6:279-289 ( 1994)).
Cells which express TR14 polypeptide and that are believed to have a potent
cellular
response to TR14 ligands include activated T-cell, endometrial, thymus, and 12
week early
stage human tissue. By "a cellular response to a TNF-family ligand" is
intended any genotypic,
25 phenotypic, and/or morphologic change to a cell, cell line, tissue, tissue
culture or patient that is
induced by a TNF-family ligand (such as, for example, a TNF-ligand described
herein). As
indicated, such cellular responses include not only normal physiological
responses to TNF-
family ligands, but also diseases associated with dysregulation of these
physiological
responses, such as, for example, diseases associated with increased apoptosis
or the inhibition
30 of apoptosis. Apoptosis-programmed cell death is a physiological mechanism
involved in the
deletion of peripheral T lymphocytes of the immune system, and its
dysregulation can lead to a
number of different pathogenic processes (J.C. Ameisen, AIDS 8:1197-1213
(1994); P.H.
Krammer et al., Curr. Opin. Immunol. 6:279-289 (1994)).
Diseases associated with increased cell survival, or the inhibition of
apoptosis, and that
35 may be treated or prevented by the TR13 polynucleotides, polypeptides
and/or agonists or
antagonists of the invention include, but are not limited to, 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,
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
222
glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach
cancer, neuroblastoma,
myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma,
osteosarcoma,
chondrosarcoma, adenoma, breast cancer, prostrate cancer, Kaposi's sarcoma and
ovarian
cancer); autoimmune disorders (such as, multiple sclerosis, Sjogren's
syndrome, Hashimoto's
thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease,
polymyositis, systemic lupus
erythematosus and immune-related glomerulonephritis rheumatoid arthritis);
viral infections
(such as herpes viruses, pox viruses and adenoviruses); inflammation; graft
vs. host disease;
acute graft rejection and chronic graft rejection. In preferred embodiments,
TR13 nucleic acids,
polypeptides, agonists and/or antagonists of the invention are used to inhibit
growth,
progression, and/or metasis of cancers, in particular those listed above, or
in the paragraphs
that follow.
Additional diseases or conditions associated with increased cell survival and
that may be
treated or prevented by the TR13 polynucleotides, polypeptides, agonists
and/or antagonists of
the invention include, but are not limited to, progression, and/or metastases
of malignancies
and related disorders such as leukemia (including acute leukemias (e.g., acute
lymphocytic
leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic,
myelomonocytic,
monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic
myelocytic
(granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera,
lymphomas
(e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma,
Waldenstrom's
2o macroglobulinemia, heavy chain disease, and solid tumors including, but not
limited to,
sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma,
chondrosarcoma,
osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,
lymphangiosarcoma,
lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,
leiomyosarcoma,
rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian
cancer, prostate
cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat
gland
carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary
adenocarcinomas,
cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell
carcinoma,
hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma,
Wilm's
tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung
carcinoma, bladder
carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma,
craniopharyngioma,
ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma,
menangioma, melanoma, neuroblastoma, and retinoblastoma. In preferred
embodiments, TR13
nucleic acids, polypeptides, agonists and/or antagonists are used to treat the
diseases and
disorders listed above.
In additional embodiments, TR13 nucleic acids, polypeptides, agonists and/or
antagonists are used to treat pancreas tumor, endometrial tumor, colon cancer,
breast cancer,
prostate BPH and/or osteosarcoma.
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
223
Thus, in preferred embodiments TR13 polynucleotides or polypeptides of the
invention
and agonists or antagonists thereof, are used to treat or prevent autoimmune
diseases and/or
inhibit the growth, progression, and/or metastasis of cancers, including, but
not limited to,
those cancers disclosed herein, such as, for example, pancreatic cancer,
endometrial cancer,
colon cancer, breast cancer, osteocarcoma, and lymphocytic leukemias
(including, for example,
MLL and chronic lymphocytic leukemia (CLL)) and follicular lymphomas. In
another
embodiment TR13 polynucleotides or polypeptides of the invention and/or
agonists or
antagonists thereof, are used to activate, differentiate or proliferate
cancerous cells or tissue
(e.g., T cell lineage related cancers and B cell lineage related cancers
(e.g., CLL and MLL),
to lymphocytic leukemia, or lymphoma) and thereby render the cells more
vulnerable to cancer
therapy (e.g., chemotherapy or radiation therapy).
Diseases associated with increased apoptosis and that may be treated or
prevented by
the polynucleotides, polypeptides and/or agonists or antagonists of the
invention include, but
are not limited to, AIDS; neurodegenerative disorders (such as Alzheimer's
disease,
Parkinson's disease, Amyotrophic lateral sclerosis, Retinitis pigmentosa,
Cerebellar
degeneration and brain tumor or prior associated disease); autoimmune
disorders (such as,
multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary
cirrhosis, Behcet's
disease, Crohn's disease, polymyositis, systemic lupus erythematosus and
immune-related
glomerulonephritis and rheumatoid arthritis); myelodysplastic syndromes (such
as aplastic
2o anemia), graft v. host disease, ischemic injury (such as that caused by
myocardial infarction,
stroke and reperfusion injury), liver injury (such as hepatitis related liver
injury,
ischemialreperfusion injury, cholestosis (bile duct injury) and liver cancer);
toxin-induced liver
disease (such as that caused by alcohol), septic shock, cachexia and anorexia.
In preferred
embodiments, TR13 nucleic acids, polypeptides, agonists and/or antagonists are
used to treat
the diseases and disorders listed above.
Diseases associated with increased cell survival, or the inhibition of
apoptosis, and that
may be treated or prevented by the TR14 polynucleotides, polypeptides and/or
agonists or
antagonists of the invention include, but are not limited to, cancers (such as
follicular
lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors,
including, but
3o not limited to colon cancer, cardiac tumors, pancreatic cancer, melanoma,
retinoblastoma,
glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach
cancer, neuroblastoma,
myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma,
osteosarcoma,
chondrosarcoma, adenoma, breast cancer, prostrate cancer, Kaposi's sarcoma and
ovarian
cancer); autoimmune disorders (such as, multiple sclerosis, Sjogren's
syndrome, Hashimoto's
thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease,
polymyositis, systemic lupus
erythematosus and immune-related glomerulonephritis rheumatoid arthritis);
viral infections
(such as herpes viruses, pox viruses and adenoviruses); inflammation; graft
vs. host disease;
acute graft rejection and chronic graft rejection. In preferred embodiments,
TR14 nucleic acids,
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
224
polypeptides, agonists and/or antagonists of the invention are used to inhibit
growth,
progression, and/or metasis of cancers, in particular those listed above, or
in the paragraphs
that follow.
Additional diseases or conditions associated with increased cell survival and
that may be
treated or prevented by the TR14 polynucleotides, polypeptides and/or agonists
or antagonists
of the invention include, but are not limited to, progression, and/or
metastases of malignancies
and related disorders such as leukemia (including acute leukemias (e.g., acute
lymphocytic
leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic,
myelomonocytic,
monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic
myelocytic
(granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera,
lymphomas
(e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma,
Waldenstrom's
macroglobulinemia, heavy chain disease, and solid tumors including, but not
limited to,
sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma,
chondrosarcoma,
osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,
lymphangiosarcoma,
lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,
leiomyosarcoma,
rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian
cancer, prostate
cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat
gland
carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary
adenocarcinomas,
cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell
carcinoma,
hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma,
Wilm's
tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung
carcinoma, bladder
carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma,
craniopharyngioma,
ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma,
menangioma, melanoma, neuroblastoma, and retinoblastoma. In preferred
embodiments, TR14
nucleic acids, polypeptides, agonists and/or antagonists are used to treat the
diseases and
disorders listed above.
Thus, in preferred embodiments TR14 polynucleotides or polypeptides of the
invention
and agonists or antagonists thereof, are used to treat or prevent autoimmune
diseases and/or
inhibit the growth, progression, and/or metastasis of cancers, including, but
not limited to,
those cancers disclosed herein, such as, for example, lymphocytic leukemias
(including, for
example, MLL and chronic lymphocytic leukemia (CLL)) and follicular lymphomas.
In
another embodiment TRI4 polynucleotides or polypeptides of the invention
and/or agonists or
antagonists thereof, are used to activate, differentiate or proliferate
cancerous cells or tissue
(e.g., T cell lineage cancers and B cell lineage related cancers (e.g., CLL
and MLL),
lymphocytic leukemia, or lymphoma) and thereby render the cells more
vulnerable to cancer
therapy (e.g., chemotherapy or radiation therapy).
Diseases associated with increased apoptosis and that may be treated or
prevented by
the TR14 polynucleotides, polypeptides and/or agonists or antagonists of the
invention include,
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
225
but are not limited to, AIDS; neurodegenerative disorders (such as Alzheimer's
disease,
Parkinson's disease, Amvotrophic lateral sclerosis, Retinitis pi~mentosa,
Cerebellar
degeneration and brain tumor or prior associated disease); autoimmune
disorders (such as,
multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary
cirrhosis, Behcet's
disease, Crohn's disease, polymyositis, systemic lupus erythematosus and
immune-related
glomerulonephritis and rheumatoid arthritis); myelodysplastic syndromes (such
as aplastic
anemia), graft v. host disease, ischemic injury (such as that caused by
myocardial infarction,
stroke and reperfusion injury), liver injury (such as 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, TR14 nucleic acids, polypeptides, agonists and/or antagonists are
used to treat
the diseases and disorders listed above.
Many of the pathologies associated with HIV are mediated by apoptosis,
including
HIV-induced nephropathy and HIV encephalitis. Thus, in additional preferred
embodiments,
~5 TR13 nucleic acids, polypeptides, and/or TR13 agonists or antagonists of
the invention are
used to treat AIDS and pathologies associated with AIDS.
Many of the pathologies associated with HIV are mediated by apoptosis,
including
HIV-induced nephropathy and HIV encephalitis. Thus, in additional preferred
embodiments,
TR14 nucleic acids, polypeptides, and/or TR14 agonists or antagonists of the
invention are
2o used to treat AIDS and pathologies associated with AIDS.
Another embodiment of the present invention is directed to the use of TR13 to
reduce
cell death dependent upon a TNF family member, of T cells in HN-infected
patients. The state
of immunodeficiency that defines AIDS is secondary to a decrease in the number
and function
of CD4+ T-lymphocytes. Recent reports estimate the daily loss of CD4+ T cells
to be between
25 3.5 x 10' and 2 x 109 cells (Wei et al., Nature 373:117-122 (1995)). One
cause of CD4+ T cell
depletion in the setting of HIV infection is believed to be HIV-induced
apoptosis (see, for
example, Meyaard et al., Science 257:217-219, 1992; Groux et al., J Exp. Med.,
175:331,
1992; and Oyaizu et al., in Cell Activation and Apoptosis in HIV Infection,
Andrieu and Lu,
Eds., Plenum Press, New York, 1995, pp. 101-114). Indeed, HIV-induced
apoptotic cell
3o death has been demonstrated not only in vitro but also, more importantly,
in infected
individuals (J.C. Ameisen, AIDS 8:1197-1213 (1994); T.H. Finkel and N.K.
Banda, Curr.
Opin. Immunol. 6:605-615(1995); C.A. Muro-Cacho et al., J. Immunol. 154:5555-
5566
(1995)). Furthermore, apoptosis and CD4+ T-lymphocyte depletion is tightly
correlated in
different animal models of AIDS (T. Brunner et al., Nature 373:441-444 (1995);
M.L.
35 Gougeon et al., AIDS Res. Hum. Retroviruses 9:553-563 (1993)) and,
apoptosis is not
observed in those animal models in which viral replication does not result in
AIDS. Id.
Further data indicates that uninfected but primed or activated T lymphocytes
from HIV-infected
individuals undergo apoptosis after encountering the TNF-family ligand Fast.
Using
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
226
monocytic cell lines that result in death following HIV infection, it has been
demonstrated that
infection of U937 cells with HIV results in the de novo expression of Fast and
that Fast
mediates HIV-induced apoptosis (A.D. Badley et al., J. Virol. 70:199-206
(1996)). Further,
the TNF-family ligand was detectable in uninfected macrophages and its
expression was
upregulated following HIV infection resulting in selective killing of
uninfected CD4 T-
lymphocytes. Id. Thus, by the invention, a method for treating HIV+
individuals is provided
which involves administering TR13 polynucleotides, polypeptides and/or TR13
agonists or
antagonists of the present invention to reduce selective killing of CD4+ T-
lymphocytes. Modes
of administration and dosages are discussed in detail below.
Activated human T cells are induced to undergo programmed cell death
(apoptosis)
upon triggering through the CD3/T cell receptor complex, a process termed
activated-induced
cell death (AICD). AICD of CD4+ T cells isolated from HIV-Infected
asymptomatic individuals
has been reported (Groux et al., supra). Thus, A1CD may play a role in the
depletion of CD4+
T cells and the progression to AIDS in HIV-infected individuals. Thus, the
present invention
provides a method of inhibiting a tumor-necrosis factor family member-mediated
T cell death in
HIV patients, comprising administering a TR13 polypeptide of the invention
(preferably, a
soluble TR13 polypeptide) to the patients. In one embodiment, the patient is
asymptomatic
when treatment with TR13 commences. If desired, prior to treatment, peripheral
blood T cells
may be extracted from an HIV patient, and tested for susceptibility to cell
death mediated by a
tumor necrosis factor family member, by procedures known in the art. In one
embodiment, a
patient's blood or plasma is contacted with TR13 ex vivo. The TR13 may be
bound to a
suitable chromatography matrix by procedures known in the art. The patient's
blood or plasma
flows through a chromatography column containing TR13 bound to the matrix,
before being
returned to the patient. In the event the immobilized TR13 bound to TRAIL, or
another TNF
family member(s), TRAIL and/or other TNF family member protein would be
removed from
the patient's blood.
In additional embodiments a TR13 polypeptide, polynucleotide, and/or agonist
or
antagonist of the invention is administered in combination with inhibitors of
T cell apoptosis.
For example, TRAIL-mediated apoptosis and Fas-mediated apoptosis have been
implicated in
loss of T cells in HIV individuals (See, e.g., Katsikis et al., J. Exp. Med.
181:2029-2036
(1995)). Thus, a patient susceptible to both Fas ligand mediated and/or TRAIL
mediated T cell
death may be treated as an agent that blocks TRAILJTRAIL receptor interactions
and/or 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 transduce the
biological signal that results in apoptosis. Preferably, the antibodies
employed according to
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
227
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, hereby incorporated
by reference.
Suitable agents, which block binding of TRAIL to a TRAIL receptor or FAS
ligand to
FAS that may be administered with the nucleic acids, polypeptides, and/or
agonists or
antagonists of the present invention include, but are not limited to, soluble
TRAIL receptor
polypeptides (e.g., a soluble form of OPG, DR4 (International application
publication number
WO 98/32856); TR5 (International application publication number WO 98/30693);
and DRS
(International application publication number WO 98/41629)); multimeric forms
of soluble
to TRAIL receptor polypeptides; and TRAIL receptor antibodies that bind the
TRAIL receptor
without transducing the biological signal that results in apoptosis, anti-
TRAIL antibodies that
block binding of TRAIL to one or more TRAIL receptors, and muteins of TRAIL
that bind
TRAIL receptors but do not transduce the biological signal that results in
apoptosis. Preferably,
the antibodies employed according to this method are monoclonal antibodies.
Another embodiment of the present invention is directed to the use of TR14 to
reduce
cell death dependent upon a TNF family member, of T cells in HIV-infected
patients. The state
of immunodeficiency that defines AIDS is secondary to a decrease in the number
and function
of CD4+ T-lymphocytes. Recent reports estimate the daily loss of CD4+ T cells
to be between
3.5 x 10' and 2 x 109 cells (Wei et al., Nature 373:117-122 (1995)). One cause
of CD4+ T cell
2o depletion in the setting of HIV infection is believed to be HIV-induced
apoptosis (see, for
example, Meyaard et al., Science 257:217-219, 1992; Groux et al., J Exp. Med.,
175:331,
1992; and Oyaizu et al., in Cell Activation and Apoptosis in HIV Infection,
Andrieu and Lu,
Eds., Plenum Press, New York, 1995, pp. 101-114). Indeed, HIV-induced
apoptotic cell
death has been demonstrated not only in vitro but also, more importantly, in
infected
individuals (J.C. Ameisen, AIDS 8:1197-1213 (1994); T.H. Finkel and N.K.
Banda, Curr.
Opin. Immunol. 6:605-615(1995); C.A. Muro-Cacho et al., J. Immunol. 154:5555-
5566
(1995)). Furthermore, apoptosis and CD4+ T-lymphocyte depletion is tightly
correlated in
different animal models of AIDS (T. Brunner et al., Nature 373:441-444 (1995);
M.L.
Gougeon et al., AIDS Res. Hum. Retroviruses 9:553-563 (1993)) and, apoptosis
is not
observed in those animal models in which viral replication does not result in
AIDS. Id.
Further data indicates that uninfected but primed or activated T lymphocytes
from HIV-infected
individuals undergo apoptosis after encountering the TNF-family ligand Fast.
Using
monocytic cell lines that result in death following HIV infection, it has been
demonstrated that
infection of U937 cells with HIV results in the de novo expression of Fast and
that Fast
mediates HIV-induced apoptosis (A.D. Badley et al., J. Virol. 70:199-206
(1996)). Further,
the TNF-family ligand was detectable in uninfected macrophages and its
expression was
upregulated following HIV infection resulting in selective killing of
uninfected CD4 T-
lymphocytes. Id. Thus, by the invention, a method for treating HIV+
individuals is provided
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
228
which involves administering TR14 polynucleotides, polypeptides, and/or TR14
agonists or
antagonists of the present invention to reduce selective killing of CD4+ T-
lymphocytes. Modes
of administration and dosages are discussed in detail below.
Activated human T cells are induced to undergo programmed cell death
(apoptosis)
upon triggering through the CD3/T cell receptor complex, a process termed
activated-induced
cell death (AICD). AICD of CD4+ T cells isolated from HIV-Infected
asymptomatic individuals
has been reported (Groux et al., supra). Thus, AICD may play a role in the
depletion of CD4+
T cells and the progression to AIDS in HIV-infected individuals. Thus, the
present invention
provides a method of inhibiting a tumor-necrosis factor family member-mediated
T cell death in
1o HIV patients, comprising administering a TRI4 polypeptide of the invention
(preferably, a
soluble TR14 polypeptide) to the patients. In one embodiment, the patient is
asymptomatic
when treatment with TR14 commences. If desired, prior to treatment, peripheral
blood T cells
may be extracted from an HIV patient, and tested for susceptibility to cell
death mediated by a
member of the TNF-family, by procedures known in the art. In one embodiment, a
patient's
blood or plasma is contacted with TR14 ex vivo. The TR14 may be bound to a
suitable
chromatography matrix by procedures known in the art. The patient's blood or
plasma flows
through a chromatography column containing TR14 bound to the matrix, before
being returned
to the patient. In the event the immobilized TR14 bound to TRAIL, or another
TNF family
member(s), TRAIL and/or other TNF family member protein would be removed from
the
patient's blood.
In additional embodiments a TR14 polypeptide, polynucleotide, and/or agonist
or
antagonist of the invention is administered in combination with inhibitors of
T cell apoptosis.
For example, TRAIL-mediated apoptosis and Fas-mediated apoptosis have been
implicated in
loss of T cells in HIV individuals (See e.g., 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 as an agent that blocks TRAILITRAIL receptor interactions
and/or 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-(igand that bind Fas but do
not transduce the
biological signal that results in apoptosis. Preferably, the antibodies
employed according to
this method are monoclonal antibodies. Examples of suitable agents for
blocking Fas-
ligand/Fas interactions, including blocking anti-Fas monoclonal antibodies,
are described in
International application publication number WO 95/10540, hereby incorporated
by reference.
Suitable agents, which block binding of TRAIL to a TRAIL receptor or FAS
ligand to
FAS that may be administered with the nucleic acids, polypeptides, and/or
agonists or
antagonists of the present invention include, but are not limited to, soluble
TRAIL receptor
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
229
polypeptides (e.g., a soluble form of OPG, DR4 (International application
publication number
WO 98/32856); TR5 (International application publication number WO 98/30693);
and DRS
(International application publication number WO 98/41629)); multimeric forms
of soluble
TRAIL receptor polypeptides; and TRAIL receptor antibodies that bind the TRAIL
receptor
without transducing the biological signal that results in apoptosis, anti-
TRAIL antibodies that
block binding of TRAIL to one or more TRAIL receptors, and muteins of TRAIL
that bind
TRAIL receptors but do not transduce the biological signal that results in
apoptosis. Preferably,
the antibodies employed according to this method are monoclonal antibodies.
TR13 polypeptides, nucleic acids, and/or agonists or antagonists of the
invention may
1o be used to treat cardiovascular disorders, including peripheral artery
disease, such as limb
ischemia.
TR14 polypeptides, nucleic acids, and/or agonists or antagonists of the
invention may
be used to treat cardiovascular disorders, including peripheral artery
disease, such as limb
ischemia.
Cardiovascular disorders include cardiovascular abnormalities, such as arterio-
arterial
fistula, arteriovenous fistula, cerebral arteriovenous malformations,
congenital heart defects,
pulmonary atresia, and Scimitar Syndrome. Congenital heart defects include
aortic coarctation,
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, and conditions characterized by clotting of
small blood vessels.
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.
Arrhythmias include sinus arrhythmia, atrial fibrillation, atrial flutter,
bradycardia,
extrasystole, Adams-Stokes Syndrome, bundle-branch block, sinoatrial block,
long QT
syndrome, parasystole, Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation
syndrome, Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias,
and
ventricular fibrillation. Tachycardias include paroxysmal tachycardia,
supraventricular
CA 02381327 2002-O1-07
WO 01/05834 PCT/US00/19343
230
tachycardia, accelerated idioventricular rhythm, atrioventricular nodal
reentry tachycardia,
ectopic atrial tachycardia, ectopic functional tachycardia, sinoatrial nodal
reentry tachycardia,
sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.
Heart valve disease include aortic valve insufficiency, aortic valve stenosis,
hear
murmurs, aortic valve prolapse, mitral valve prolapse, tricuspid valve
prolapse, mural valve
insufficiency, mitral valve stenosis, pulmonary atresia, pulmonary valve
insufficiency,
pulmonary valve stenosis, tricuspid atresia, tricuspid valve insufficiency,
and tricuspid valve
stenosis.
Myocardial diseases include alcoholic cardiomyopathy, congestive
cardiomyopathy,
to hypertrophic cardiomyopathy, aortic subvalvular stenosis, pulmonary
subvalvular stenosis,
restrictive cardiomyopathy, Chagas cardiomyopathy, endocardial fibroelastosis,
endomyocardial fibrosis, Kearns Syndrome, myocardial reperfusion injury, and
myocarditis.
Myocardial ischemias include coronary disease, such as angina pectoris,
coronary
aneurysm, coronary arteriosclerosis, coronary thrombosis, coronary vasospasm,
myocardial
infarction and myocardial stunning.
Cardiovascular diseases also include vascular diseases such as aneurysms,
angiodysplasia, angiomatosis, bacillary angiomatosis, Hippel-Lindau Disease,
Klippel-
Trenaunay-Weber Syndrome, Sturge-Weber Syndrome, angioneurotic edema, aortic
diseases,
Takayasu's Arteritis, aortitis, Leriche's Syndrome, arterial occlusive
diseases, arteritis,
enarteritis, polyarteritis nodosa, cerebrovascular disorders, diabetic
angiopathies, diabetic
retinopathy, embolisms, thrombosis, erythromelalgia, hemorrhoids, hepatic veno-
occlusive
disease, hypertension, hypotension, ischemia, peripheral vascular diseases,
phlebitis,
pulmonary veno-occlusive disease, Raynaud's disease, CREST syndrome, retinal
vein
occlusion, Scimitar syndrome, superior vena cava syndrome, telangiectasia,
atacia
telangiectasia, hereditary hemorrhagic telangiectasia, varicocele, varicose
veins, varicose ulcer,
vasculitis, thrombotic microangiopathies (e.g., thrombotic thrombocytopenic
purpura (TTP)
and hemolytic-uremic syndrome (HUS)), and venous insufficiency.
Aneurysms include dissecting aneurysms, false aneurysms, infected aneurysms,
ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary aneurysms,
heart
3o aneurysms, and iliac aneurysms.
Arterial occlusive diseases include arteriosclerosis, intermittent
claudication, carotid
stenosis, fibromuscular dysplasias, mesenteric vascular occlusion, Moyamoya
disease, renal
artery obstruction, retinal artery occlusion, and thromboangiitis obliterans.
Cerebrovascular disorders include carotid artery diseases, cerebral amyloid
angiopathy,
~ cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral
arteriovenous
malformation, cerebral artery diseases, cerebral embolism and thrombosis,
carotid artery
thrombosis, sinus thrombosis, Wallenberg's syndrome, cerebral hemorrhage,
epidural
hematoma, subdural hematoma, subaraxhnoid hemorrhage, cerebral infarction,
cerebral
DEMANDES OU BREVETS VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVETS
COMPRI~:ND PLUS D'UN TOME.
CECI EST L,E TOME 1 DE 2
NOTE: Pour les tomes additionels, veillez contacter 1e Bureau Canadien des
Brevets.
JUMBO APPLICATIONS / PATENTS
THIS SECTION OF THE APPLICATION / PATENT CONTAINS MORE
THAN ONE VOLUME.
THIS IS VOLUME 1 OF 2
NOTE: For additional valumes please contact the Canadian Patent Office.
