Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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ERYTHROPOIETIN RECEPTOR BINDING ANTIBODIES
Technical Field of the Invention
The present invention relates to antibodies that recognize, bind to and,
preferably,
activate the erythropoietin receptor.
Background of the Invention
Erythropoietin ("EPO") is a glycoprotein that is the primary regulator of
erythropoiesis.
Specifically, EPO is responsible for promoting the growth, differentiation and
survival of
erythroid progenitors, which give rise to mature red blood cells. In response
to changes in the
level of oxygen in the blood and tissues, erythropoietin appears to stimulate
both proliferation
and differentiation of immature erythroblasts. It also functions as a growth
factor, stimulating
the mitotic activity of erythroid progenitor. cells, such as erythrocyte burst
forming and colony-
forming units. It also acts as a differentiation factor, triggering
transformation of an
erythrocyte colony-forming-unit into a proerythroblast (See Erslev, A., New
Eng. J. Med.,
316:101-103 (1987)).
EPO has a molecular weight of about 34,000 daltons and can occur in three
forms -
alpha, beta and asialo. During mid- to late gestation, EPO is synthesized in
the fetal liver.
Subsequently, EPO is synthesized in the kidney, circulates in the plasma and
is excreted in the
urine.
Human urinary EPO has been isolated and purified (See, Miyake et al., J. Biol.
Chem.,
252:5558 (1977)). Moreover, methods for identifying, cloning and expressing
genes encoding
EPO (See U.S. Patent 4,703,008) as well as purifying recombinant EPO from a
cell medium
(See U.S. Patent 4,667,016) are known in the art.
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The activity of EPO is mediated through the binding and activation of a cell
surface
receptor referred to as the erythropoietin receptor. The EPO receptor belongs
to the cytokine
receptor superfamily and is believed to contain at least two distinct
polypeptides, a 55-72 kDa
species and a 85-100 kDa species (See U.S. Patent 6,319,499, Mayeux et al., J.
Biol. Chem,
266:23380 (1991), McCaffery et al., J Biol. Chem., 264:10507 (1991)). Other
studies have
revealed other polypeptide complexes of EPO receptor having molecular weights
such as 110,
130 and 145 kDa (See U.S. Patent 6,319,499).
Both the murine and human EPO receptors have been cloned and expressed (See
D'Andrea et al., Cell, 57:277 (1989); Jones et al., Blood, 76:31 (1990);
Winkelmann et al.,
Blood, 76:24 (1990); WO 90/08822/ U.S. Patent 5,278,065). The full length
human EPO
receptor is a 483 amino acid transmembrane protein with an approximately 25
amino acid
signal peptide (See U.S. Patent 6,319,499). The human receptor demonstrates
about a 82%
amino acid sequence homology with the murine receptor. Id.
In the absence of ligand the EPO receptor exists in a preformed dimer. The
binding of
EPO to its receptor causes a conformational change such that the cytoplasmic
domains are
placed in close proximity. While not completely understood, it is believed
that this
"dimerization" plays a role in the activation of the receptor. The activation
of the EPO receptor
results in a number of biological effects. Some of these activities include
stimulation of
proliferation, stimulation of differentiation and inhibition of apoptosis (See
U.S. Patent
6,319,499, Liboi et al., PNAS USA, 90:11351 (1993), Koury, Science, 248:378
(1990)).
It is the relationship between the EPO receptor dimerization and activation
that can be
used to identify compounds (i.e. such as antibodies) other than EPO that are
capable of: (1)
dimerizing the EPO receptor; and (2) activating the receptor. These compounds
would be
useful in treating mammals suffering from anemia and in identifying mammals
having a
dysfunctional EPO receptor.
Summary of the Invention
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In one embodiment, the invention relates to antibodies that bind to the human
erythropoietin receptor. In one embodiment, the antibodies comprise a heavy
chain variable
region that is selected from the group consisting of SEQ ID NOS: 3, 7, 11, 15,
19, 31, 35, 39,
43, 47, 51, 55 and fragments thereof. In another embodiment, the antibodies
comprise a light
chain variable region that is selected from the group consisting of SEQ ID
NOS: 5, 9, 13, 17,
21, 23, 25, 27, 29, 33, 37, 41, 45, 49, 53, 57 and fragments thereof.
In another embodiment, the present invention relates to an isolated antibody
that is
capable of binding a human erythropoietin receptor in a mammal. Such an
antibody comprises
a heavy chain variable region or a light chain variable region that comprises
a continuous
sequence from CDRl through CDR3. The amino acid sequence of the heavy chain
variable
region comprising the continuous sequence from CDRl through CDR3 is selected
from the
group consisting of: SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61
and
fragments thereof. The amino acid sequence of the light chain variable region
comprising the
continuous sequence from CDRl through CDR3 is selected from the group
consisting of. SEQ
ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID
NO:67,
SEQ ID NO:68 and fragments thereof.
In another embodiment, the present invention relates to an antibody that
activates an
endogenous activity of a human erythropoietin receptor in a mammal but does
not interact with
a peptide having an amino acid sequence of PGNYSFSYQLEDEPWKLCRLHQAPTARGAV
(SEQ ID NO:1).
In another embodiment, the present invention relates to an antibody that is
capable of
activating an endogenous activity of a human erythropoietin receptor in a
mammal, wherein
said antibody or antibody fragment thereof exhibits a binding affinity within
one hundred fold
of the binding affinity of endogenous human erythropoietin to the
erythropoietin receptor.
In yet another embodiment, the present invention relates to an antibody or
antibody
fragment thereof that activates an endogenous activity of a human
erythropoietin receptor in a
mammal. The antibody or antibody fragment thereof comprises at least one human
heavy
chain variable region having the amino acid sequence of SEQ ID NO:3 or
antibody fragment
thereof, and/or at least one human light chain variable region having the
amino acid sequence
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of SEQ ID NO:5 or antibody fragment thereof, provided that said antibody or
antibody
fragment thereof does not interact with a peptide having an amino acid
sequence of
PGNYSFSYQLEDEPWKLCRLHQAPTARGAV (SEQ ID NO:1).
In yet another embodiment, the present invention relates to an antibody or
antibody
fragment thereof that activates an endogenous activity of a human
erythropoietin receptor in a
mammal. The antibody or antibody fragment thereof comprises at least one heavy
chain
variable region having the amino acid sequence of SEQ ID NO:7 or antibody
fragment thereof,
and/or at least one light chain variable region having the amino acid sequence
of SEQ ID NO:9
or antibody fragment thereof, provided that said antibody or antibody fragment
thereof does
not interact with a peptide having an amino acid sequence of
PGNYSFSYQLEDEPWKLCRLHQAPTARGAV (SEQ ID NO:1).
This embodiment also includes other heavy chain variable regions selected from
the
group consisting of SEQ ID NO: 11, 15, 19, 31, 35, 39, 43, 47, 51, and 55 or
an antibody
fragment of any of these aforementioned SEQ ID NOS, wherein said antibody or
antibody
fragment thereof does not interact with a peptide having an amino acid
sequence of SEQ ID
NO: 1. Other light chain variable regions included in this embodiment may be
selected from
the group consisting of SEQ ID NO: 13, 17, 21, 23, 25, 27, 29, 33, 37, 41, 45,
49, 53 and 57 or
an antibody fragment of any of these aforementioned SEQ ID NOS, wherein said
antibody or
antibody fragment thereof does not interact with a peptide having an amino
acid sequence of
SEQ ID NO: 1.
In yet another embodiment, the invention provides an antibody or antibody
fragment
thereof that activates an endogenous activity of a human erythropoietin
receptor in a mammal,
the antibody comprising the amino acid sequences of at least one heavy chain
variable region
and at least one light chain variable region selected from the group
consisting of SEQ ID
NO:11/SEQ ID NO:13, SEQ ID NO:15/SEQ ID NO:17, SEQ ID NO:19/SEQ ID NO:21, SEQ
ID NO:11/SEQ ID NO:23,'SEQ ID NO: 11/SEQ ID NO:25, SEQ ID NO:11/SEQ ID NO:27,
SEQ ID NO:11/SEQ ID NO:29, SEQ ID NO:31/SEQ ID NO:33, SEQ ID NO:35/SEQ ID
NO:37, SEQ ID NO:39/SEQ ID NO:41, SEQ ID NO:43/SEQ ID NO:45, SEQ ID NO:47/SEQ
ID NO:49, SEQ ID NO:51/SEQ ID NO:53 and SEQ ID NO:55/SEQ ID NO:57 or antibody
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fragment thereof, wherein said antibody or antibody fragment thereof does not
interact with a
peptide having an amino acid sequence of SEQ ID NO: 1.
In yet another embodiment, the present invention relates to a method of
activating an
endogenous activity of a human erythropoietin receptor in a mammal. The method
involves
the step of administering to a mammal a therapeutically effective amount of an
antibody or
antibody fragment thereof to activate the EPO receptor. The antibody or
antibody fragment
thereof does not interact with a peptide having an amino acid sequence of
PGNYSFSYQLEDEPWKLCRLHQAPTARGAV (SEQ ID NO:1).
In yet a further embodiment, the present invention relates to a method of
modulating an
endogenous activity of a human erythropoietin receptor in a mammal. The method
involves
administering to a mammal a therapeutically effective amount of an antibody or
antibody
fragment thereof to modulate the endogenous activity of a human erythropoietin
receptor in a
mammal but does not interact with a peptide having an amino acid sequence of
PGNYSFSYQLEDEPWKLCRLHQAPTARGAV (SEQ ID NO:1).
In yet a further embodiment, the present invention relates to a method of
treating a mammal suffering from pure red cell aplasia induced by neutralizing
anti-
erythropoietin antibodies. The method involves administering to a mammal in
need of
treatment a therapeutically effective amount of an antibody or antibody
fragment thereof to
activate said receptor, wherein said antibody or antibody fragment thereof
does not interact
with a peptide having an amino acid sequence of
PGNYSFSYQLEDEPWKLCRLHQAPTARGAV (SEQ ID NO:1).
In yet a further embodiment, the present invention relates to pharmaceutical
compositions. The pharmaceutical compositions of the present invention contain
a
therapeutically effective amount of an antibody or antibody fragment thereof
and a
pharmaceutically acceptable excipient. The antibody or antibody fragment
contained in the
pharmaceutical composition activates an endogenous activity of a human
erythropoietin
receptor in a mammal but does not interact with a peptide having an amino acid
sequence of
PGNYSFSYQLEDEPWKLCRLHQAPTARGAV (SEQ ID NO:1).
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In yet a further embodiment, the present invention relates to an IgG2 antibody
or
antibody fragment that binds to and activates the erythropoietin receptor. The
IgG2 antibodies
or antibody fragments of this embodiment bind to and interact with any epitope
that is involved
in activating the EPO receptor. Such antibodies may be polyclonal or
monoclonal antibodies
or any antibody fragment thereof. The IgG2 antibodies may be chimeric,
humanized or human
antibodies.
In yet a further embodiment, the present invention provides a method of
activating an
endogenous activity of a human erythropoietin receptor in a mammal comprising
the step of
administering to a mammal a therapeutically effective amount of an IgG2
antibody or antibody
fragment of the invention to activate the receptor.
In yet a further embodiment, the present invention provides a method of
modulating an
endogenous activity of a human erythropoietin receptor in a mammal comprising
the step of
administering to a mammal a therapeutically effective amount of an IgG2
antibody or antibody
fragment of the invention to modulate the receptor.
In yet another embodiment, the present invention provides a method of treating
a
mammal suffering aplasia, the method comprising the step of administering to a
mammal in
need of treatment a therapeutically effective amount of an IgG2 antibody or
antibody fragment
of the invention to activate the receptor.
In yet another embodiment, the present invention provides a method of treating
a
mammal suffering aplasia, the method comprising the step of administering to a
mammal in
need of treatment a therapeutically effective amount of an IgG2 antibody or
antibody fragment
of the invention to modulate the receptor.
In yet another embodiment, the present invention provides a pharmaceutical
composition comprising a therapeutically effective amount of an IgG2 antibody
or antibody
fragment of the invention and a pharmaceutically acceptable excipient.
Finally, the present invention relates to isolated and purified polynucleotide
and amino
acid sequences. The isolated and purified polynucleotide sequences can be
selected from the
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group consisting of. SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ
ID
NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20,
SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID
NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42,
SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID
NO:54, SEQ ID NO:56 and fragments, complements and degenerate codon
equivalents
thereof.
The present invention further relates to isolated and purified amino acid
sequences
selected from the group consisting of. SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7,
SEQ ID
NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19,
SEQ
ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:31, SEQ ID
NO:33,
SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID
NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55,
SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID
NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67,
SEQ ID NO:68 and fragments and complements and thereof.
Brief Description of the Figures
Figure 1 shows the isolated and purified polynucleotide (top strand and bottom
strands,
SEQ ID NO:69 and SEQ ID NO:70, respectively) and amino acid sequence of the
heavy chain
of human antibody Ab12. The amino acid sequence comprises SEQ ID NOS:71
through 74.
The sequence of the constant region alone is shown as SEQ ID NO:75. The
variable chain
ends at nucleotide 1283. The variable/constant joining region (underlined) is
at nucleotides
1284-1289. The constant region is from nucleotides 1290-2826.
Figure 2 shows the isolated and purified polynucleotide (top strand and bottom
strands,
SEQ ID NO:76 and SEQ ID NO:77, respectively) and amino acid sequence of the
light chain
of human antibody Ab12. The amino acid sequence comprises SEQ ID NOS:78. The
sequence of the constant region alone is shown as SEQ ID NO: 79. The variable
chain ends at
nucleotidel363. The variable/constant joining region (underlined) is at
nucleotides 1364-1369.
The constant region is from nucleotides 1370-1618.
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Figure 3 shows the isolated and purified polynucleotide (top strand and bottom
strands,
SEQ ID NO:80 and SEQ ID NO:81, respectively) and amino acid sequence of the
heavy chain
of human antibody Ab198. The amino acid sequence comprises SEQ ID NOS:82 and
SEQ ID
NOS 72 through 74. The variable chain ends at nucleotide 1304. The
variable/constant
joining region (underlined) is at nucleotides 1305-1310. The constant region
is from
nucleotides 1311-2847.
Figure 4 shows the isolated and purified polynucleotide (top strand and bottom
strands,
SEQ ID NO:83 and SEQ ID NO:84, respectively) and amino acid sequence of the
light chain
of human antibody Ab198. The amino acid sequence comprises SEQ ID NOS:78. The
variable chain ends at nucleotide 1351. The variable/constant joining region
(underlined) is at
nucleotides 1352-1357. The constant region is from nucleotides 1358-1606.
Figure 5 shows the competition of Ab12 with 125I-labeled EPO for binding to
Chinese
Hamster Ovary cells expressing recombinant EPO receptor.
Figure 6 shows the results of an EPO dependent human cell proliferation assay
using
Abl2 and Ab198.
Figure 7 shows that Ab12 remains active in inducing the proliferation of F36E
cells
after storage at 4 C for up to 20 days.
Figure 8 shows that Ab 12 induces the formation of CFU-E (colony forming unit-
erythroid) from human 36+ progenitor cells.
Figure 9 shows the induction of proliferation of human erythroid producing
cells with
Ab198.
Figure 10 shows that Ab198 induces the formation of CFU-E colonies from
cynomologous bone marrow-derived erythroid progenitor cells.
Figure 11 shows that Ab 12 does not interact with the peptide SE-3. Ab71A
interacts
with the SE-3 peptide.
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Figure 12 shows that human Abs secreted by primary hybridomas induce the
proliferation of F36E cells.
Figure 13 shows that human Ab supernatants secreted by primary hybridomas
interact
with intact EPO receptor, but not with peptide SE-3.
Figure 14 shows the activity of various concentrations of Ab12 on the
proliferation of
UT7/EPO cells.
Figure 15 shows the activity of various concentrations of Ab198 on the
proliferation of
UT7/EPO cells.
Figure 16 shows the activity of various concentrations of Ab198 (with or
without the
addition of a secondary goat anti-human FC antibody) on the growth and
proliferation of
UT7/EPO cells.
Figure 17 shows the activity of various concentrations of Ab12 (with or
without the
addition of a secondary goat anti-human FC antibody) on the growth and
proliferation of
UT7/EPO cells.
Figure 18 is a series of representations of the heavy chain and light chain
variable
region nucleotide and amino acid sequences of the human anti-EPO-R antibody
expressed by
the cell line designated ABT2-SCX-003 of the invention, with Figure 18A (SEQ
ID NO: 10)
representing the nucleotide sequence encoding the variable region of the heavy
chain, Figure
18B (SEQ ID NO:11) representing the amino acid sequence encoded by the
nucleotide
sequence shown in Figure 18A, Figure 18C (SEQ ID NO:12) representing the
nucleotide
sequence encoding the variable region of the light chain, and Figure 18D (SEQ
ID NO:13)
representing the amino acid sequence encoded by the nucleotide sequence shown
in Figure
18C.
Figure 19 is a series of representations of the heavy chain and light chain
variable
region nucleotide and amino acid sequences of the human anti-EPO-R antibody
expressed by
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the cell line designated ABT2-SCX-012 (also referred to herein as Ab12) of the
invention, with
Figure 19A (SEQ ID NO:2) representing the nucleotide sequence encoding the
variable region
of the heavy chain, Figure 19B (SEQ ID NO:3) representing the amino acid
sequence encoded
by the nucleotide sequence shown in Figure 19A, Figure 19C (SEQ ID NO:4)
representing the
nucleotide sequence encoding the variable region of the light chain, and
Figure 19D (SEQ ID
NO:5) representing the amino acid sequence encoded by the nucleotide sequence
shown in
Figure 19C.
Figure 20 is a series of representations of the heavy chain and light chain
variable
region nucleotide and amino acid sequences of the human anti-EPO-R antibody
expressed by
the cell line designated ABT2-SCX-022 of the invention, with Figure 20A (SEQ
ID NO:14)
representing the nucleotide sequence encoding the variable region of the heavy
chain, Figure
20B (SEQ ID NO: 15) representing the amino acid sequence encoded by the
nucleotide
sequence shown in Figure 20A, Figure 20C (SEQ ID NO: 16) representing the
nucleotide
sequence encoding the variable region of the light chain, and Figure 20D (SEQ
ID NO:17)
representing the amino acid sequence encoded by the nucleotide sequence shown
in Figure
20C.
Figure 21 is a series of representations of the heavy chain and light chain
variable
region nucleotide and amino acid sequences of the human anti-EPO-R antibody
expressed by
the cell line designated ABT2-SCX-054 of the invention, with Figure 21A (SEQ
ID NO:18)
representing the nucleotide sequence encoding the variable region of the heavy
chain, Figure
21B (SEQ ID NO:19) representing the amino acid sequence encoded by the
nucleotide
sequence shown in Figure 21A, Figure 21C (SEQ ID NO:20) representing the
nucleotide
sequence encoding the variable region of the light chain, and Figure 21D (SEQ
ID NO:21)
representing the amino acid sequence encoded by the nucleotide sequence shown
in Figure
21 C.
Figure 22 is a series of representations of the heavy chain and light chain
variable
region nucleotide and amino acid sequences of the human anti-EPO-R antibody
expressed by
the cell line designated ABT2-SCX-060 of the invention, with Figure 22A (SEQ
ID NO:10)
representing the nucleotide sequence encoding the variable region of the heavy
chain, Figure
22B (SEQ ID NO: 11) representing the amino acid sequence encoded by the
nucleotide
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sequence shown in Figure 22A, Figure 22C (SEQ ID NO:22) representing the
nucleotide
sequence encoding the variable region of the light chain, and Figure 22D (SEQ
ID NO:23)
representing the amino acid sequence encoded by the nucleotide sequence shown
in Figure
22C.
Figure 23 is a series of representations of the heavy chain and light chain
variable
region nucleotide and amino acid sequences of the human anti-EPO-R antibody
expressed by
the cell line designated ABT2-SCX-102 of the invention, with Figure 23A (SEQ
ID NO:10)
representing the nucleotide sequence encoding the variable region of the heavy
chain, Figure
23B (SEQ ID NO: 11) representing the amino acid sequence encoded by the
nucleotide
sequence shown in Figure 23A, Figure 23C (SEQ ID NO:24) representing the
nucleotide
sequence encoding the variable region of the light chain, and Figure 23D (SEQ
ID NO:25)
representing the amino acid sequence encoded by the nucleotide sequence shown
in Figure
23C.
Figure 24 is a series of representations of the heavy chain and light chain
variable
region nucleotide and amino acid sequences of the human anti-EPO-R antibody
expressed by
the cell line designated ABT2-SCX-135 of the invention, with Figure 24A (SEQ
ID NO:10)
representing the nucleotide sequence encoding the variable region of the heavy
chain, Figure
24B (SEQ ID NO: 11) representing the amino acid sequence encoded by the
nucleotide
sequence shown in Figure 24A, Figure 24C (SEQ ID NO:26) representing the
nucleotide
sequence encoding the variable region of the light chain, and Figure 24D (SEQ
ID NO:27)
representing the amino acid sequence encoded by the nucleotide sequence shown
in Figure
24C.
Figure 25 is a series of representations of the heavy chain and light chain
variable
region nucleotide and amino acid sequences of the human anti-EPO-R antibody
expressed by
the cell line designated ABT2-SCX-145 of the invention, with Figure 25A (SEQ
ID NO:10)
representing the nucleotide sequence encoding the variable region of the heavy
chain, Figure
25B (SEQ ID NO: 11) representing the amino acid sequence encoded by the
nucleotide
sequence shown in Figure 25A, Figure 25C (SEQ ID NO:28) representing the
nucleotide
sequence encoding the variable region of the light chain, and Figure 25D (SEQ
ID NO:29)
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representing the amino acid sequence encoded by the nucleotide sequence shown
in Figure
25C.
Figure 26 is a series of representations of the heavy chain and light chain
variable
region nucleotide and amino acid sequences of the human anti-EPO-R antibody
expressed by
the cell line designated ABT2-SCX-198 (also referred to herein as Ab198) of
the invention,
with Figure 26A (SEQ ID NO:6) representing the nucleotide sequence encoding
the variable
region of the heavy chain, Figure 26B (SEQ ID NO:7) representing the amino
acid sequence
encoded by the nucleotide sequence shown in Figure 26A, Figure 26C (SEQ ID
NO:8)
representing the nucleotide sequence encoding the variable region of the light
chain, and
Figure 26D (SEQ ID NO:9) representing the amino acid sequence encoded by the
nucleotide
sequence shown in Figure 26C.
Figure 27 is a series of representations of the heavy chain and light chain
variable
region nucleotide and amino acid sequences of the human anti-EPO-R antibody
expressed by
the cell line designated ABT2-SCX-254 of the invention, with Figure 27A (SEQ
ID NO:30)
representing the nucleotide sequence encoding the variable region of the heavy
chain, Figure
27B (SEQ ID NO:3 1) representing the amino acid sequence encoded by the
nucleotide
sequence shown in Figure 27A, Figure 27C (SEQ ID NO:32) representing the
nucleotide
sequence encoding the variable region of the light chain, and Figure 27D (SEQ
ID NO:33)
representing the amino acid sequence encoded by the nucleotide sequence shown
in Figure
27C.
Figure 28 is a series of representations of the heavy chain and light chain
variable
region nucleotide and amino acid sequences of the human anti-EPO-R antibody
expressed by
the cell line designated ABT2-SCX-267 of the invention, with Figure 28A (SEQ
ID NO:34)
representing the nucleotide sequence encoding the variable region of the heavy
chain, Figure
28B (SEQ ID NO:35) representing the amino acid sequence encoded by the
nucleotide
sequence shown in Figure 28A, Figure 28C (SEQ ID NO:36) representing the
nucleotide
sequence encoding the variable region of the light chain, and Figure 28D (SEQ
ID NO:37)
representing the amino acid sequence encoded by the nucleotide sequence shown
in Figure
28C.
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Figure 29 is a table showing amino acid sequence alignments of heavy chain
variable
regions of anti-EPOr mAbs generated according to the invention with their
associated germline
variable region sequences and identifying framework regions and
complementarity
determining regions.
Figure 30 is a table showing amino acid sequence alignments of light chain
variable
regions of anti-EPOr mAbs generated according to the invention with their
associated germline
variable region sequences and identifying framework regions and
complementarity
determining regions.
Figure 31 is a graph comparing the erythropoietic activity, at various
concentrations, of
a gamma-1 Ab 12 monoclonal antibody (Mab) and a gamma-2 Ab 12 Mab on an F36e
human
erythroleukemic cell line.
Figure 32 is a graph showing the increase in percent reticulocyte and percent
hematocrit in trangenic mice subjected to a multiple dosing regimen of
vehicle, Epogen (5U)
or Ab 12 antibody (5 or 50 g).
Figure 33 is a graph showing the increase in percent hematocrit in transgenic
mice
subjected to a weekly dosing regimen (over 3 weeks) of various concentrations
of AranespTM
or Ab 12.
Figure 34 is a graph showing the increase in percent hematocrit in transgenic
mice
subjected to single versus weekly dosing regimens of various concentrations of
AranespTM or
Ab 12.
Figure 35 is a series of representations of the heavy chain and light chain
variable
region nucleotide and amino acid sequences of the human anti-EPO-R antibody
expressed by
the cell line designated ABT2-SCX-390 of the invention, with Figure 35A (SEQ
ID NO:38)
representing the nucleotide sequence encoding the variable region of the heavy
chain, Figure
35B (SEQ ID NO:39) representing the amino acid sequence encoded by the
nucleotide
sequence shown in Figure 35A, Figure 35C (SEQ ID NO:40) representing the
nucleotide
sequence encoding the variable region of the light chain, and Figure 35D (SEQ
ID NO:41)
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representing the amino acid sequence encoded by the nucleotide sequence shown
in Figure
35C.
Figure 36 is a series of representations of the heavy chain and light chain
variable
region nucleotide and amino acid sequences of the human anti-EPO-R antibody
expressed by
the cell line designated ABT2-SCX-412 of the invention, with Figure 36A (SEQ
ID NO:42)
representing the nucleotide sequence encoding the variable region of the heavy
chain, Figure
36B (SEQ ID NO:43) representing the amino acid sequence encoded by the
nucleotide
sequence shown in Figure 36A, Figure 36C (SEQ ID NO:44) representing the
nucleotide
sequence encoding the variable region of the light chain, and Figure 36D (SEQ
ID NO:45)
representing the amino acid sequence encoded by the nucleotide sequence shown
in Figure
36C.
Figure 37 is a series of representations of the heavy chain and light chain
variable
region nucleotide and amino acid sequences of the human anti-EPO-R antibody
expressed by
the cell line designated ABT2-SCX-430/432 of the invention, with Figure 37A
(SEQ ID
NO:46) representing the nucleotide sequence encoding the variable region of
the heavy chain,
Figure 37B (SEQ ID NO:47) representing the amino acid sequence encoded by the
nucleotide
sequence shown in Figure 37A, Figure 37C (SEQ ID NO:48) representing the
nucleotide
sequence encoding the variable region of the light chain, and Figure 37D (SEQ
ID NO:49)
representing the amino acid sequence encoded by the nucleotide sequence shown
in Figure
37C.
Figure 38 is a series of representations of the heavy chain and light chain
variable
region nucleotide and amino acid sequences of the human anti-EPO-R antibody
expressed by
the cell line designated ABT2-SCX-467 of the invention, with Figure 38A (SEQ
ID NO:50)
representing the nucleotide sequence encoding the variable region of the heavy
chain, Figure
38B (SEQ ID NO:51) representing the amino acid sequence encoded by the
nucleotide
sequence shown in Figure 38A, Figure 38C (SEQ ID NO:52) representing the
nucleotide
sequence encoding the variable region of the light chain, and Figure 38D (SEQ
ID NO:53)
representing the amino acid sequence encoded by the nucleotide sequence shown
in Figure
38C.
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Figure 39 is a series of representations of the heavy chain and light chain
variable
region nucleotide and amino acid sequences of the human anti-EPO-R antibody
expressed by
the cell line designated ABT2-SCX-484 of the invention, with Figure 39A (SEQ
ID NO:54)
representing the nucleotide sequence encoding the variable region of the heavy
chain, Figure
39B (SEQ ID NO:55) representing the amino acid sequence encoded by the
nucleotide
sequence shown in Figure 39A, Figure 39C (SEQ ID NO:56) representing the
nucleotide
sequence encoding the variable region of the light chain, and Figure 39D (SEQ
ID NO:57)
representing the amino acid sequence encoded by the nucleotide sequence shown
in Figure
39C.
Figure 40 is a table showing amino acid sequence alignments of heavy chain
variable
regions of anti-EPOr mAbs generated according to the invention with their
associated germline
variable region sequences and identifying framework regions and
complementarity
determining regions.
Figure 41 is a table showing amino acid sequence alignments of light chain
variable
regions of anti-EPOr mAbs generated according to the invention with their
associated germline
variable region sequences and identifying framework regions and
complementarity
determining regions.
Figure 42 shows the isolated and purified polynucleotide (top strand and
bottom
strands, SEQ ID NO:86 and SEQ ID NO:87, respectively) and amino acid sequence
of the
heavy chain of human antibody Ab390. The amino acid sequence comprises SEQ ID
NOS:88 and SEQ ID NOS 72 through 74. The variable chain ends at nucleotide
463. The
variable/constant joining region (underlined) is at nucleotides 464-469. The
constant region is
from nucleotides 470-2006.
Figure 43 shows the isolated and purified polynucleotide (top strand and
bottom
strands, SEQ ID NO:89 and SEQ ID NO:90, respectively) and amino acid sequence
of the light
chain of human antibody Ab390. The amino acid sequence comprises SEQ ID
NOS:91. The
variable chain ends at nucleotide 463. The variable/constant joining region
(underlined) is at
nucleotides 464-469. The constant region is from nucleotides 470-718.
CA 02501984 2005-04-11
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Figure 44 shows the isolated and purified polynucleotide (top strand and
bottom
strands, SEQ ID NO:92 and SEQ ID NO:93, respectively) and amino acid sequence
of the
heavy chain of human antibody Ab412. The amino acid sequence comprises SEQ ID
NOS:94 and SEQ ID NOS 72 through 74. The variable chain ends at nucleotide
469. The
variable/constant joining region (underlined) is at nucleotides 470-475. The
constant region is
from nucleotides 476-2012.
Figure 45 shows the isolated and purified polynucleotide (top strand and
bottom
strands, SEQ ID NO:95 and SEQ ID NO:96, respectively) and amino acid sequence
of the light
chain of human antibody Ab412. The amino acid sequence comprises SEQ ID
NOS:97. The
variable chain ends at nucleotide 463. The variable/constant joining region
(underlined) is at
nucleotides 464-469. The constant region is from nucleotides 470-718.
Figure 46 shows the isolated and purified polynucleotide (top strand and
bottom
strands, SEQ ID NO:98 and SEQ ID NO:99, respectively) and amino acid sequence
of the
heavy chain of human antibody Ab432. The amino acid sequence comprises SEQ ID
NOS: 100 and SEQ ID NOS 72 through 74. The variable chain ends at nucleotide
463. The
variable/constant joining region (underlined) is at nucleotides 464-469. The
constant region is
from nucleotides 470-2006.
Figure 47 shows the isolated and purified polynucleotide (top strand and
bottom
strands, SEQ ID NO:101 and SEQ ID NO:102, respectively) and amino acid
sequence of the
light chain of human antibody Ab430. The amino acid sequence comprises SEQ ID
NOS:103.
The variable chain ends at nucleotide 463. The variable/constant joining
region (underlined) is
at nucleotides 464-469. The constant region is from nucleotides 470-718.
Figure 48 shows the isolated and purified polynucleotide (top strand and
bottom
strands, SEQ ID NO:104 and SEQ ID NO:105, respectively) and amino acid
sequence of the
heavy chain of human antibody Ab467. The amino acid sequence comprises SEQ ID
NOS:106 and SEQ ID NOS 72 through 74. The variable chain ends at nucleotide
463. The
variable/constant joining region (underlined) is at nucleotides 464-469. The
constant region is
from nucleotides 470-2006.
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Figure 49 shows the isolated and purified polynucleotide (top strand and
bottom
strands, SEQ ID NO:107 and SEQ ID NO:108, respectively) and amino acid
sequence of the
light chain of human antibody Ab467. The amino acid sequence comprises SEQ ID
NOS:109.
The variable chain ends at nucleotide 463. The variable/constant joining
region (underlined) is
at nucleotides 464-469. The constant region is from nucleotides 470-718.
Figure 50 shows the isolated and purified polynucleotide (top strand and
bottom
strands, SEQ ID NO:110 and SEQ ID NO: 111, respectively) and amino acid
sequence of the
heavy chain of human antibody Ab484. The amino acid sequence comprises SEQ ID
NOS: 112 and SEQ ID NOS 72 through 74. The variable chain ends at nucleotide
469. The
variable/constant joining region (underlined) is at nucleotides 470-475. The
constant region is
from nucleotides 470-2012.'
Figure 51 shows the isolated and purified polynucleotide (top strand and
bottom
strands, SEQ ID NO:113 and SEQ ID NO: 114, respectively) and amino acid
sequence of the
light chain of human antibody Ab484. The amino acid sequence comprises SEQ ID
NOS: 115.
The variable chain ends at nucleotide 463. The variable/constant joining
region (underlined) is
at nucleotides 464-469. The constant region is from nucleotides 470-718.
Detailed Description of the Invention
Definitions
As used herein, the term "antibody" or "immunoglobulin" refers to single
chain, two-
chain, and multi-chain proteins and glycoproteins that belong to the classes
of polyclonal,
monoclonal, chimeric and human or humanized. The term "antibody" also includes
synthetic
and genetically engineered variants thereof.
As used herein, the term "antibody fragment" refers to Fab, Fab', F(ab')2 and
Fv
fragments, as well as any portion of an antibody having specificity toward at
least one desired
epitope.
As used herein, the term "gamma-2", "gamma-2 isotype" or "IgG2" refers to
subclass 2
of immunoglobulin G (IgG), as well as any antibody fragment thereof. The four
subclasses of
IgG molecules are well characterized and well known to those of ordinary skill
in the art. (See
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for example, Molecular Biology of the Cell, 2nd Edition by Bruce Alberts et
al., 1989) Panels
of monoclonal antibodies are available that recognize all human isotypes
(IgA,IgG, IgD IgE,
and IgM) and subisotypes (IgA1,IgA2, IgGI, IgG2, IgG3, and IgG4) of human
immunoglobulins.
As used herein, the term "humanized antibody" refers to an antibody that is
derived
from a non-human antibody (i.e marine) that retains or substantially retains
the antigen-binding
properties of the parent antibody but is less immunogenic in humans.
As used herein, the term "human antibody" refers to an antibody that possesses
a
sequence that is derived from a human germ-line immunoglobulin sequence, such
as antibodies
derived from transgenic mice having human immunoglobulin genes (e.g.,
XenoMouse(M mice),
human phage display libraries, or human B cells.
As used herein, the term "epitope" refers to any protein determinate capable
of
specifically binding to an antibody or T-cell receptors. Epitopic determinants
usually consist
of chemically active surface groupings of molecules such as amino acids or
sugar side chains
and usually have specific three dimensional structural characteristics, as
well as specific charge
characteristics.
As used herein, the term "endogenous" refers to a product or activity arising
in the
body or cell as opposed to a product or activity coming from outside.
As used herein the phrase, a polynucleotide "derived from" or "specific for a
designated sequence refers to a polynucleotide sequence that comprises a
contiguous sequence
of approximately at least 6 nucleotides, preferably at least about 8
nucleotides, more preferably
at least about 10-12 nucleotides, and even more preferably at least about 15-
20 nucleotides
corresponding, i.e., identical or complementary to, a region of the designated
nucleotide
sequence. The sequence may be complementary or identical to a sequence that is
unique to a
particular polynucleotide sequence as determined by techniques known in the
art. Regions
from which sequences may be derived, include but are not limited to, regions
encoding specific
epitopes, as well as non-translated and/or non-transcribed regions.
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The derived polynucleotide will not necessarily be derived physically from the
nucleotide sequence of interest under study, but may be generated in any
manner, including,
but not limited to, chemical synthesis, replication, reverse transcription or
transcription, that is
based on the information provided by the sequence of bases in the region(s)
from which the
polynucleotide is derived. As such, it may represent either a sense or an
antisense orientation
of the original polynucleotide. In addition, combinations of regions
corresponding to that of
the designated sequence may be modified in ways known in the art to be
consistent with the
intended use.
As used herein, the phrase "encoded by" refers to a nucleic acid sequence that
codes for
a polypeptide sequence, wherein the polypeptide sequence or a portion thereof
contains an
amino acid sequence of at least 3 to 5 amino acids, more preferably at least 8
to 10 amino
acids, and even more preferably at least 15 to 20 amino acids from a
polypeptide encoded by
the nucleic acid sequence. Also encompassed are polypeptide sequences that are
immunologically identifiable with a polypeptide encoded by the sequence. Thus,
a
"polypeptide," "protein" or "amino acid" sequence has at least about 50%, 60%,
70%, 75%,
80%, 85%, 90%, 95% or more identity to the antibodies of the present
invention. Further, the
antibodies of the present invention may have at least about 60%, 70%, 75%,
80%, 85%, 90%
or 95% similarity to a polypeptide or amino sequences of the antibodies of the
present
invention. The amino acid sequences of the antibodies of the present invention
can be selected
from the group consisting of SEQUENCE ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19,
21, 23, 25,
27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55 and 57. Preferred
amino acid sequences
of the antibodies of the present invention are selected from the group
consisting of SEQ ID
NOS: 3, 5, 7, 9, 51 and 53.
As used herein, the phrase "recombinant polypeptide," "recombinant protein,"
or "a
polypeptide produced by recombinant techniques", which terms may be used
interchangeably
herein, describes a polypeptide that by virtue of its origin or manipulation
is not associated
with all or a portion of the polypeptide with which it is associated in nature
and/or is linked to
a polypeptide other than that to which it is lined in nature. A recombinant or
encoded
polypeptide or protein is not necessarily translated from a designated nucleic
acid sequence. It
also may be generated in any manner, including chemical synthesis or
expression of a
recombinant expression system.
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As used herein, the phrase "synthetic peptide" refers to a polymeric form of
amino
acids of any length, which may be chemically synthesized by methods well known
in the art
(See U.S. Patents 4,816,513, 5,854,389, 5,891,993 and 6,184,344).
As used herein, the term "polynucleotide" refers to a polymeric form of
nucleotides of
any length, either ribonucleotides or deoxyribonucleotides. This term refers
only to the
primary structure of the molecule. Thus, the term includes double and single-
stranded DNA as
well as double- and single-stranded RNA. It also includes modifications, such
as methylation
or capping and unmodified forms of the polynucleotide. The terms
"polynucleotide",
"oligomer," "oligonucleotide," and "oligo," are used interchangeably herein.
As used herein the phrase "purified polynucleotide" refers to a polynucleotide
of
interest or fragment thereof that is essentially free, e.g. contains less than
about 50%, 60%,
70%, 75%, 80%, 85%, 90%, 95% of the protein with which the polynucleotide is
naturally
associated. Techniques for purifying polynucleotides of interest are well
known in the art and
include, for example, disruption of the cell containing the polynucleotide
with a chaotropic
agent and separation of the polynucleotide(s) and proteins by ion-exchange
chromatography,
affinity chromatography and sedimentation according to density.
As used herein, the phrase "purified polypeptide" or "purified protein" means
a
polypeptide of interest or fragment thereof which is essentially free of,
e.g., contains less than
about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, cellular components with which
the
polypeptide of interest is naturally associated. Methods for purifying
polypeptides of interest
are known in the art.
As used herein, the term "isolated" refers to material that is removed from
its original
environment (e.g., the natural environment if it is naturally occurring). For
example, a
naturally occurring polynucleotide or polypeptide present in a living animal
is not isolated, but
the same polynucleotide or DNA or polypeptide, separated from some or all of
the coexisting
materials in the natural system, is isolated. Such polynucleotide could be
part of a vector
and/or such polynucleotide or polypeptide could be part of a composition, and
still be isolated
in that the vector or composition is not part of its natural environment.
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As used herein, the term "polypeptide" and "protein" are used interchangeably
and
refer to at least one molecular chain of amino acids linked through covalent
and/or non-
covalent bonds. The terms do not refer to a specific length of the product.
Thus, peptides,
oligopeptides and proteins are included within the definition of polypeptide.
The terms include
post-translational modifications of the polypeptide, including, but not
limited to,
glycosylations, acetylations, phosphorylations and the like. In addition,
protein fragments,
analogs, mutated or variant proteins, fusion proteins and the like are
included within the
meaning of polypeptide.
As used herein, the phrase "recombinant host cells," "host cells," "cells,"
"cell lines,"
"cell cultures," and other such terms denoting microorganisms or higher
eukaryotic cell lines
cultured as unicellular entities refer to cells that can be, or have been,
used as recipients for
recombinant vector or other transferred DNA, and include the original progeny
of the original
cell that has been transfected.
As used herein, the term "replicon" refers to any genetic element, such as a
plasmid, a
chromosome or a virus, that behaves as an autonomous unit of polynucleotide
replication
within a cell.
As used herein, the term "operably linked" refers to a situation wherein the
components
described are in a relationship permitting them to function in their intended
manner. Thus, for
example, a control sequence "operably linked" to a coding sequence is ligated
in such a
manner that expression of the coding sequence is achieved under conditions
compatible with
the control sequence.
As used herein, the term "vector" refers to a replicon in which another
polynucleotide
segment is attached, such as to bring about the replication and/or expression
of the attached
segment.
As used herein, the term "control sequence" refers to a polynucleotide
sequence that is
necessary to effect the expression of a coding sequence to which it is
ligated. The nature of
such control sequences differs depending upon the host organism. In
prokaryotes, such control
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sequences generally include a promoter, a ribosomal binding site and
terminators and, in some
instances, enhancers. The term "control sequence" thus is intended to include
at a minimum
all components whose presence is necessary for expression, and also may
include additional
components whose presence is advantageous, for example, leader sequences.
The term "transfection" refers to the introduction of an exogenous
polynucleotide into a
prokaryotic or eucaryotic host cell, irrespective of the method used for the
introduction. The
term "transfection" refers to both stable and transient introduction of the
polynucleotide, and
encompasses direct uptake of polynucleotides, transformation, transduction and
f-mating. -
Once introduced into the host cell, the exogenous polynucleotide may be
maintained as a non-
integrated replicon, for example, a plasmid, or alternatively, may be
integrated into the host
genome.
As used herein, the term "treatment" refers to prophylaxis and/or therapy.
As used herein, the term "purified product" refers to a preparation of the
product which
has been isolated from the cellular constituents with which the product is
normally associated
and from other types of cells that may be present in the sample of interest.
As used herein, the phrase "activation of an erythropoietin (EPO) receptor"
refers to
one or more molecular processes which an EPO receptor undergoes that result in
the
transduction of a signal to the interior of a receptor-bearing cell.
Ultimately, this signal brings
about one or more changes in cellular physiology. Activation of the EPO
receptor typically
results in the proliferation or differentiation of EPO receptor-bearing cells,
such as, but not
limited to, erythroid progenitor cells. A number of events are involved in the
activation of the
EPO receptor, such as, but not limited to, the dimerization of the receptor.
The structural unit of an antibody is a tetramer. Each tetramer is composed of
two
identical pairs of polypeptide chains, each pair having one "light" (25 kDa)
and one "heavy"
chain (about 50-70 kDa). The amino-terminal portion of each chain includes a
variable region
that is primarily responsible for antigen recognition. The carboxy-terminal
portion of the chain
defines a constant region that is responsible for the effector function of the
antibody. Human
light chains are classified as kappa and lambda light chains. Heavy chains are
classified as mu,
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delta, gamma, alpha, or epsilon and define the antibody's isotype as IgM, IgD,
IgG, IgA, and
IgE. IgG immunoglobulins are classified further into four subclasses (IgGi,
IgG2, IgG3 and
IgG4) having gamma-1, gamma-2, gamma-3 and gamma-4 heavy chains, respectively.
Most
of the therapeutic human, chimeric or humanized antibodies available are of
the IgGi antibody
type including Herceptin for breast cancer, Rituxan for Non-Hodgkins lymphoma
and Humira
and Remicade for rheumatoid arthritis (See Glennie, M.J. et al., Drug
Discovery Today, 8:503
(2003).
Within the light and heavy chains, the variable and constant regions are
joined by a "J"
region with the heavy chain also include a "D" region. The variable regions of
each
light/heavy chain pair form the antigen binding site. Thereupon, an intact
antibody has two
binding sites, which, except in bifunctional or bispecific antibodies, are the
same. Bifunctional
or bispecific antibodies are artificial hybrid antibodies that have two
different heavy/light chain
pairs and two different binding sites. Bifunctional or bispecific antibodies
can be produced
using routine techniques known in the art.
The structure of the chains of an antibody exhibit the same general structure
of
relatively conserved framework regions (FR) joined by three hyper variable
regions, also
called complementarity determining regions or CDRs. The CDRs from the two
chains of each
pair are aligned by the framework regions, enabling binding to a specific
epitope. From N-
terminal to C-terminal, both the light and heavy chains comprise the domains
FR1, CDR1,
FR2, CDR2, FR3, CDR3 and FR4.
U.S. Patent 6,319,499 describes antibodies that bind to and activate an
erythropoietin
receptor (EPO-R). The antibodies specifically identified in this patent are
Mabs 71 and 73.
Mab 71 binds to a peptide designated "SE-3" having the amino acid sequence of
PGNYSFSYQLEDEPWKLCRLHQAPTARGAV (SEQ ID NO:1) (See Example 3). SE-3 is
located on the human EPO-R between amino acid residues 49-78. According to
U.S. Patent
6,319,499, when this region of the EPO-R (i.e. amino acid residues 49-79) is
bound with a
cross linker such as Mab 71, this results in the activation of the EPO
receptor. Example 6 in
U.S. Patent 6,319,499 states that Mab 71 binds "significant amounts of peptide
SE-3"
compared to other peptides tested. This example further states that this
"indicates that Mab 71
binds to a region of the human EPO-R containing or overlapping residues 49 to
78." Mabs 71
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and 73 are murine antibodies. Although rodent and human antibodies may both
provide
precision for target specificity, human antibodies interact far more
effectively with the natural
defenses of the body and do not elicit anti-antibody responses to the same
extent as rodent
antibodies (Winter, G. and Milstein, C. Nature 349: 293 (1991). Additionally,
the flexibility of
human IgG subclasses differ (Roux, K.H. et al., J. Immunol. 159: 3372 (1997)
and this
difference also extends to rodent IgG isotypes since rodent IgG isotypes
differ from their
human counterparts. Since protein flexibility may affect antibody-antigen
recognition
(Jimenez, R., et al. Proc. Natl. Acad Sci. USA, 100: 92 (2003), human IgG2
isotypes may
result in antigen recognition mechanisms distinct from those of murine
antibodies. Murine IgG
isotypes generally differ from those of humans.
In one embodiment, the present invention relates to an antibody or antibody
fragment
that binds to the erythropoietin receptor. The antibody or antibody fragment
that binds to the
erythropoietin receptor comprises at least one heavy chain having an amino
acid sequence
selected from the group consisting of. SEQ ID NOS: 3, 7, 11, 15, 19, 31, 35,
39, 43, 47, 51, 55
and fragments thereof. In a second embodiment, the antibody or antibody
fragment that binds
to the erythropoietin receptor comprises at least one light chain having an
amino acid sequence
selected from the group consisting of. SEQ ID NOS: 5, 9, 13, 17, 21, 23, 25,
27, 29, 33, 37, 41,
45, 49, 53, 57 and fragments thereof.
In a third embodiment, the present invention relates to an isolated antibody
that
is capable of binding a human erythropoietin receptor in a mammal. More
specifically, the
antibody comprises a heavy chain variable region or a light chain variable
region which
comprises a continuous sequence from CDR1 through CDR3. The amino acid
sequence of the
heavy chain variable region comprising the continuous sequence from CDR1
through CDR3 is
selected from the group consisting of. SEQ ID NO:58, SEQ ID NO:59,, SEQ ID
NO:60 and
SEQ ID NO:61, and fragments thereof. The amino acid sequence of the light
chain variable
region comprising the continuous sequence from CDR1 through CDR3 is selected
from the
group consisting of. SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65,
SEQ
ID NO:66, SEQ ID NO:67, and SEQ ID NO:68, and fragments thereof. In addition,
the
present invention relates to an isolated antibody which comprises a heavy
chain variable region
or a light chain variable region which comprises at least one CDR. More
specifically, the
antibody comprises a heavy chain variable region comprising at least one CDR
selected from
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the group consisting of amino acid residues 99-112 of SEQ ID NO: 11, 26-35 of
SEQ ID NO:3,
50-65 of SEQ ID NO:3, 98-105 of SEQ ID NO:3, 26-35 of SEQ ID NO:19, 50-66 of
SEQ ID
NO:19, 99-105 of SEQ ID NO:19, 50-66 of SEQ ID NO:31, 99-105 of SEQ ID NO:31,
26-35
of SEQ ID NO:39, 50-65 of SEQ ID NO:39, 98-105 of SEQ ID NO:39, 26-37 of SEQ
ID
NO:43, 52-67 of SEQ ID NO:43, 100-107 of SEQ ID NO:43, 26-35 of SEQ ID NO:47,
50-65
of SEQ ID NO:47, 26-35 of SEQ ID NO:51, 50-65 of SEQ ID NO:51, 98-105 of SEQ
ID
NO:51, 26-37 of SEQ ID NO:55 and 52-67 of SEQ ID NO:55 or a light chain
variable region
comprising at least one CDR selected from the group consisting of amino acid
residues 24-34
of SEQ ID NO:13, 50-56 of SEQ ID NO:13, 89-97 of SEQ ID NO:5, 24-34 of SEQ ID
NO:27,
50-56 of SEQ ID NO:9, 24-39 of SEQ ID NO:33, 55-61 of SEQ ID NO:33, 24-34 of
SEQ ID
NO:41, 89-97 of SEQ ID NO:41, 24-34 of SEQ ID NO:45, 50-56 of SEQ ID NO:45, 89-
97 of
SEQ ID NO:45, 89-97 of SEQ ID NO:49 and 24-34 of SEQ ID NO:57.
In a fourth embodiment, the present invention relates to an antibody or
antibody
fragment that binds to and activates the erythropoietin receptor. The
antibodies of the present
invention bind to at least one epitope that is involved in activating the EPO
receptor (Example
4). Unlike other antibodies or fragments known in the art that bind to and
activate an
erythropoietin receptor, such as the antibodies described in U.S. Patent
6,319,499, the
antibodies of the present invention do not interact with the peptide
designated SE-3.
Surprisingly, the antibodies of the present invention are erythropoietic even
though the
antibodies do not bind to the SE-3 peptide. Therefore, the human antibodies of
the present
invention interact with at least one different epitope on the human EPO
receptor than the
antibodies described in U.S. Patent 6,319,499.
In a fifth embodiment, the present invention relates to an IgG2 antibody or
antibody
fragment that binds to and activates the erythropoietin receptor. The IgG2
antibodies or
antibody fragments of this embodiment bind to and interact with any epitope
that is involved in
activating the EPO receptor.
Additionally, as demonstrated by the BlAcore results shown in Example 1, the
antibodies of the present invention exhibit a binding affinity to the
erythropoietin receptor
within one hundred fold of the binding affinity of endogenous human
erythropoietin to the
erythropoietin receptor. A high (-InM) and low (-l M) affinity of the EPO
receptor for EPO
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has been reported resulting from two nonequivalent receptor binding sites on
EPO (See Philo,
J.S. et al., Biochemistry, 35:1681 (1996)).
The antibodies of the present invention can be polyclonal antibodies,
monoclonal
antibodies, chimeric antibodies (See U.S. Patent 6,020,153) or human or
humanized antibodies
or antibody fragments thereof. Synthetic and genetically engineered variants
(See U.S. Patent
6,331,415) of any of the foregoing are also contemplated by the present
invention. Preferably,
however, the antibodies of the present invention are human or humanized
antibodies. The
advantage of human or humanized antibodies is that they potentially decrease
or eliminate the
immunogenicity of the antibody in a host recipient, thereby permitting an
increase in the
bioavailability and a reduction in the possibility of adverse immune reaction,
thus potentially
enabling multiple antibody administrations.
Humanized antibodies include chimeric or CDR-grafted antibodies. Also, human
antibodies can be produced using genetically engineered strains of animals in
which the
antibody gene expression of the animal is suppressed and functionally replaced
with human
antibody gene expression.
Methods for making humanized and human antibodies are known in the art. One
method for making human antibodies employs the use of transgenic animals, such
as a
transgenic mouse. These transgenic animals contain a substantial portion of
the human
antibody producing genome inserted into their own genome and the animal's own
endogenous
antibody production is rendered deficient in the production of antibodies.
Methods for making
such transgenic animals are known in the art. Such transgenic animals can be
made using
XenoMouse technology or by using a "minilocus" approach. Methods for making
XenomiceTM are described in U.S. Patent Nos. 6,162,963, 6,150,584, 6,114,598
and 6,075,181.
Methods for making transgenic animals using the "minilocus" approach are
described in U.S.
Patents 5,545,807, 5,545,806 and 5,625,825. Also see International Publication
No.
W093/12227.
Using the XenoMouse technology, human antibodies can be obtained by
immunizing
a XenoMouse mouse (Abgenix, Fremont, California) with an antigen of interest.
The
lymphatic cells (such as B-cells) are recovered from the mice that express
antibodies. These
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recovered cells can be fused with myeloid-type cell line to prepare immortal
hybridoma cell
lines. These hybridoma cell lines can be screened and selected to identify
hybridoma cell lines
that produce antibodies specific to the antigen of interest. Alternatively,
the antibodies can be
expressed in cell lines other than hybridoma cell lines. More specifically,
sequences encoding
particular antibodies can be cloned from cells producing the antibodies and
used for
transformation of a suitable mammalian host cell.
Transformation can be by any known method for introducing polynucleotides into
a
host cell, including, for example, packaging the polynucleotide in a virus or
into a viral vector
and transducing a host cell with a virus or vector or by transfection
procedures known in the art
such as those described in U.S. Patents 4,399,216, 4,912,040, 4,740,461 and
4,959,455. For
example, one or more genes encoding the heavy chain can be expressed in a cell
and one or
more genes encoding the light chain can be expressed in a second cell. The
resulting heavy
and light chains can then be fused together to form the antibodies of the
present invention
using techniques known in the art. Alternatively, genes encoding for parts of
the heavy and
light chains can be ligated using restriction endonucleases to reconstruct the
gene coding for
each chain. Such a gene can then be expressed in a cell to produce the
antibodies of the
present invention.
The transformation procedure used will depend upon the host to be transformed.
Methods for introducing heterologous polynucleotides into mammalian cells are
well known in
the art and include dextran-mediated transfection, calcium phosphate
precipitation, polybrene
mediated transfection, protoplast fusion, electroporation, encapsulation of
the
polynucleotide(s) into liposomes and direct microinjection of the DNA
molecule.
Mammalian cell lines that can be used as hosts for expression are well known
in the art
and include, but are not limited to, Chinese hamster ovary (CHO) cells, HeLa
cells, baby
hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular
carcinoma
cells bacterial cells, such as E. coli, yeast cells, such as Saccharomyces
cerevisiae, etc.
Humanized antibodies can also be made using a CDR-grafted approach. Such
humanized antibodies are well known in the art. Generally, humanized
antibodies are
produced by obtaining nucleic acid sequences that encode the variable heavy
and variable light
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sequences of an antibody that binds to the EPO receptor, identifying the
complementary
determining region or "CDR" in the variable heavy and variable light sequences
and grafting
the CDR nucleic acid sequences on to human framework nucleic acid sequences.
(See, for
example, U.S. Patent Nos. 4,816,567 and 5,225,539).
The human framework that is selected is one that is suitable for in vivo
administration,
meaning that it does not exhibit immunogenicity. For example, such a
determination can be
made by prior experience with in vivo usage of such antibodies and studies of
amino acid
similarities.
Methods for cloning nucleic acids are known in the art. These methods involve
amplification of the antibody sequences to be cloned using appropriate primers
by polymerase
chain reaction (PCR). Primers that are suitable for amplifying antibody
nucleic acid sequences
and specifically murine variable heavy and variable light sequences are known
in the art.
Once the CDRs and FRs of the cloned antibody sequences that are to be
humanized are
identified, the amino acid sequences encoding the CDRs are identified and the
corresponding
nucleic acid sequences grafted on to selected human FRs. This can be done
using known
primers and linkers, the selection of which are known in the art.
After the CDRs are grafted onto selected human FRs, the resulting "humanized"
variable heavy and variable light sequences are expressed to produce a
humanized Fv or
humanized antibody that binds to the EPO receptor. Typically, the humanized
variable heavy
and light sequences are expressed as a fusion protein with human constant
domain sequences
so an intact antibody that binds to the EPO receptor is obtained. However, a
humanized Fv
antibody can be produced that does not contain the constant sequences. Fusion
of the human
constant sequence to the humanized variable region is preferred.
The EPO receptor that is bound by and preferably activated using the
antibodies of the
present invention is preferably a mammalian EPO receptor, most preferably a
human EPO
receptor. The present invention also contemplates the use of analogs of the
EPO receptor, such
as those described in U.S. Patent 5,292,654. Human EPO receptor can be
purchased from R &
D Systems (Minneapolis, Minnesota).
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An example of two (2) antibodies that (1) bind to and activate the EPO
receptor; (2) do
not interact with a peptide having an amino acid sequence of
PGNYSFSYQLEDEPWKLCRLHQAPTARGAV (SEQ ID NO: 1); and (3) exhibit a binding
affinity within one hundred fold of the binding affinity of endogeous human
EPO to the EPO
receptor, are the human antibodies designated Ab12 and Ab198. Ab12 and Ab198
are human
antibodies that were developed using the XenoMouse XenoMax technology
described herein
(See Example 1).
In another embodiment, the present invention relates to polynucleotide and
polypeptide
sequences that encode for the antibodies described herein. Preferably, such
polynucleotides
encode for both the variable and constant regions of each of the heavy and
light chains,
although other combinations are also contemplated by the present invention.
The present invention also contemplates oligonucleotide fragments derived from
the
disclosed polynucleotides and nucleic acid sequences complementary to these
polynucleotides.
The polynucleotides can be in the form of RNA or DNA. Polynucleotides in the
form of DNA,
cDNA, genomic DNA, nucleic acid analogs and synthetic DNA are within the scope
of the
present invention. The DNA may be double-stranded or single-stranded, and if
single
stranded, may be the coding (sense) strand or non-coding (anti-sense) strand.
The coding
sequence that encodes the polypeptide may be identical to the coding sequence
provided herein
or may be a different coding sequence which coding sequence, as a result of
the redundancy or
degeneracy of the genetic code, encodes the same polypeptide as the DNA
provided herein.
Preferably, the polynucleotides encode at least one heavy chain variable
region and at
least one light chain variable region of the present invention. Examples of
such
polynucleotides are shown in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14,16, 18, 20,
22, 24, 26, 28, 30,
32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54 and 56 as well as fragments,
complements and
degenerate codon equivalents thereof. For example, SEQ ID NO: 2 encodes for
the heavy
chain of Ab12 (variable region) and SEQ ID NO:4 encodes for the light chain of
Ab12
(variable region). SEQ ID NO:6 encodes for the heavy chain of Ab198 (variable
region) and
SEQ ID NO: 8 encodes for the light chain of Ab 198 (variable region).
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The present invention also includes variant polynucleotides containing
modifications
such as polynucleotide deletions, substitutions or additions, and any
polypeptide modification
resulting from the variant polynucleotide sequence. A polynucleotide of the
present invention
may also have a coding sequence that is a naturally occurring variant of the
coding sequence
provided herein.
It is contemplated that polynucleotides will be considered to hybridize to the
sequences
provided herein if there is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%
identity
between the polynucleotide and the sequence.
The present invention further relates to polypeptides that encode for the
antibodies of
the present invention as well as fragments, analogs and derivatives of such
polypeptides. The
polypeptides of the present invention may be recombinant polypeptides,
naturally purified
polypeptides or synthetic polypeptides. The fragment, derivative or analogs of
the
polypeptides of the present invention may be one in which one or more of the
amino acid
residues is substituted with a conserved or non-conserved amino acid residue
(preferably a
conserved amino acid residue) and such substituted amino acid residue may or
may not be one
encoded by the genetic code; or it may be one in which one or more of the
amino acid residues
includes a substituent group; or it may be on in which the polypeptide is
fused with another
compound, such as a compound to increase the half-life of the polypeptide (for
example,
polyethylene glycol); or it may be one in which the additional amino acids are
fused to the
polypeptide, such as a leader or secretory sequence or a sequence that is
employed for
purification of the polypeptide or a proprotein sequence. Such fragments,
derivatives and
analogs are within the scope of the present invention.
A polypeptide of the present invention may have an amino acid sequence that is
identical to that of the antibodies described herein or that is different by
minor variations due to
one or more amino acid substitutions. The variation may be a "conservative
change" typically
in the range of about 1 to 5 amino acids, wherein the substituted amino acid
has similar
structural or chemical properties, e.g., replacement of leucine with
isoleucine or threonine with
serine. In contrast, variations may include nonconservative changes, e.g.,
replacement of a
glycine with a tryptophan. Similar minor variations may also include amino
acid deletions or
insertions or both. Guidance in determining which and how many amino acid
residues may be
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substituted, inserted, or deleted without changing biological or immunological
activity may be
found using computer programs well known in the art, for example DNASTAR
software
(DNASTAR, Inc., Madison, Wisconsin).
Preferably, the polypeptides encode at least one heavy chain variable region
or at least
one light chain variable region of the antibodies of the present invention.
More preferably, the
polypeptides encode at least one heavy chain variable region and one light
chain variable
region of the antibodies of the present invention. Examples of such
polypeptides are those
having the amino acid sequences shown in SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15,
17, 19, 21, 23,
25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 46, 47, 49, 51, 53, 55, 57, 58,
59, 60, 61, 62, 63, 64,
65, 66, 67, 68, and fragments thereof. Specifically, the heavy chain of Ab12
has the amino
acid sequence shown in SEQ ID NO: 3 and the light chain has the amino acid
sequence shown
in SEQ ID NO:5. The amino acid sequence of the heavy chain of Ab198 is shown
in SEQ ID
NO:7 and the light chain has the amino acid sequence shown in SEQ ID NO:9.
The present invention also provides vectors that include the polynucleotides
of the
present invention, host cells which are genetically engineered with vectors of
the present
invention and the production of the antibodies of the present invention by
recombinant
techniques.
Host cells are genetically engineered (transfected, transduced or transformed)
with
vectors, such as, cloning vectors or expression vectors. The vector may be in
the form of a
plasmid, a viral particle, a phage, etc. The engineered host cells can be
cultured in
conventional nutrient media modified as appropriate for activating promoters,
selecting
transfected cells, etc. The culture conditions, such as temperature, pH and
the like, are those
previously used with the host cell selected for expression, and will be
apparent to those of
skilled in the art.
The polynucleotides of the present invention can be employed to produce the
polypeptides and hence the antibodies of the present invention. The
polynucleotide sequences
of the present invention can be included in any one of a variety of expression
vehicles, in
particular, vectors or plasmids for expressing a polypeptide. Such vectors
include
chromosomal, nonchromosomal and synthetic DNA sequences, derivatives of SV40,
bacterial
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plasmids, phage DNA, yeast plasmids, vectors derived from combinations of
plasmids and
phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus and
pseudorabies.
However, any other plasmid or vector may be used so long as it is replicable
and viable in the
host.
The appropriate DNA sequence may be inserted into the vector by a variety of
procedures. In general, the DNA sequence is inserted into appropriate
restriction endonuclease
sites by procedures known in the art. The polynucleotide sequence in the
expression vector is
operatively linked to an appropriate expression control sequence (i.e.
promoter) to direct
mRNA synthesis. Examples of such promoters include, but are not limited to,
the LTR or the
SV40 promoter, the E. coli lac or trp, the phage lambda PL promoter and other
promoters
known to control expression of genes in prokaryotic or eukaryotic cells or
their viruses. The
expression vector also contains a ribosome binding site for translation
initiation and a
transcription terminator. The vector may also include appropriate sequences
for amplifying
expression. For example, the vector can contain enhancers, which are
transcription-stimulating
DNA sequences of viral origin, such as those derived form simian virus such as
SV40,
polyoma virus, bovine papilloma virus or Moloney sarcoma virus, or genomic,
origin. The
vector preferably also contains an origin of replication. The vector can be
constructed to
contain an exogenous origin of replication or, such an origin of replication
can be derived from
SV40 or another viral source, or by the host cell chromosomal replication
mechanism.
In addition, the vectors preferably contain a marker gene for selection of
transfected
host cells such as dihydrofolate reductase or antibiotics, such as G-418
(geneticin, a neomycin-
derivative) or hygromycin, or genes which complement a genetic lesion of the
host cells such
as the absence of thymidine kinase, hypoxanthine phosphoribosyl transferase,
dihydrofolate
reductase, etc.
Suitable vectors for use in the present invention are known in the art. Any
plasmid or
vector can be used in the present invention as long as it is replicable and is
viable in the host.
Examples of vectors that can be used include those that are suitable for
mammalian hosts and
based on viral replication systems, such as simian virus 40 (SV40), Rous
sarcoma virus (RSV),
adenovirus 2, bovine papilloma virus (BPV), papovavirus BK mutant (BKV), or
mouse and
human cytomegalovirus (CVM).
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In a further embodiment, the present invention relates to host cells
containing the
above-described constructs. The host cell can be a higher eukaryotic cell,
such as a
mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host
cell can be a
prokaryotic cell, such as a bacterial cell. Preferably, the host cells provide
a suitable
environment for the production of active antibodies, since the biosynthesis of
functional
tetrameric antibody molecules requires correct nascent polypeptide chain
folding,
glycosylation, and assembly. Example of suitable host cells, include mammalian
cells, such as
COS-7 cells, Bowes melanoma cells, Chinese hamster ovary (CHO) cells,
embryonic lung
cells L-132, and mammalian cells of lymphoid origin, such as myeloma or
lymphoma cells.
The host cells can be transfected with a vector containing a polynucleotide
sequence encoding
the H-chain alone, with a second vector encoding the light chain alone (such
as by using two
different vectors as discussed previously). Preferably, the host cells are
transfected with two
different vectors.
Introduction of the vectors into the host cell can be effected by calcium
phosphate
transfection, DEAE-Dextran mediated transfection or electroporation (L. David
et al., Basic
Methods in Molecular Biology 2nd Edition, Appleton and Lang, Paramount
Publishing, East
Norwalk, Conn. (1994)).
In order to obtain the antibodies of the present invention, one or more
polynucleotide
sequences that encode for the light and heavy chain variable regions and light
and heavy chain
constant regions of the antibodies of the present invention should be
incorporated into a vector.
Polynucleotide sequences encoding the light and heavy chains of the antibodies
of the present
invention can be incorporated into one or multiple vectors and then
incorporated into the host
cells.
Cell lines expressing Ab12 and Ab467 antibodies were deposited with the
American
Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Virginia
20110,
under the terms of the Budapest Treaty, on September 30, 2003 and were
accorded accession
numbers PTA-5554 and PTA-5555. These deposits are provided for the convenience
of those
skilled in the art and are neither an admission that such deposits are
required to practice the
invention nor that equivalent embodiments are not within the skill of the art
in view of the
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present disclosure. The public availability of these deposits is not a grant
of a license to make,
use or sell the deposited materials under this or any other patents. The
nucleic acid sequences
of the deposited materials are
controlling if in conflict with any sequence described herein.
The antibodies of the present invention have a number of uses. In general, the
antibodies may be used to treat any condition treatable by erythropoietin or a
biologically
active variant or analog thereof. For example, antibodies of the invention are
useful for
treating disorders characterized by low red blood cell levels and/or decreased
hemoglobin
levels (e.g. anemia). In addition, the antibodies of the invention may be used
for treating
disorders characterized by decreased or subnormal levels of oxygen in the
blood or tissue, such
as, for example, hypoxemia or chronic tissue hypoxia and/or diseases
characterized by
inadequate blood circulation or reduced blood flow. Antibodies of the
invention also may, be
useful in promoting wound healing or for protecting against neural cell and/or
tissue damage,
resulting from brain/spinal cord injury, stroke and the like. Non-limiting
examples of
conditions that may be treatable by the antibodies of the invention include
anemia, such as
chemotherapy-induced anemia, cancer associated anemia, anemia of chronic
disease, HIV-
associated anemia, bone marrow transplant-associated anemia and the like,
heart failure,
ischemic heart disease and renal failure. As such, the invention includes
methods of treating
any of the aforementioned diseases or conditions comprising the step of
administering to a
mammal a therapeutically effective amount of said antibody. Preferably, the
mammal is a
human.
The antibodies of the present invention also can be used to identify and
diagnose
mammals that have a dysfunctional EPO receptor. Mammals that have a
dysfunctional EPO
receptor are characterized by disorders such as anemia. Preferably, the mammal
being
identified and diagnosed is a human. Additionally, the antibodies of the
present invention can
be used in the treatment of anemia in mammals suffering from red blood cell
aplasia. Red
blood cell aplasia may result from the formation of neutralizing anti-
erythropoietin antibodies
in patients during treatment with recombinant erythropoietin (Casadevall, N.
et al., n. Eng. J.
Med. 346: 469 (2002)). The method involves the step of administering to a
mammal
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suffering from said aplasia and in need of treatment a therapeutically
effective amount of the
antibodies of the present invention.
In another embodiment of the invention, the EPO receptor antibodies and
antibody
fragments of the invention also can be used to detect EPO receptor (e.g., in a
biological
sample, such as tissue specimens, intact cells, or extracts thereof), using a
conventional
immunoassay, such as an enzyme linked immunosorbent assay (ELISA), a
radioimmunoassay
(RIA) or tissue immunohistochemistry. The invention provides a method for
detecting EPO
receptor in a biological sample comprising contacting a biological sample with
an antibody or
antibody fragment of the invention and detecting either the antibody (or
antibody portion), to
thereby detect EPO receptor in the biological sample. The antibody or antibody
fragment is
directly or indirectly labeled with a detectable substance to facilitate
detection of the bound or
unbound antibody or antibody fragment. A variety of immunoassay formats may be
practiced
(such as competitive assays, direct or indirect sandwich immunoassays and the
like) and are
well known to those of ordinary skill in the art.
Suitable detectable substances include various enzymes, prosthetic groups,
fluorescent
materials, luminescent materials and radioactive materials. Examples of
suitable enzymes
include horseradish peroxidase, alkaline phosphatase, B-galactosidase, or
acetylcholinesterase;
examples of suitable prosthetic group complexes include streptavidin/biotin
and avidin/biotin;
examples of suitable fluorescent materials include umbelliferone, fluorescein,
fluorescein
isothiocyanate, rhodamine, dichlorotriazinylamine, dansyl chloride or
phycoerythrin; and an
example of a luminescent material includes luminol; and examples of suitable
radioactive
material include 1251, 131I335S. or 3H.
In yet another embodiment, the present invention relates to a pharmaceutical
composition containing a therapeutically effective amount of the antibody of
the present
invention along with a pharmaceutically acceptable carrier or excipient. As
used herein,
"pharmaceutically acceptable carrier" or "pharmaceutically acceptable
excipient" includes any
and all solvents, dispersion media, coating, antibacterial and antifungal
agents, isotonic and
absorption delaying agents, and the like that are physiologically compatible.
Examples of
pharmaceutically acceptable carriers or excipients include one or more of
water, saline,
phosphate buffered saline, dextrose, glycerol, ethanol and the like as well as
combinations
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thereof. In many cases, it will be preferable to include isotonic agents, for
example, sugars,
polyalcohols such as mannitol, sorbitol, or sodium chloride in the
composition.
Pharmaceutically acceptable substances such as wetting or minor amounts of
auxiliary
substances such as wetting or emulsifying agents, preservatives or buffers,
which enhance the
shelf life or effectiveness of the of the antibody or antibody portion also
may be included.
Optionally, disintegrating agents can be included, such as cross-linked
polyvinyl pyrrolidone,
agar, alginic acid or a salt thereof, such as sodium alginate and the like. In
addition to the
excipients, the pharmaceutical composition can include one or more of the
following, carrier
proteins such as serum albumin, buffers, binding agents, sweeteners and other
flavoring agents;
coloring agents and polyethylene glycol.
The compositions of this invention may be in a variety of forms. They include,
for
example, liquid, semi-solid and solid dosage forms, such as liquid solutions
(e.g. injectable and
infusible solutions), dispersions or suspensions, tablets, pills, powders,
liposomes and
suppositories. The preferred form depends on the intended mode of
administration and
therapeutic application. Typical preferred compositions are in the form of
injectable or
infusible solutions, such as compositions similar to those used for passive
immunization of
humans with other antibodies. The preferred mode of administration is
parenteral (e.g.,
intravenous, subcutaneous, intraperitoneal, intramuscular). In a preferred
embodiment, the
antibody is administered by intravenous infusion or injection. In another
preferred
embodiment, the antibody or antibody fragment is administered by intramuscular
or
subcutaneous injection.
Other suitable routes of administration for the pharmaceutical composition
include, but are not
limited to, rectal, transdermal, vaginal, transmucosal or intestinal
administration.
Therapeutic compositions typically must be sterile and stable under the
conditions of
manufacture and storage. The composition can be formulated as a solution,
microemulsion,
dispersion, liposome, or other ordered structure suitable to high drug
concentration. Sterile
injectable solutions can be prepared by incorporating the active compound
(i.e. antibody or
antibody fragment) in the required amount in an appropriate solvent with one
or a combination
of ingredients enumerated above, as required, followed by filtered
sterilization. Generally,
dispersions are prepared by incorporating the active compound into a sterile
vehicle that
contains a basic dispersion medium and the required other ingredients from
those enumerated
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above. In the case of sterile powders for the preparation of sterile
injectable solutions, the
preferred methods of preparation are vacuum drying and freeze-drying that
yields a powder of
the active ingredient plus any additional desired ingredient from a previously
sterile-filtered
solution thereof. The proper fluidity of a solution can be maintained, for
example, by the use
of a coating such as lecithin, by the maintenance of the required particle
size in the case of
dispersion and by the use of surfactants. Prolonged absorption of injectable
compositions can
be brought about by including in the composition an agent that delays
absorption, for example,
monostearate salts and gelatin.
The antibodies and antibody fragments of the invention can be administered by
a
variety of methods known in the art, although for many therapeutic
applications, the preferred
route/mode of administration is intravenous injection or infusion. As will be
appreciated by
the skilled artisan, the route and/or mode of administration will vary
depending upon the
desired results. In certain embodiments, the active compound may be prepared
with a carrier
that will protect the compound against rapid release, such as a controlled
release formulation,
including implants, transdermal patches, and microencapsulated delivery
systems.
Biodegradable, biocompatible polymers can be used, such as ethylene vinyl
acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic
acid. Many
methods for the preparation of such formulations are patented or generally
known to those
skilledin the art. See, e.g. Sustained and Controlled Release Drug Delivery
Systems, J. R.
Robinson, ed., Marcel Dekker, Inc., New York, 1978).
In certain embodiments, an antibody or antibody portion of the invention may
be orally
administered, for example, with an inert diluent or an assimilable edible
carrier. The
compound (and other ingredients if desired) may also be enclosed in a hard or
soft shell gelatin
capsule, compressed into tablets, buccal tablets, troches, capsules, elixiers,
suspensions, syrups,
wafers, and the like. To administer an antibody or antibody fragment of the
invention by other
than parenteral administration, it may be necessary to coat the compound with,
or co-
administer the compound with, a material to prevent its inactivation.
Supplementary active compounds also can be incorporated into the compositions.
In
certain embodiments, an antibody or antibody fragment of the invention is
coformulated with
and/or coadministered with one or more additional therapeutic agents. For
example, an EPO
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receptor antibody or antibody fragment of the invention may be coformulated
and/or
coadministerd with one or more additional antibodies that bind other targets
(e.g. , antibodies
that bind other cytokines or that bind cell surface molecules) or one or more
cytokines.
Furthermore, one or more antibodies of the invention may be used in
combination with two or
more of the foregoing therapeutic agents. Such combination therapies may
advantageously
utilize lower dosages of the administered therapeutic agents, thus avoiding
possible toxicities
or complications associated with the various monotherapies.
As used herein, the term "therapeutically effective amount" means an amount of
antibody or antibody fragment that produces the effects for which it is
administered. The exact
dose will be ascertainable by one skilled in the art. As known in the art,
adjustments based on
age, body weight, sex, diet, time of administration, drug interaction and
severity of condition
may be necessary and will be ascertainable with routine experimentation by
those skilled in the
art. A therapeutically effective amount is also one in which the
therapeutically beneficial
effects outweigh any toxic or detrimental effects of the antibody or antibody
fragment. A
"prophylactically effective amount" refers to an amount effective, at dosages
and for periods of
time necessary to achieve the desired prophylactic result. Typically, since a
prophylactic dose
is used in subjects prior to or at an earlier stage of disease, the
prophylactically effective
amount will be less than the therapeutically effective amount.
Dosage regimens may be adjusted to provide the optimum desired response (e.g.,
a
therapeutic or prophylactic response). For example, a single bolus may be
administered,
several divided doses may be administered over time or the dose may be
proportionally
reduced or increased as indicated by the exigencies of the therapeutic
situation. It is especially
advantageous to formulate parenteral compositions in dosage unit form for ease
of
administration and uniformity of dosage. Dosage unit form as used herein
refers to physically
discrete units suited as unitary dosages for the mammalian subjects to be
tested; each unit
containing a predetermined quantity of active compound calculated to produce
the desired
therapeutic effect in association with the required pharmaceutical carrier.
The specification for
the dosage unit forms of the invention are dictated by and directly dependent
on (a) the unique
characteristics of the active compound and the particular therapeutic or
prophylactic effect to
be achieved and (b) the limitations inherent in the art of compounding such an
active
compound for the treatment of sensitivity in individuals.
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An exemplary, non-limiting range for a therapeutically or prophylactically
effective
amount of an antibody or antibody portion of the invention is 0.1-20 mg/kg,
more preferably 1-
mg/kg. It is to be noted that dosage values may vary with the type and
severity of the
5 condition to be alleviated. It is to be further understood that for any
particular subject, specific
dosage regimens should be adjusted over time according to the individual need
and the
professional judgment of the person administering or supervising the
administration of the
compositions, and that dosage ranges set forth herein are exemplary only and
are not intended
to limit the scope or practice of the claimed composition.
By way of example, and not of limitation, examples of the present invention
shall now
be given.
EXAMPLE 1: Generation of human Erythropoietin Receptor antibodies
Antigen Preparation. The antigen used for immunization of XenoMouse animals
was
coupled to a universal T-cell epitope (TCE) (Jlmmunol., 148(5):1499 (1992))
using two
different methods. A mixture containing an equal amount of each was used as
the immunogen.
1) 2.3 mg of Dithiothreitol (DTT), and 200 mcg of cysteine coupled TCE
(J.Immunol.,
148(5):1499 (1992)) are mixed at room temperature for 30 minutes. DTT is
removed by
centrifugation through a SephadexMG10 (Pharmacia, Upsala, Sweden)
chromatography column.
The reduced cysteine coupled TCE is added to 200 mcg soluble extracellular
domain of human
EpoR (R&D Systems, Minneapolis, MN) re-suspended in Phosphate Buffered Saline
(PBS)
(8.1 mM Na2HP04, 1.6 mM NaH2PO4i 136 mM NaCl, 2.6 mM KCI, pH 7.4) and 33 mcg
of
Sulfosuccinimidyl 4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (sulfo
SMCC), and
mixed 4 C over night. Un-reacted EpoR was removed by centrifugation through a
IOKDa cut
off Centricon column (Millipore, Bedford, MA).
2) The soluble extracellular domain (200 mcg) of human EpoR (R&D Systems,
Minneapolis, MN) was re-suspended in PBS and mixed with 4 mcg of TCE-BPA (p-
Benzoyl
Phenylalanine) and incubated under UV light (362 nM) at room temperature for
45 minutes.
The un-reacted EpoR was removed by centrifugation through a IOKDa cut off
CentriconTM
column (Millipore, Bedford, MA).
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Immunization of animals. Monoclonal antibodies of the invention, including
Ab12 and
Ab198 (also referred to herein as AB-ABT2-XG2-012 and AB-ABT2-XG2-198,
respectively)
were developed by immunizing XenoMouse mice (XenoMouse XG2, Abgenix, Inc.,
Fremont, CA and Vancouver, BC) with soluble EpoR coupled to a TCE as described
above.
The initial immunization was with 20 mcg of antigen and mixed 1:1 v/v with
Complete
Freund's Adjuvant (CFA) (Sigma, St Louis, MO) per mouse. The subsequent
immunizations
were with 20 mcg of antigen mixed 1:1 v/v with incomplete Freund's (IFA). In
particular, each
animal was immunized at the base of tail and by intraperitoneal injection on
days 0, 14, 28 and
42.
Biotinylation of EpoR. 300 mcg of EpoR (Abbott CHO cell derived ref.#
RB69084:4)
was re-suspended in 990 mcL of PBS pH 8.6 and added to 100 mcg of biotin-NHS
(Pierce,
Rockford, 111) dissolved in DMSO (Dimethyl Sulfoxide) incubated for forty
minutes at room
temperature (RT). Free biotin and buffer was removed by centrifugation through
a 5kDa
Centricon column with several washes with PBS pH 7.4 and re-suspended in an
appropriate
volume to a final concentration was 600 mcg/mL.
Selection of animals for harvest. Anti-EpoR antibody titers were determined by
ELISA. 0.7 mcg/ml biotin EpoR (described above) was coated onto streptavadin
plates (Sigma,
St Louis, MO) at room teperature for 1 hour. The solution containing unbound
biotin EpoR
was removed and all plates were washed five times with dH2O. XenoMouse sera
from the
EpoR immunized animals, or naive XenMouse animals, were titrated in
2%milk/PBS at a 1:2
dilution in duplicate from a 1:100 initial dilution. The last well was left
blank, and plates were
washed five times with dH2O. A goat anti-human IgG Fe-specific horseradish
peroxidase
(HRP)(Pierce, Rockford, IL) conjugated antibody was added at a final
concentration of 1
mcg/mL for 1 hour at room temperature. The plates were washed five times with
dH2O. The
plates were developed with the addition of TMB chromogenic substrate (KPL,
Gaithersburg,
MD) for 30 minutes and the ELISA was stopped by the addition of 1 M phosphoric
acid. The
specific titers obtained from XenoMouse animals were determined from the
optical density
at 450 nm and are shown in Table 1. The titer represents the reciprocal
dilution of the serum
and therefore the higher the number the greater the humoral immune response to
EpoR.
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Table 1
Mouse I.D. Titer
11 1600
12 12800
13 51200
14 102400
15 102400
16 0
17 102400
18 3200
19 102400
20 2560
XenoMouse animal 14 was selected for harvest based on the serology data in
Table 1.
Culture and selection of B cells. B cells from the harvested animals were
cultured and
those secreting EpoR-specific antibodies were isolated essentially as
described in Babcook et
al., Proc. Natl. Acad. Sci. USA, 93:7843-7848 (1996). ELISA, performed as
described above
for sera titers, was used to identify EpoR-specific wells. Fifty plates
cultured at 500 cells/well
were screened on biotin EpoR to identify the antigen-specific wells. The data
as shown in
Table 2 demonstrated the presence of 701 wells with ODs significantly over
background
(0.05).
Table 2
Optical Density Number of Positives
0.1 701
0.2 273
0.3 163
0.4 130
0.5 102
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0.6 91
0.7 76
0.8 70
0.9 67
1.0 65
2.0 25
3.0 7
These data indicated a very low frequency of hits and indicated that the wells
were
monoclonal for antigen-specificity. These 701 positive wells were re-screened
on biotin EpoR
and 137 wells (shown in bold in Table 3 below) were found to repeat as real
antigen-specific
wells with ODs significantly over background (0.05).
Table 3
Optical Density Number of Positives
0.1 207
0.15 137
0.2 110
0.3 94
0.4 85
0.5 79
0.6 71
0.7 63
0.8 57
0.9 53
1.0 50
2.0 32
3.0 13
Agonist activity assay. Proliferation of an Epo responsive cell line was used
as the
basis for the agonist screen. These 137 wells were then screened for agonist
activity using the
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human erythroleukemia cell line UT-7/Epo (Abbott ref#.RB29454-174). 12.5 mcL
of
supernatant were added to 1 x 105 cells per well in RPMI 1640 (10% FCS) to a
final volume of,
50 mcL in a half-area 96 well plate. The well size is half the area of a
typical 96 well plate.
Proliferation was identified visually and compared to cells in media
containing a titration of
human Epo or no Epo as a base line control. Eleven wells with proliferation
activity were
identified.
EpoR-specific Hemolytic Plaque Assay. A number of specialized reagents are
needed
to conduct the assay. These reagents were prepared as follows.
Biotinylation of Sheep red blood cells (SRBC): SRBC are stored in RPMI media
as a
25% stock. A 250u1 SRBC packed-cell pellet was obtained by aliquoting 1.0 ml
of the stock
into a 15-m1 falcon tube, spinning down the cells and removing the
supernatant. The cell pellet
was then re-suspended in 4.75 ml PBS at pH 8.6 in a 50 ml tube. In a separate
50ml tube, 2.5
mg of Sulfo-NHS biotin was added to 45 ml of PBS at pH 8.6. Once the biotin
had completely
dissolved, 5 ml of SRBCs were added and the tube rotated at RT for 1 hour. The
SRBCs were
centrifuged at 3000g for 5 min, the supernatant drawn off and 25 mis PBS at pH
7.4 as a wash.
The wash cycle was repeated 3 times, then 4.75 ml immune cell media (RPMI 1640
with 10%
FCS) was added to the 250 ul biotinylated-SRBC (B-SRBC) pellet to gently re-
suspend the B-
SRBC (5% B-SRBC stock). Stock was stored at 4 C until needed.
Streptavidin (SA) coating of B-SRBC: One ml of the 5% B-SRBC stock was
transferred
into to a fresh eppendorf tube. The B-SRBC cells were pelleted with a pulse
spin at 8000 rpm
(6800 ref) in a microfuge, the supernatant drawn off, the pellet re-suspended
in 1.0 ml PBS at
pH 7.4, and the centrifugation repeated. The wash cycle was repeated 2 times,
then the B-
SRBC pellet was resuspended in 1.0 ml of PBS at pH 7.4 to give a final
concentration of 5%
(v/v). 10 ul of a 10mg/ml streptavidin (CalBiochem, San Diego, CA) stock
solution was added
and the tube mixed and rotated at RT for 20min. The washing steps were
repeated and the SA-
SRBC were re-suspended in lml PBS pH 7.4 (5% (v/v)).
EpoR coating of SA-SRBC: The SA-SRBC were coated with biotinylated EpoR at
IOug/ml, the mixed and rotated at RT for 20 min. The SRBC were washed twice
with 1.0 ml
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of PBS at pH 7.4 as above. The EpoR-coated SRBC were re-suspended in RPM!
(+10%FCS)
to a final concentration of 5% (v/v).
Determination of the quality of EpoR-SRBC by immunofluorescence (In: 10 ul of
5%
SA-SRBC and 10 ul of 5% PTH-coated SRBC were each added to separate fresh 1.5
ml
eppendorf tube containing 40u1 of PBS. The murine anti-EpoR antibody (R&D
Systems Cat.#
MAB307) was added to each sample of SRBCs at 20ug/ml. The tubes were rotated
at RT for
25 min, and the cells were then washed three times with 100ul of PBS. The
cells were re-
suspended in 50ul of PBS and incubated with 40 mcg/mL Gt-anti mouse IgG Fc
antibody
conjugated to A1exa4887(Molecular Probes, Eugene, OR). The tubes were rotated
at RT for 25
min, and then washed with 100ul PBS and the cells re-suspended in 10 ul PBS.
lOul of the
stained cells were spotted onto a clean glass microscope slide, covered with a
glass coverslip,
observed under fluorescent light, and scored on an arbitrary scale of 0-4.
Preparation of plasma cells: The contents of a single microculture well
identified by
the previous assays as containing a B cell clone secreting the immunoglobulin
of interest were
harvested. Using a 100-1000 ul PipettmanTM, the contents of the well were
recovered by adding
37C RPMI (+10% FCS). The cells were re-suspended by pipetting and then
transfered to a
fresh 1.5 ml eppendorf tube (final vol. approx 500-700u1). The cells were
centrifuged in a
microfuge at 1500 rpm (240 rcf) for 2 minutes at room temperature, then the
tube rotated 180
degrees and spun again for 2 minutes at 1500 rpm. The freeze media was drawn
off and the
immune cells resuspended in 100 ul RPMI (10% FCS), then centrifuged. This
washing with
RPMI (10% FCS) was repeated and the cells re-suspended in 60u1 RPMI (FCS) and
stored on
ice until ready to use.
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Plaque assay: Glass slides (2 x 3 inch) were prepared in advance with silicone
edges
and allowed to cure overnight at RT. Before use the slides were treated with
approx. 5u1 of
SigmaCoat (Sigma, Oakville, ON) wiped evenly over glass surface, allowed to
dry and then
wiped vigorously. To a 60u1 sample of cells was added 60u1 each of EpoR-coated
SRBC (5%
v/v stock), 4x guina pig complement (Sigma, Oakville, ON) stock prepared in
RPMI with 10%
FCS, and 4x enhancing sera stock (1:900 in RPMI with 10% FCS). The mixture (3 -
5u1) was
spotted onto the prepared slides and the spots covered with undiluted paraffin
oil. The slides
were incubated at 37 C for a minimum of 45 minutes.
Plaque assay results: The coating was determined qualitatively by
immunofluorescent
microscopy to be very high (4/4) using MAB307 to detect coating compared to a
secondary
detection reagent alone (0/4). There was no signal detected using the MAB307
antibody on
red blood cells that were only coated with streptavidin (0/4). These red blood
cells were then
used to identify antigen-specific plasma cells from the fourteen wells
identified in Table 4.
After micromanipulation to rescue the antigen-specific plasma cells, the genes
encoding the
variable region genes were rescued by RT-PCR on a single plasma cell.
Table 4
Plate ID Single Cell numbers
11 G10 ABT2-SCX-251-260
21D1 ABT2-SCX-54
25C3 ABT2-SCX-134-144
29G8 ABT2-SCX-1-l 1
33G8 ABT2-SCX-12-18
37A11 ABT2-SCX-19-44
431112 ABT2-SCX-185-201,233-239
16F7 ABT2-SCX-267-278
24C3 ABT2-SCX-55-77
24F8 ABT2-SCX-82-102
34D4 ABT2-SCX-145-168
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Expression. After isolation of the single plasma cells, mRNA was extracted and
reverse transcriptase PCR was conducted to generate cDNA. The cDNA encoding
the variable
heavy and light chains was specifically amplified using polymerase chain
reaction. The
variable heavy chain region was cloned into an IgG2 expression vector. This
vector was
generated by cloning the constant domain of human IgG2 into the multiple
cloning site of
pcDNA3. I +/Hygro (Invitrogen, Burlington, ON). The variable light chain
region was cloned
into an IgK expression vector. This vector was generated by cloning the
constant domain of
human IgK into the multiple cloning site of pcDNA3.1+/Neo (Invitrogen,
Burlington, ON).
The appropriate pairs of heavy chain and the light chain expression vectors
were then co-
lipofected into a 60 mm dish of 70% confluent human embryonal kidney 293 cells
and the
transfected cells were left to secrete a recombinant antibody for 24 hours.
The supernatant (3
mL) was harvested from the HEK 293 cells and the secretion of an intact
antibody (AB-ABT2-
XG2-012 and AB-ABT2-XG2-198) was demonstrated with a sandwich ELISA to
specifically
detect human IgG (Table 5, fourth column). The specificity of AB-ABT2-XG2-012
and AB-
ABT2-XG2-198 was assessed through binding of the recombinant antibody to
biotinylated
EpoR using ELISA (Table 5, fifth column).
Table 5
Well ID Single cell number Secretion Binding
11G10 ABT2-SCX-254 1:4 1:8
21D1 ABT2-SCX-054 >1:64 >1:64
2503 ABT2-SCX-135 1:4 1:4
29G8 ABT2-SCX-003 >1:64 >1:64
33G8 ABT2-SCX-012 >1:64 >1:64
37A11 ABT2-SCX-022 >1:64 >1:64
43H12 ABT2-SCX-198 >1:64 >1:64
16F7 ABT2-SCX-267 >1:64 >1:64
24C3 ABT2-SCX-060 >1:64 >1:64
24F8 ABT2-SCX-102 >1:64 >1:64
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34D4 ABT2-SCX-145 >1:64 >1:64
The ELISA for antigen specific antibody secretion was performed as follows.
Control
plates were coated with 2mg/mL Goat anti-human IgG H+L 0/N. For the binding
plates,
biotin-EpoR (0.7 mcg/mL) was coated onto streptavadin 96 well plates (Sigma,
St Louis, MO)
for one hour at room temperature. The plates were washed five times with dH2O.
Recombinant antibodies were titrated 1:2 for 7 wells from the undiluted
minilipofection
supernatant. The plates were washed five times with dH2O. A goat anti-human
IgG Fc-
specific HRP-conjugated antibody was added at a final concentration of 1 ug/mL
for 1 hour at
RT for the secretion and the binding ELISA. The plates were washed five times
with dH20.
The plates were developed with the addition of TMB chromogenic substrate (KPL,
Gaithersburg, MD) for 30 minutes and the ELISA was stopped by the addition of
1 M
phosphoric acid. Each ELISA plate was analyzed to determine the optical
density of each well
at 450 nm.
Purification of AB-ABT2-XG2-012 and AB-ABT2-XG2-198. For larger scale
production, the heavy and light chain expression vectors (2.5 ug of each
chain/dish) were
lipofected into ten 100 mm dishes that were 70% confluent with HEIR 293 cells.
The
transfected cells were incubated at 37 C for 4 days, the supernatant (6 mL)
was harvested and
replaced with 6 mL of fresh media. At day 7, the supernatant was removed and
pooled with
the initial harvest (120 mL total from 10 plates). The ABT2-XG2-012 and ABT2-
XG2-198
antibody were purified from the supernatant using a Protein-A Sepharose
(Amersham
Biosciences, Piscataway, NJ) affinity chromatography (1 mL). The antibody was
eluted from
the Protein-A column with 500 mcL of 0.1 M Glycine pH 2.5. The eluate was
dialysed in PBS
pH 7.4 and filter sterilized. The antibody was analyzed by non-reducing SDS-
PAGE to assess
purity and yield.
Agonist activity of recombinant antibodies. The ability of these recombinant
antibodies
to stimulate the proliferation of Epo responsive cells was examined using the
UT-7/Epo cells
with proliferation quantitated by MTS reagent (Promega, Madison, WI) measured
at 490 nm as
described in the Agonist Activity Assay above. ABT2-SCX-012 and ABT2-SCX-198
induced
proliferation in comparison to cells in media without antibody and are shown
below (Fig. 14
and 15 respectively).
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Effect of anti-Human Fc. It is possible that the agonist activity of ABT2-SCX-
012 and
ABT2-SCX-198 are due to self-aggregation. In order to address this issue we
induced
aggregation by the addition of an anti-human Fc secondary antibody and the
effect on the
agonist activity of ABT2-SCX-012 and ABT2-SCX-198 was determined using the UT-
7/Epo
cells. As shown below the addition of a secondary antibody had no effect on
the activity of
ABT2-SCX-198 (Fig. 16) and inhibited the activity of ABT2-SCX-012 (Fig. 4 17).
Since the addition of secondary Ab inhibited the activity of ABT2-SCX-012 we
concluded that aggregation of this antibody interferes with it's activity and
thus it is unlikely
that ABT2-SCX-012 has agonist activity due to aggregation. However, the
results of ABT2-
SCX-198 are more difficult to interpret. The lack of an effect could suggest
that ABT2-SCX-
198 is fully aggregated and thus the addition of secondary Ab has no further
effects on its
activity. Alternatively, the lack of effect suggests the activity of ABT2-SCX-
198 is not
perturbed by the conformational restrictions applied by a secondary antibody.
Sequence analysis of ABT2-SCX-012 and ABT2-SCX-198 The variable heavy chains
and the variable light chains for antibodies ABT2-SCX-012 and ABT2-SCX-198
were
sequenced to determine their DNA sequences. The complete sequence information
for the
anti-EpoR antibodies shown in Figures 1, 2, and 18-30 with nucleotide and
amino acid
sequences for each variable region of the heavy chain gamma and kappa light
chains. Figures
1 and 2 provide full-length sequences, including the constant regions.
The variable heavy sequences were analyzed to determine the VH family, the D-
region
sequence and the J-region sequence. The sequences were then translated to
determine the
primary amino acid sequence (Fig.29) and compared to the germline VH, D and J-
region
sequences to assess somatic hypermutations. The primary amino acid sequences
of all the anti-
EpoR antibody gamma chains are shown in Figure 16. The germline sequences are
shown
above and the mutations are indicated with the new amino acid sequence.
Unaltered amino
acids are indicated with a dash (-). The light chain was analyzed similarly to
determine the V
and the J-regions and to identify any somatic mutations from germline kappa
sequences
(Figure 30). The heavy chain of ABT2-SCX-012 was shown to utilize the VH 4-59
(DP-71),
DIR4rc and the JH4a gene segments, while the light chain was shown to use the
VkI (A30) and
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the Jkl gene segments. The heavy chain of ABT2-SCX-198 was shown to utilize
the VH 3-30
(V3-30), D4-23 and the JH6b gene segments, while the light chain was shown to
use the VkI
(L5) and the Jk3 gene segments.
EXAMPLE 2: Competition of Ab12 with 125I-Labeled EPO for Binding
CHO Cells Expressing Recombinant EPO Receptor
CHO cells expressing the full length recombinant human EPO receptor were
plated at
5x105 cells/well in 24 well plates 72 hours prior to the assay. On the day of
the assay, 95u1 of
Abl2, Ab198, or EPO at indicated concentrations (shown in Figure 5) diluted in
RPMI 1640,
0.5% BSA, 1mM Na N3 and 5 ul (6ng) of 125I-EPO (Amersham Cat. #IM178,
Arlington
Heights,IL 486ci/mM) were added to the wells. After incubating at 37 C for 1.5
hours, the
wells were washed three times with cold HBSS and harvested using 0.5m10.IN
NaOH.
Samples were counted in a Micromedic ME Plus gamma counter. The results are
shown in
Figure 5. Specifically, the results show that Abs 12 and 198 competed with EPO
for binding to
the erythropoietin receptor.
EXAMPLE 3: Biacore Studies
The studies described below were performed on a Biacore 2000 utilizing the
Biacontrol
software version 3.1. (Biacore, Uppsala, Sweden). Binding analyses were
performed with
antibody immobilized directly to the chip surface and followed by injection of
varying receptor
concentrations.
Immobilization of Antibody
Immobilizations of antibody were performed using the default immobilization
program
in the BiacoreM
software package. Antibodies were diluted to 10 ug/mL in the supplied acetate
buffers to prescreen for the appropriate pH at which to conduct the
immobilizations. For
inuuobilizations, antibodies were diluted into the appropriate acetate buffer
(10 mM acetate pH
4.0) and coupled directly to the chip surface using standard EDC chemistry at
three different
protein levels (500, 1000, and 1500 RU). The fourth flow cell was mock coupled
with EDC to
cap the carboxyl groups and provide a background surface as a negative
control.
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Binding studies
Binding studies were performed by successive injections of varying
concentrations of
soluble human EPO receptor over the chip surface (500 RU immobilized protein).
Binding
analyses were performed in the supplied HBS-EP buffer [HBS buffer -10 mM HEPES
pH -
7.4, 150 mM NaCl, 3 mM EDTA, 0.005% Polysorbate 20 (v/v), Biacore] using
receptor
diluted to the desired concentrations (10 - 200 nM) using the running buffer
(HBS-EP).
Experiments were performed at a flow rate of 30 uL/min. The receptor was
injected over a
period of 3 minutes followed by a 15 minute dissociation period. Simultaneous
injections over
the flow cell created as a negative control were also performed. All
injections were performed
in triplicate.
Model fitting
Data were fit to the models available in the BiaEvaluation 3Ø2 software
package
(Biacore). The data points from the experimental injections were corrected by
subtraction of
data points from simultaneous over the negative control surface. The corrected
data were used
to fit to the 1:1 (Langmuir) binding model as well as the bivalent analyte
model available in the
BiaEvaluation software package. Dissociation constants were calculated
directly from fitting
to the Langmuir binding model. For the bivalent analyte model, the
dissociation constants
were calculated indirectly using the calculated values for the kinetic
dissociation and kinetic
association constants, kd and ka.
Table 6
Antibody kD
Ab12 17.5nM
Ab 198 13.9 nM
EXAMPLE 4: EPO Dependent Human Cell Proliferation Assay
Stock cultures of the human erythroleukemic cell line, F36E cells were
maintained in
RPMI 1640 media with 10% fetal bovine serum and 1 unit per mL of recombinant
human
erythropoietin. Prior to assays, cells were cultured overnight at a density of
4.0 to 5.0 x 105
cells per mL in growth medium without EPO. Cells were recovered, washed and
resuspended
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at a density of 1.0 x 106 cells per mL in assay medium (RPMI 1640 + 10% FBS)
and 50 uL of
cells added to wells of a 96 well microtiter plate. 50 uL of each of Ab12, Ab
390, Ab 412, Ab
467, Ab 484, Ab 430/432 and Ab198 or EPO standards (recombinant human EPO
(rHuEPO))
in assay medium were added to wells and the plates were incubated in a
humidified incubator
at 37 C with a 5% CO2 atmosphere. After 72 hours, 20 gL of Promega Cell Titer
96
Aqueous reagent (as prepared per manufacturer's instructions, Madison,
Wisconin) was
added to all wells. Plates were incubated at 37 C with a 5% CO2 atmosphere for
4 hours and
the optical density at 490 nm was determined using a microplate reader (Wallac
Victor 1420
Multilabel Counter, Wallac Company, Boston, MA). The results are shown in
Figure 6. All
Abs stimulated proliferation of the F36E cell line. Maximal proliferative
activity was similar to
that observed with the EPO control and shown by a bell shaped curve as
concentration
increased. The results in Figure 7 demonstrate that Abl2, after storage at 4 C
for up to 20 days,
is active in inducing the proliferation of F36E cells. Proliferative activity
was similar to that
observed with the EPO control with the maximal response differing about ten-
fold on a molar
equivalent basis
EXAMPLE 5: Human CD36+ CFUe Assay
Frozen human CD36+ erythroid progenitor cells obtained from Poietics
(Biowhittaker
(Walkersville, MD)) were thawed and 104 cells/ml in IMDM-2%FBS. Cells (0.3 ml)
were
added to 0.3 ml tubes containing 2.4 ml Methocult (StemCell Technologies,
Vancouver,
Canada) Cat. #04230), 0.3 ml stem cell growth factor (Sigma, St. Louis,
Missouri Cat. #S7901,
100ug/ml), and 0.3 ml EPO (R&D Systems), Ab 12, or IMDM-2% FBS. After mixing,
1.1 ml
of the Methocult suspension was added to a 35 mm non tissue culture treated
sterile petri dish
and incubated at 37 C, 5% CO2 for 2 weeks. Colonies were identified
microscopically. The
results are shown in Figure 8. Specifically, Ab12 induced the formation of CFU-
E colonies
from human
CD 36+ progenitor cells. The colonies, identified microscopically, were red in
color. The size
and number of the colonies is reduced compared to those observed with the EPO
control
probably due to a reduced proliferative signal.
EXAMPLE 6: Demonstration of Erythopoietic Activity in Liquid Cultures
CD34+ cells were enriched from human peripheral blood using a Direct CD34+
Progenitor Cell Isolation Kit (Miltenyi, Auburn, CA). Recovered cells were
washed twice with
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alpha-medium and re-suspended in suspension culture media (alpha-media
supplemented with
30% FCS, 1% deionized BSA, 10-5M (3-mercaptoethanol, 10"6 M dexamethasone, 0.3
mg/mL
human hollo-transferrin and 10 ng/mL human recombinant stem cell factor).
Cells were plated
out at a density of 1 x 104 cells/mL in duplicates in 6-well microplates with
test antibody at
concentrations ranging from 0.1 - 100 ng/mL. Plates were incubated at 37 C and
5% CO2 for
two weeks. Duplicate samples from each well were recovered for cell counts and
staining with
benzidine (Reference Fibach, E., 1998 Hemoglobin, 22:5-6, 445-458).
The results are shown in Figure 9. Specifically, Ab198 induced the
proliferation of
human erythroid producing cells derived from progenitor cells in a dose
dependent manner.
The number of proliferating cells and the percentage expressing hemoglobin, as
indicated by
staining with benzidine, was reduced compared to the EPO treated controls
again probably due
to a reduced proliferative signal.
EXAMPLE 7: Cynomolgus Bone Marrow CFUe Assay
Bone marrow was harvested from cynoinolgus monkeys and diluted 1:2 with PBS.
Three ml of the diluted bone marrow was layered over six ml of Lymphoprep
(Gibco
(Invitrogen), Carlsbad, CA Cat. #1001967), centrifuged at 2700rpm for 20
minutes and the
buffy coat recovered and diluted in l Oml IMDM-2%FBS. Cells were centrifuged
and
resuspended at 106cells/ml in IMDM-2%FBS. Cells (0.3 ml) were added to tubes
containing
2.4 ml Methocult (StemCell Technologies, Vancouver, Canada) Cat. #04230), 0.3
ml stem cell
growth factor (Sigma, Cat. #S7901, 100 ug/ml), 0.3 ml EPO (R& D Systems,
Minneapolis,
Minnesota), test antibody (Ab198), or IMDM-2%FBS. After mixing, 1.lml of the
Methocult
suspension was added to a 35mm non tissue culture treated sterile petri dish
and incubated at
37 C, 5%C02 for 2 weeks. Colonies were identified microscopically. The results
of this assay
are shown in Figure 10 demonstrate that Ab 198 induced the formation of CFU-E
colonies
(although the number of colonies was reduced compared to that observed with
the EPO
control).
EXAMPLE 8: ELISA to Measure Binding of SE-3 Peptide
96 well polystyrene plates (Dynatec (Elk Grove Village, IL) Immunolon 4) were
coated
with 80 ul of 5ug/ml soluble EPO receptor (sEPOR) (R&D Systems (Minneapolis,
MN) Cat.
#307-ER/LF), or peptide SE-3 (PGNYSFSYQLEDEPWKLCRLHWAPTARGAV) (described
52
CA 02501984 2009-04-30
WO 2004/035603 PCT/1JS2003/032243
in U.S. Patent 6,319,499) diluted in 0.015M Na2CO3, 0.035M NaHCO3, pH 9.4 for
2 hours at
room temperature and overnight at 4 C. Plates were blocked for 30 minutes at
room
temperature with 100 ul of 5%BSA in PBS (Gibco (Invitrogen (Carlsbad, CA))
Cat.#10010).
After removal of blocking solution, 50 ul of Ab12 at 5 ug/ml in PBS with 1%
BSA was added
to wells and plates were incubated at room temperature for 2 hours. Plates
were washed three
TM
times using a Skatron 400 Plate Washer with PBS/0.05% Tween 20 and 50 ul of
secondary
TM
antibody diluted in PBS/0.25%BSA/0.05%Tween 20 added to the wells. For Ab12,
goat anti-
human IgG (Fc)-HRP (Caltag (Burlingame, CA) Cat.#H 10507) diluted 1:1000 was
used and
for Ab 71A (available from the American Type Culture Collection
HB11689, also described in U.S. Patent 6,319,499), goat anti mouse IgG (Fc)-
HRP (Jackson
Laboratories (West Grove, PA) Cat.# 115-03 5-164) diluted 1:5000 was used.
After a 1 hour
incubation at room temperature, plates were washed three times as before and
50 ul of OPD
Developing Reagent (Sigma #P9187) added to each well. Color development was
stopped by
addition of 50ul of IN HC1 to the wells and optical density measured at 490nm
on a Victor
1420 Multi-Label Counter.
Figure 11 shows that Ab12 does not interact (i.e. bind) with SE-3 peptide. Ab
71A
does interact (i.e. binds) with the SE-3 peptide Both Abs 12, and 71A
interacted with
immobilized erythropoietin receptor.
EXAMPLE 9: EPO Dependent Proliferation Assay
Primary hybridoma supernatants were diluted in assay medium and tested for
their
ability to stimulate the proliferation of the F36E human erythroleukemic cells
as described in
EXAMPLE 5. Results with five primary supernatants are shown in Figure 12.
These samples
stimulated the proliferation of F36E cells .
EXAMPLE 10: ELISA to Measure Binding of Hybridoma Supernatants to SE-3 Peptide
Forty-two primary hybridoma supernatants were tested for their ability to bind
to either
immobilized EPO receptor or peptide SE-3 as described in EXAMPLE 10. Figure 13
shows
that whereas all the hybridoma supernatants tested interact with immobilized
EPO receptor,
only sample 16 interacted with SE-3 peptide at levels above background.
EXAMPLE 11: Comparison of Erythropoietic Activity of
53
CA 02501984 2005-04-11
WO 2004/035603 PCT/US2003/032243
Gamma-1 Ab12 versus Gamma-2 Ab12
Proliferation assays (as described in Example 4) were performed to compare the
erythropoietic activity of gamma-1 Ab 12 and gamma-2 Ab 12 on F36e human
erythroleukemic cells. The results are shown in Figure 31. As Figure 31 shows,
gamma-2 Ab
12 was more effective at stimulating proliferation of the F3 6E cell line than
gamma-1 Ab 12.
EXAMPLE 12: Effect of Ab 12 on Erythropoiesis in vivo
(a) Construction of mEpoR -/-, hEopR + transgenic mice: Transgenic mice that
produced only human EpoR (hEpoR+, single allele) and no endogenous mouse EpoR
(mEpoR
-/-, double allele mutation) were generated as described in Liu, C. et al.,
Jounal of Biological
Chemistry, 272:32395 (1997) and Yu, X., et al., Blood, 98(2):475 (2001).
Breeding colonies
were established to generate mice for in vivo studies of eryhthropoiesis.
(b) Multiple dosing regimen: In initial experiments, animals were subjected to
a
multiple dosing regimen of Ab 12 to determine whether the antibody would cause
an increase
15, in reticulocyte counts and/or % hematocrit. Five transgenic mice (mEpoR -/-
, hEpoR+, were
injected subcutaneously with either 5 gg or 50 gg of Ab 12 in 0.2 mL vehicle
(phosphate
buffered saline [PBS] containing 0.1% bovine serum albumin ([BSA]). Control
animals also
were injected in the same manner with equal volumes of the vehicle alone or
vehicle
containing 5U Epogen (Amgen , Thousand Oaks, CA). All animals were dosed over
a three-
week period in accordance with the following schedule:
Week 1 Week 2 Week 3
Monday, Tuesday, Wednesday, Friday Monday, Wednesday, Friday Monday, Wednesday
Sample bleeds were taken on day 4 (Thursday of week 1) for determining
reticulocyte counts
and on day 19 (Friday of week 3) for determining hematocrits. Reticulocyte
counts and
hematocrit determinations were made using methods well known in the art. As
Figure 32
shows, Ab 12 caused a statistically significant increase (over controls) in
reticulocyte count
and % hematocrit in animals receiving either 5 or 50 g of Ab 12 antibody.
(c) Weekly dosing regimen: To assess whether the results seen under a multiple
dosing regimen still would be observed in animals receiving fewer doses of Ab
12, transgenic
mice were injected (as described in (b) above) with varying concentrations
(0.5, 2.5, 5.0, 50
and 250 g) of Ab 12 or a control, AranespTM (Amgen, Thousand Oaks, CA), a
more active
variant of Epogen on days 1, 8 and 15 and bled on days 4 and 19 for
determination of
54
CA 02501984 2009-04-30
WO 2004/035603 PCT/US2003/032243
reticulocyte count and hematocrit, respectively. Control animals received a
single dose of
vehicle only or a human IgG2 isotype control. Figure 33 shows that Ab 12
caused a
statistically significant increase (over vehicle and isotype controls) in
pecent hematocrit with
all but the lowest concentrations tested.
(d) Single versus weekly dosing regimens: To determine whether a single dose
of Ab-
12 would have an effect on erythropoiesis after 3 weeks, 'transgenic mice were
dosed with Ab
12 (50 g), at one week intervals for 3 weeks or with a single dose of Ab 12
(150 g) and bled
on day 19 for determination of percent hematocrit. Control animals received
vehicle alone, a
single dose of AranespTM (900 ng) or 3 total doses of AranespTM injected at
weekly intervals
(300 ng x 3). Figure 34 shows that both dosing regimens of Ab 12 caused a
statistically
significant increase in percent hematocrit over the vehicle control. In
contrast, the single dose
regimen of AranespTM did not have this effect.
The present invention is illustrated by way of the foregoing description and
examples.
The foregoing description is intended as a non-limiting illustration, since
many variations will
become apparent to those skilled in the art in view thereof.
Changes can be made to the composition, operation and arrangement of the
method of
the present invention described herein without departing from the concept and
scope of the
invention.
CA 02501984 2005-09-29
1/44
SEQUENCE LISTING
<110> Abbott Laboratories
<120> Erythropoietin Receptor Binding Antibodies
<130> 31760-2200
<140> CA 2,501,984
<141> 2003-10-14
<150> US 10/269,711
<151> 2002-10-14
<150> US 10/684,109
<151> 2003-10-10
<160> 115
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Leu Cys Arg Leu His Gln Ala Pro Thr Ala Arg Gly Ala Val
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<210> 2
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<212> DNA
<213> Homo sapiens
<400> 2
caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60
acctgcactg tctctggtgc ctccatcagt agttactact ggagctggat ccggcagccc 120
ccagggaagg gactggagtg gattgggtat atctattaca gtgggagcac caactacaac 180
ccctccctca agagtcgagt caccatatca gtagacacgt ccaagaacca gttctccctg 240
aagctgaggt ctgtgaccgc tgcggacacg gccgtgtatt actgtgcgag agagcgactg 300
gggatcgggg actactgggg ccaaggaacc ctggtcaccg tctcctcag 349
<210> 3
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Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
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2/44
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ala Ser Ile Ser Ser Tyr
20 25 30
Tyr Trp Ser Trp Ile Arg Gin Pro Pro Gly Lys Gly Leu Glu Trp Ile
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Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys
50 55 60
Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu
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Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Glu Arg Leu Gly Ile Gly Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 4
<211> 322
<212> DNA
<213> Homo sapiens
<400> 4
gacatccagc tgacccaatc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60
atcacttgcc gggcaagtca gggcattaga aatgatttag gctggtatca gcagaaacca 120
gggaaagccc ctaagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180
aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240
gaagattttg caacttatta ctgtctacag cataatactt accctccgac gttcggccaa 300
gggaccaagg tggaaatcaa ac 322
<210> 5
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<213> Homo sapiens
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Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp
20 25 30
Leu Gly Trp Tyr Gln Gin Lys Pro G1y Lys Ala Pro Lys Arg Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Thr Tyr Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 6
<211> 370
<212> DNA
<213> Homo sapiens
<400> 6
caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60
tcctgtgtag cctctggatt caccttcagt agctatggca tgcactgggt ccgccaggct 120
ccaggcaagg ggctggagtg ggtggcagtt atatcatatg atggaagtaa taaatactat 180
gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240
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ctgcaaatga acagcctgag agttgaggac acggctgtgt attactgtgc gagagatcac 300
ggtgggaggt acgtctacga ctacggtatg gacgtctggg gccaagggac cacggtcacc 360
gtctcctcag 370
<210> 7
<211> 123
<212> PRT
<213> Homo sapiens
<400> 7
Gin Val Gin Leu Val Glu Ser Gly Gly Gly Val Val Gin Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Gly Met His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gin Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp His Gly Gly Arg Tyr Val Tyr Asp Tyr Gly Met Asp Val
100 105 110
Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 8
<211> 322
<212> DNA
<213> Homo sapiens
<400> 8
gacatccaga tgacccaatc tccatcttcc gtgtctgcat ctataggaga cagagtctcc 60
atcacttgtc gggcgagtca gggtattagc agctggttag cctggtatca gcagaaacca 120
gggaaagccc ctacgctcct tatctatgct gcatccactt tgcaacgtgg ggtcccatca 180
aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240
gaagattttg caacttactt ttgtcaacag gctaacagtt tcccattcac tttcggccct 300
gggaccaaag tggatatcaa ac 322
<210> 9
<211> 107
<212> PRT
<213> Homo sapiens
<400> 9
Asp Ile Gin Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Ile Gly
1 5 10 15
Asp Arg Val Ser Ile Thr Cys Arg Ala Ser Gin Gly Ile Ser Ser Trp
20 25 30
Leu Ala Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Thr Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Thr Leu Gin Arg Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gin Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Phe Cys Gin Gin Ala Asn Ser Phe Pro Phe
85 90 95
Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys
100 105
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<210> 10
<211> 370
<212> DNA
<213> Homo sapiens
<400> 10
caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60
tcctgtgcag cctctggatt caccttcagt agctatggca tgcactgggt ccgccaggct 120
ccaggcaagg ggctggagtg ggtggcagtt atatcatatg atggaagtaa taaatactat 180
gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agttgaggac acggctgtgt attactgtgc gagagatcac 300
ggtgggaggt acgtctacga ctacggtatg gacgtctggg gccaagggac cacggtcacc 360
gtctcctcag 370
<210> 11
<211> 123
<212> PRT
<213> Homo sapiens
<400> 11
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp His Gly Gly Arg Tyr Val Tyr Asp Tyr Gly Met Asp Val
100 105 110
Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 12
<211> 322
<212> DNA
<213> Homo sapiens
<400> 12
gacatccaga tgacccaatc tccatcttcc gtgtctgcat ctgtaggaga cagagtctcc 60
atcacttgtc gggcgagtca gggtattagc agctggttag tctggtatca gcagaaacca 120
gggaaagccc ctgcgctcct aatctatgct gcatccagtt tgcagcgtgg ggtcccatca 180
aggttcagcg gcagtggatc tgggacagac ttcactctca ccatcagcag cctgcagcct 240
gaagattttg caacttactt ttgtcaacag gctaacagtt tcccattcac tttcggccct 300
gggaccaaag tggatatcaa ac 322
<210> 13
<211> 107
<212> PRT
<213> Homo sapiens
<400> 13
Asp Ile Gln Met Thr Gin Ser Pro Ser Ser Val Ser Ala Ser Val Gly
1 5 10 15
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Asp Arg Val Ser Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp
20 25 30
Leu Val Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Ala Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Arg Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Phe Cys Gin Gln Ala Asn Ser Phe Pro Phe
85 90 95
Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys
100 105
<210> 14
<211> 370
<212> DNA
<213> Homo sapiens
<400> 14
caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60
tcctgtgcag cctctggatt caccttcagt agctatggca tgcactgggt ccgccaggct 120
ccaggcaagg ggctggagtg ggtggtagtt atatcatatg atggaagtaa taaatactat 180
gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agttgaggac acggctgtgt attactgtgc gagagatcac 300
ggtgggaggt acgtctacga ctacggtatg gacgtctggg gccaagggac cacggtcacc 360
gtctcctcag 370
<210> 15
<211> 123
<212> PRT
<213> Homo sapiens
<400> 15
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Sex Ser Tyr
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Val Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp His Gly Gly Arg Tyr Val Tyr Asp Tyr Gly Met Asp Val
100 105 110
Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 16
<211> 322
<212> DNA
<213> Homo sapiens
<400> 16
gacatccaga tgacccaatc tccatcttcc gtgtctgcat ctgtaggaga cagagtctcc 60
atcacttgtc gggcgagtca gggtattagc agctggttag cctggtatca gcagaaacca 120
gggaaagccc ctacgctcct aatctatgct gcatccagtt tgcaacgtgg ggtcccatca 180
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aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240
gaagattttg caacttactt ttgtcaacag gctaacagtt tcccattcac tttcggccct 300
gggaccaaag tggatatcaa ac 322
<210> 17
<211> 107
<212> PRT
<213> Homo sapiens
<400> 17
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Ser Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Thr Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Arg Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Ala Asn Ser Phe Pro Phe
85 90 95
Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys
100 105
<210> 18
<211> 349
<212> DNA
<213> Homo sapiens
<400> 18
caggtgcagc tggtggagtc ggggggaggc gtggtccagc ctgggaggtc cctgagactc 60
tcctgtgcag cgtctggatt caccttcagt aaatatggca tgcactgggt ccgccaggct 120
ccaggcaagg ggctggagtg ggtggcagtt ttatggtatg atggaagtaa taaatactat 180
gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc gagaggtccg 300
tactactttg actactgggg ccagggaacc ctggtcaccg tctcctcag 349
<210> 19
<211> 116
<212> PRT
<213> Homo sapiens
<400> 19
Gln Val Gln Leu Val Glu Ser Gly Gly-Gly Val Val Gin Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Sex Lys Tyr
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Leu Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
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<210> 20
<211> 325
<212> DNA
<213> Homo sapiens
<400> 20
gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60
ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120
cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180
gacaggttca gtggcagtgg gtctgggaca gacttcactg tcaccatcag cagtctggaa 240
cctgaagatt ttgcagtgta ttactgtcag cagtatggta gttcaccgtg gacgttcggc 300
caagggacca aggtggaaat caaac 325
<210> 21
<211> 108
<212> PRT
<213> Homo sapiens
<400> 21
Glu Ile Val Leu Thr Gin Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser
20 25 30
Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45
Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser
50 55 60
Gly Ser Gly Her Gly Thr Asp Phe Thr Val Thr Ile Ser Arg Leu Glu
65 70 75 80
Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gin Gin Tyr Gly Ser Ser Pro
85 90 95
Trp Thr Phe G1y Gin Gly Thr Lys Val Glu Ile Lys
100 105
<210> 22
<211> 322
<212> DNA
<213> Homo sapiens
<400> 22
gacatccaga tgacccaatc tccatcttcc gtgtccgcat ctgtaggaga cagagtctcc 60
atcacttgtc gggcgagtca gggtattagc agctggttag cctggtatca gcagaaacca 120
gggaaagccc ctacgctcct aatctatgct gcatccagtt tgcaacgtgg ggtcccatca 180
aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240
gaagattttg caacttactt ttgtcaacag gctaacagtt tcccattcac tttcggccct 300
gggaccaaag tggatatcaa ac 322
<210> 23
<211> 107
<212> PRT
<213> Homo sapiens
<400> 23
Asp Ile Gin Met Thr Gin Ser Pro Ser Ser Val Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Ser Ile Thr Cys Arg Ala Ser Gin Gly Ile Ser Ser Trp
20 25 30
Leu Ala Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Thr Leu Leu Ile
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35 4G 45
Tyr Ala Ala Ser Ser Leu Gln Arg Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Ala Asn Ser Phe Pro Phe
85 90 95
Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys
100 105
<210> 24
<211> 322
<212> DNA
<213> Homo sapiens
<400> 24
gacatccaga tgacccaatc tccatcttcc gtgtctgcat ctgtaggaga cagagtctcc 60
atcacttgtc gggcgagtca gggtattagc agctggttag cctggtatca gcagaaacca 120
gggaaagccc ctaagcgcct gatctatgct gcatccagtt tgcaacgtgg ggtcccatca 180
aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240
gaagattttg caacttactt ttgtcaacag gctaacagtt tcccattcac tttcggccct 300
gggaccaaag tggatatcaa ac 322
<210> 25
<211> 107
<212> PRT
<213> Homo sapiens
<400> 25
Asp Ile Gln Net Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Ser Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Arg Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser G1y Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Ala Asn Ser Phe Pro Phe
85 90 95
Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys
100 105
<210> 26
<211> 322
<212> DNA
<213> Homo sapiens
<400> 26
gacatccaga tgacccagtc tccatcttcc gtgtctacat ctgtaggaga cagagtctcc 60
atcacttgtc gggcgagtca gggtattggc agctggttag cctggtatca gcagaaacca 120
gggcaagccc ctacgctcct aatctatgct gcatccagtt tgcaacgtgg ggtcccatca 180
agattcagcg gcagtggatc tgggacagat ttcactctca ccatcaacag cctgcagcct 240
gaagattttg caacttactt ttgtcaacag gctaacagtt tcccattcac tttcggccct 300
gggaccaaag tggatgtcaa ac 322
<210> 27
<211> 107
CA 02501984 2005-04-11
WO 2004/035603 PCT/US2003/032243
9/44
<212> PRT
<213> Homo sapiens
<400> 27
Asp Ile Gln Met Thr Gin Ser Pro Ser Ser Val Ser Thr Ser Val Gly
1 5 10 15
Asp Arg Val Ser Ile Thr Cys Arg Ala Ser Gln Gly Ile Gly Ser Trp
20 25 30
Leu Ala Trp Tyr Gln Gin Lys Pro Gly Gln Ala Pro Thr Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Arg Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Ala Asn Ser Phe Pro Phe
85 90 95
Thr Phe Gly Pro Gly Thr Lys Val Asp Val Lys
100 105
<210> 28
<211> 322
<212> DNA
<213> Homo sapiens
<400> 28
gacatccaga tgacccagtc tccatcttcc gtgtctgcat ctgtaggaga cagagtctcc 60
atcacttgtc gggcgagtca gggtattggc agctggttag cctggtatca gcagaaacca 120
gggcaagccc ctacgctcct aatctatgct gcatccagtt tgcaacgtgg ggtcccatca 180
agattcagcg gcagtggatc tgggacagat ttcactctca ccatcaacag cctgcagcct 240
gaagattttg caacttactt ttgtcaacag gctaacagtt tcccattcac tttcggccct 300
gggaccaaag tggatgtcaa ac 322
<210> 29
<211> 107
<212> PRT
<213> Homo sapiens
<400> 29
Asp Ile Gin Met Thr Gin Ser Pro Ser Ser Val Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Ser Ile Thr Cys Arg Ala Ser Gln Gly Ile Gly Ser Trp
20 25 30
Leu Ala Trp Tyr Gln Gin Lys Pro Gly Gln Ala Pro Thr Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Arg Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Ala Asn Ser Phe Pro Phe
85 90 95
Thr Phe Gly Pro Gly Thr Lys Val Asp Val Lys
100 105
<210> 30
<211> 349
<212> DNA
<213> Homo sapiens
<400> 30
CA 02501984 2005-04-11
WO 2004/035603 PCT/US2003/032243
10/44
caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60
tcctgtgcag cgtctggatt caccttcagt agctatggca tgcactgggt ccgccaggct 120
ccaggcaagg ggctggagtg ggtggcagtt atatggtttg atggaaataa taaattctat 180
gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agtcgaggac acggctgtgt attactgtgc gcgaggcggg 300
agctactggg actactgggg ccagggaacc ctggtcaccg tctcctcag 349
<210> 31
<211> 116
<212> PRT
<213> Homo sapiens
<400> 31
Gin Val Gin Leu Val Glu Ser Gly Gly Gly Val Val Gin Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30,
Gly Net His Trp Val Arg Gln Ala Pro Gly Lys Giy Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Phe Asp Gly Asn Asn Lys Phe Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gin Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Gly Ser Tyr Trp Asp Tyr Trp Gly Gin Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 32
<211> 336
<212> DNA
<213> Homo sapiens
<400> 32
gatattgtga tgacccagac tccactcttc tcatttgtca tgattggaca gccggcctcc 60
atctcctgca ggtctaggca aagcctcgta cacagtgatg gaaacaccta cttgaattgg 120
cttcagcaga ggccaggcca gcctccaaga ctcctaattt ataagacttc taaccggttc 180
tctggggtcc cagatagatt cagtggcagt ggggcaggga cagatttcac actgaaaatc 240
agcagggtgg aagctgagga tgtcggggtt tattactgta tgcaagctac acaatttcct 300
atcacgttcg gccaagggac acgactggag attaaa 336
<210> 33
<211> 112
<212> PRT
<213> Homo sapiens
<400> 33
Asp Ile Val Met Thr Gin Thr Pro Leu Phe Ser Phe Val Met Ile Gly
1 5 10 15
Gin Pro Ala Ser Ile Ser Cys Arg Ser Arg Gin Ser Leu Val His Ser
20 25 30
Asp Gly Asn Thr Tyr Leu Asn Trp Leu Gin Gin Arg Pro Gly Gin Pro
35 40 45
Pro Arg Leu Leu Ile Tyr Lys Thr Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gin Ala
CA 02501984 2005-04-11
WO 2004/035603 PCT/US2003/032243
11/44
85 90 95
Thr Gln Phe Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys
100 105 110
<210> 34
<211> 370
<212> DNA
<213> Homo sapiens
<400> 34
caggtgcagc tggtggagtc tggtggaggc gtggtccagc ctgggaggtc cctgagactc 60
tcctgtgcag cctctggatt caccttcagt agctatggca tgcactgggt ccgccaggct 120
ccaggcaagg ggctggagtg ggtggcagtt atatcatatg atggaagtaa taaatactat 180
gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agttgaggac acggctgtgt attactgtgc gaaagatcac 300
ggtgggaggt acgtctacga ctacggtatg gacgtctggg gccaagggac cacggtcacc 360
gtctcctcag 370
<210> 35
<211> 123
<212> PRT
<213> Homo sapiens
<400> 35
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu SerCys Ala Ala Ser Gly Phe Thr Phe Sex Ser Tyr
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Asp His Gly Gly Arg Tyr Val Tyr Asp Tyr Gly Met Asp Val
100 105 110
Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 36
<211> 322
<212> DNA
<213> Homo sapiens
<400> 36
gacatccaga tgacccagtc tccatcttcc gtgtctgcat ctgtaggaga cagagtctcc 60
atcacttgtc gggcgagtca gggtattggc agctggttag cctggtatca gcagaaacca 120
gggcaagccc ctacgctcct aatctatgct gcctccagtt tgcaacgtgg ggtcccatca 180
agattcagcg gcagtggatc tgggacagat ttcactctca ccatcaacag cctgcagcct 240
gaagattttg caacttactt ttgtcaacag gctaacagtt tcccattcac tttcggccct 300
gggaccaaag tggatgtcaa ac 322
<210> 37
<211> 107
<212> PRT
<213> Homo sapiens
CA 02501984 2005-04-11
WO 2004/035603 PCT/US2003/032243
12/44
<400> 37
Asp Ile Gin Met Thr Gin Ser Pro Ser Ser Val Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Ser Ile Thr Cys Arg Ala Ser Gin Gly Ile Gly Ser Trp
20 25 30
Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Thr Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gin Arg Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp She Thr Leu Thr Ile Asn Ser Leu Gin Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Phe Cys Gin Gin Ala Asn Ser Phe Pro She
85 90 95
Thr Phe Gly Pro Gly Thr Lys Val Asp Val Lys
100 105
<210> 38
<211> 348
<212> DNA
<213> Homo sapiens
<400> 38
caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60
acctgcactg tctctggtgc ctccatcagt aattactact ggagctggat ccggcagccc 120
ccagggaagg gactggagtg gattgggtat gtctcttaca gtgggagtac gtactacaac 180
ccctccctca agggtcgagt caccatgtca gtagacacgt ccaagaacca gttctccctg 240
aagctgagct ctgtgaccgc tgcggacacg gccgtgtatt actgtgcgag agaaaaactg 300
gggattggag actactgggg ccagggaacc ctggtcaccg tctcctca 348
<210> 39
<211> 116
<212> PRT
<213> Homo sapiens
<400> 39
Gin Val Gin Leu Gin Giu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Her Gly Ala Ser Ile Ser Asn Tyr
20 25 30
Tyr Trp Ser Trp Ile Arg Gin Pro Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Val Ser Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys
50 55 60
Gly Arg Val Thr Met Ser Val Asp Thr Ser Lys Asn Gin She Ser Leu
65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Glu Lys Leu Gly Ile Giy Asp Tyr Trp Gly Gin Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 40
<211> 322
<212> DNA
<213> Homo sapiens
<400> 40
gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagattcacc 60
CA 02501984 2005-04-11
WO 2004/035603 PCT/US2003/032243
13/44
atcacttgcc gggcaagtca gggcattaaa aatgatttag gctggtatca gcagaaacca 120
gggaaagccc ctaagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180
aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240
gaagattttg caacttatta ctgtctacag cataatagtt atccgtgcag ttttggccag 300
gggaccaagc tggagatcaa ac 322
<210> 41
<211> 107
<212> PRT
<213> Homo sapiens
<400> 41
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Lys Asn Asp
20 25 30
Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile
35 40 45
Tyr Ala Ala'Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 r 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro Cys
85 90 95
Ser Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 42
<211> 354
<212> DNA
<213> Homo sapiens
<400> 42
caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60
acctgcactg tctctggtgc ctccatcagc agtggtgctt actactggag ttggatccgc 120
cagcacccag ggaagggcct ggagtggatt gggtacatct ataagagtga gacctcctac 180
tacaacccgt ccctcaagag tcgacttacc ctatcagtag acacgtctaa gaaccagttc 240
tccctgaacc tgatctctgt gactgccgcg gacacggccg tgtattattg tgcgagagat 300
aaactgggga tcgcggacta ctggggccag ggaaccctgg tcaccgtctc ctca 354
<210> 43
<211> 118
<212> PRT
<213> Homo sapiens
<400> 43
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ala Ser Ile Ser Ser Gly
20 25 30
Ala Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu
35 40 45
Trp Ile Gly Tyr Ile Tyr Lys Ser Glu Thr Ser Tyr Tyr Asn Pro Ser
50 55 60
Leu Lys Ser Arg Leu Thr Leu Ser Val Asp Thr Ser Lys Asn Gln Phe
65 70 75 80
Ser Leu Asn Leu Ile Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Ala Arg Asp Lys Leu Gly Ile Ala Asp Tyr Trp Gly Gln Gly Thr
100 105 110
CA 02501984 2005-04-11
WO 2004/035603 PCT/US2003/032243
14/44
Leu Val Thr Val Ser Ser
115
<210> 44
<211> 322
<212> DNA
<213> Homo sapiens
<400> 44
gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60
atcacttgcc gggcaagtca ggacattaga aatgatttag gctggtatca gcagaaacca 120
gggaaagccc ctaagcgcct gatctatgct gcatccaatt tgcaaagtgg ggtcccatca 180
aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240
gaagattttg caacttatta ctgtctacag cataatagct accctcccac tttcggcgga 300
gggaccaagg tggaaatcaa ac 322
<210> 45
<211> 107
<212> PRT
<213> Homo sapiens
<400> 45
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr lie Thr Cys Arg Ala Ser Gin Asp Ile Arg Asn Asp
20 25 30
Leu Gly Trp Tyr Gin Gin Lys Pro G1y Lys Ala Pro Lys Arg Leu Ile
35 40 45
Tyr Ala Ala Ser Asn Leu Gin Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gin Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro Pro
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 46
<211> 349
<212> DNA
<213> Homo sapiens
<400> 46
caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60
acctgcactg tctctggtgt ctccatcagt aattactact ggagctggat ccggcagtcc 120
ccagggaagg gactggagtg gattggatat atctattaca gtgggagtcc ctattacaac 180
ccctccctca agagtcgagt cactatatct gcagacacgt ccaagaacca attctccctg 240
aagctgagct ctgtgaccgc tgcggacacg gccatttatt actgtgcgag agaaaaactg 300
gggattggag actactgggg ccagggaacc ctggtcaccg tctcctcag 349
<210> 47
<211> 116
<212> PRT
<213> Homo sapiens
<400> 47
Gin Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Ile Ser Asn Tyr
CA 02501984 2005-04-11
WO 2004/035603 PCT/US2003/032243
15/44
20 25 30
Tyr Trp Ser Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Tyr Tyr Ser Gly Ser Pro Tyr Tyr Asn Pro Ser Leu Lys
50 55 60
Ser Arg Val Thr Ile Ser Ala Asp Thr Ser Lys Asn Gln Phe Ser Leu
65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr Cys Ala
85 90 95
Arg Glu Lys Leu Gly Ile Gly Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 48
<211> 322
<212> DNA
<213> Homo sapiens
<400> 48
gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtcggaga cagagtcacc 60
atcacttgcc gggcaagtca gggcattaga aatgatttag gctggtatca gcagaaacca 120
gggaaagccc ctaagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180
aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240
gaagattttg caacttatta ctgtctacag cataatagtt accctcccac tttcggccct 300
gggaccaagg tggatatcaa ac 322
<210> 49
<211> 107
<212> PRT
<213> Homo sapiens
<400> 49
Asp Ile Gin Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp
20 25 30
Leu Gly Trp Tyr Gln Gin Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Sex Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro Pro
85 90 95
Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys
100 105
<210> 50
<211> 349
<212> DNA
<213> Homo sapiens
<400> 50
caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60
acctgcactg tctctggtgg ctccatcagt cgttactact ggagctggat ccggcagccc 120
ccagggaagg gactggagtg gattgggtat gtctcttaca gtgggagcac ctactacaac 180
ccctccctca agagtcgagt caccatatca gtagacacgt ccaagaacca gttctccctg 240
aagctgagct ctgtgaccgc tgcggacacg gccgtgtatt actgtgcgag agataaactg 300
CA 02501984 2005-04-11
WO 2004/035603 PCT/US2003/032243
16/44
gggattggag actactgggg ccagggaacc ctggtcaccg tctcctcag 349
<210> 51
<211> 116
<212> PRT
<213> Homo sapiens
<400> 51
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Arg Tyr
20 25 30
Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Val Ser Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys
50 55 60
Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu
65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Asp Lys Leu Gly Ile Gly Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 52
<211> 322
<212> DNA
<213> Homo sapiens
<400> 52
gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60
atcacttgcc gggcaagtca gggcattaga aatgatttag gctggtatca gcagaaaccg 120
gggaaagccc ctaagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180
aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240
gaagattttg caacttatta ctgtctacag cataatagtt acccgtgcag ttttggccag 300
gggaccaagc tggagatcaa ac 322
<210> 53
<211> 107
<212> PRT
<213> Homo sapiens
<400> 53
Asp Ile Gln Net Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp
20 25 30
Leu Gly Trp Tyr Gln Gin Lys Pro Giy Lys Ala Pro Lys Arg Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro Cys
85 90 95
Ser Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
CA 02501984 2005-04-11
WO 2004/035603 PCT/US2003/032243
17/44
<210> 54
<211> 355
<212> DNA
<213> Homo sapiens
<400> 54
ctggtgcagc tgcaggagtc gggcccagga ctggtgaagc ctttacagac cctgtccctc 60
acctgcactg tctctggtgg ctccatcagc agtggtgttt actactggag ctggatccgc 120
cagcacccag ggaagggcct ggagtggatt gggtacatct ataacagtaa gacctcctat 180
tataatccgt ccctcaagag tcgacttacc ctatcagtag acacgtctaa gaaccagttc 240
tccctgaacc tgatctctgt gactgccgcg gacacggccg tgtattactg tgcgagagat 300
aaattgggga tcgcggacta ctggggccag ggaaccctgg tcaccgtctc ctcag 355
<210> 55
<211> 118
<212> PRT
<213> Homo sapiens
<400> 55
Gln Val Gin Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Leu Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly
20 25 30
Val Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu G1u
35 40 45
Trp Ile Gly Tyr Ile Tyr Asn Ser Lys Thr Ser Tyr Tyr Asn Pro Ser
50 55 60
Leu Lys Ser Arg Leu Thr Leu Ser Val Asp Thr Ser Lys Asn Gln Phe
65 70 75 80
Ser Leu Asn Leu Ile Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Ala Arg Asp Lys Leu Gly Ile Ala Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 56
<211> 322
<212> DNA
<213> Homo sapiens
<400> 56
gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60
atcacttgcc ggacaagtca gggcattaga aatgatttag gctggtatca gcagaaacca 120
gggaaagccc ctaagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180
aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240
gaagattttg caacttatta ctgtctacag,cataatagct accctcccac tttcggcgga 300
gggaccaagg tggagatcaa ac 322
<210> 57
<211> 107
<212> PRT
<213> Homo sapiens
<400> 57
Asp Ile Gin Net Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Gly Ile Arg Asn Asp
20 25 30
Leu G1y Trp Tyr Gln Gin Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile
CA 02501984 2005-04-11
WO 2004/035603 PCT/US2003/032243
18/44
35 40 45
Tyr Ala Ala Ser Ser Leu Gin Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gin Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gin His Asn Ser Tyr Pro Pro
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 58
<211> 34
<212> PRT
<213> Homo sapiens
<400> 58
Gly Ala Ser Ile Ser Ser Tyr Tyr Trp Ser Tyr Ile Tyr Tyr Ser Gly
1 5 10 15
Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser Glu Arg Leu Gly Ile Gly
20 25 30
Asp Tyr
<210> 59
<211> 41
<212> PRT
<213> Homo sapiens
<400> 59
Gly Phe Thr Phe Ser Ser Tyr Gly Met His Val Ile Ser Tyr Asp Gly
1 5 10 15
Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly Asp His Gly Gly Arg
20 25 30
Tyr Val Tyr Asp Tyr Gly Met Asp Val
35 40
<210> 60
<211> 34
<212> PRT
<213> Homo sapiens
<400> 60
Gly Phe Thr Phe Ser Lys Tyr Gly Met His Val Leu Trp Tyr Asp Gly
1 5 10 15
Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly Asp Gly His Tyr Phe
20 25 30
Asp Tyr
<210> 61
<211> 34
<212> PRT
<213> Homo sapiens
<400> 61
Gly Phe Thr Phe Ser Ser Tyr Gly Met His Val Ile Trp Phe Asp Gly
1 5 10 15
CA 02501984 2005-04-11
WO 2004/035603 PCT/US2003/032243
19/44
Asn Asn Lys Phe Tyr Ala Asp Ser Val Lys Gly Ala Pro Ala Tyr Trp
20 25 30
Asp Tyr
<210> 62
<211> 27
<212> PRT
<213> Homo sapiens
<400> 62
Arg Ala Ser Gln Gly Ile Arg Asn Asp Leu Gly Ala Ala Ser Ser Leu
1 5 10 15
Gln Ser Leu Gln His Asn Thr Tyr Pro Pro Thr
20 25
<210> 63
<211> 27
<212> PRT
<213> Homo sapiens
<400> 63
Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala Ala Ala Ser Thr Leu
1 5 10 15
Gln Arg Gln Gln Ala Asn Ser Phe Pro Phe Thr
20 25
<210> 64
<211> 29
<212> PRT
<213> Homo sapiens
<400> 64
Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Val Ala Leu Ala Ala Ser
1 5 10 15
Ser Leu Gln Arg Gln Gin Ala Asn Ser Phe Pro Phe Thr
20 25
<210> 65
<211> 27
<212> PRT
<213> Homo sapiens
<400> 65
Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala Ala Ala Ser Ser Leu
1 5 10 15'
Gln Arg Gln Gin Ala Asn Ser Phe Pro Phe Thr
20 25
<210> 66
<211> 27
<212> PRT
<213> Homo sapiens
<400> 66
Arg Ala Ser Gln Gly Ile Gly Ser Trp Leu Ala Ala Ala Ser Ser Leu
CA 02501984 2005-04-11
WO 2004/035603 PCT/US2003/032243
20/44
1 5 10 15
Gln Arg Gln Gln Ala Asn Ser Phe Pro Phe Thr
20 25
<210> 67
<211> 32
<212> PRT
<213> Homo sapiens
<400> 67
Arg Ser Arg Gln Ser Leu Val His Ser Asp Gly Asn Thr Tyr Leu Asn
1 5 10 15
Lys Thr Sex Asn Arg Phe Ser Met Gln Ala Thr Gln Phe Pro Ile Thr
20 25 30
<210> 68
<211> 28
<212> PRT
<213> Homo sapiens
<400> 68
Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala Gly Ala Ser Ser
1 5 10 15
Arg Ala Thr Gln Gln Tyr Gly Ser Ser Pro Trp Thr
20 25
<210> 69
<211> 1990
<212> DNA
<213> Homo sapiens
<400> 69
atgaagcatc tgtggttctt ccttctccta gtggcagctc ccagatgggt cctgtcccag 60
gtgcagctgc aggagtcggg cccaggactg gtgaagcctt cggagaccct gtccctcacc 120
tgcactgtct ctggtgcctc catcagtagt tactactgga gctggatccg gcagccccca 180
gggaagggac tggagtggat tgggtatatc tattacagtg ggagcaccaa ctacaacccc 240
tccctcaaga gtcgagtcac catatcagta gacacgtcca agaaccagtt ctccctgaag 300
ctgaggtctg tgaccgctgc ggacacggcc gtgtattact gtgcgagaga gcgactgggg 360
atcggggact actggggcca aggaaccctg gtcaccgtct cctcagcctc caccaagggc 420
ccatcggtct tccccctggc gccctgctct agaagcacct ccgagagcac agccgccctg 480
ggctgcctgg tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgct 540
ctgaccagcg gcgtgcacac cttcccagct gtcctacagt cctcaggact ctactccctc 600
agcagcgtgg tgaccgtgcc ctccagcaac ttcggcaccc agacctacac ctgcaacgta 660
gatcacaagc ccagcaacac caaggtggac aagacagttg gtgagaggcc agctcaggga 720
gggagggtgt ctgctggaag ccaggctcag ccctcctgcc tggacgcacc ccggctgtgc 780
agccccagcc caaggcagca aggcaggccc catctgtctc ctcccccgga ggcctctgcc 840
cgccccactc atgctcaggg agagggtctt ctggcttttt ccaccaggct ccaggcaggc 900
acaggctggg tgcccctacc ccaggccctt cacacacagg ggcaggtgct tggctcagac 960
ctgccaaaag ccatatccgg gaggaccctg cccctgccct aagccgaccc caaaggccaa 1020
actgtccact ccctcagctc ggacaccttc tctcctccca gatccgagta actcccaatc 1080
ttctctctgc agagcgcaaa tgttgtgtcg agtgcccacc gtgcccaggt aagccagccc 1140
aggcctcgcc ctccagctca aggcgggaca ggtgccctag agtagcctgc atccagggac 1200
aggccccagC tgggtgctga cacgtccacc tccatctctt cctcagcctc acctgtggca 1260
ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 1320
cctgaggtca cgtgcgtggt ggtggacgtg agccacgaag accccgaggt ccagttcaac 1380
tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccacggga ggagcagttc 1440
aacagcacgt tccgtgtggt ctgcgtcctc accgttgtgc accaggactg gctgaacggc 1500
aaggagtaca agtgcaaggt ctccagcaaa ggcctcccag cccccatcga gaaaaccatc 1560
CA 02501984 2005-04-11
WO 2004/035603 PCT/US2003/032243
21/44
tccaaaacca aaggtgggac ccgcggggta tgagggccac atggacagag gccggctcgg 1620
cccaccctct gccctgggag tgaccgctgt gccaacctct gtccctacag ggcagccccg 1680
agaaccacag gtgtacaccc tgcccccatc ccgggaggag atgaccaaga accaggtcag 1740
cctgacctgc ctggtcaaag gcttctaccc cagcgacatc gccgtggagt gggagagcaa 1800
tgggcagccg gagaacaact acaagaccac acctcccatg ctggactccg acggctcctt 1860
cttcctctac agcaagctca ccgtggacaa gagcaggtgg cagcagggga acttcttctc 1920
atgctccgtg atgcatgagg ctctgcacaa ccactacacg cagaagagcc tctccctgtc 1980
tccgggtaaa 1990
<210> 70
<211> 1990
<212> DNA
<213> Homo sapiens
<400> 70
tttacccgga gacagggaga ggctcttctg cgtgtagtgg ttgtgcagag cctcatgcat 60
cacggagcat gagaagacgt tcccctgctg ccacctgctc ttgtccacgg tgagcttgct 120
gtagaggaag aaggagccgt cggagtccag catgggaggt'gtggtcttgt agttgttctc 180
cggctgccca ttgctctccc actccacggc gatgtcgctg gggtagaagc ctttgaccag 240
gcaggtcagg ctgacctggt tcttggtcat ctcctcccgg gatgggggca gggtgtacac 300
ctgtggttct cggggctgcc ctgtagggac agaggttggc acagcggtca ctcccagggc 360
agagggtggg ccgagccggc ctctgtccat gtggccctca taccccgcgg gtcccacctt 420
tggttttgga gatggttttc tcgatggggg ctgggaggcc tttgttggag accttgcact 480
tgtactcctt gccgttcagc cagtcctggt gcacaacggt gaggacgctg accacacgga 540
acgtgctgtt gaactgctcc tcccgtggct ttgtcttggc attatgcacc tccacgccgt 600
ccacgtacca gttgaactgg acctcggggt cttcgtggct cacgtccacc accacgcacg 660
tgacctcagg ggtccgggag atcatgaggg tgtccttggg ttttgggggg aagaggaaga 720
ctgacggtcc tgccacaggt ggtgctgagg aagagatgga ggtggacgtg tcagcaccca 780
gctggggcct gtccctggat gcaggctact ctagggcacc tgtcccgcct tgagctggag 840
ggcgaggcct gggctggctt acctgggcac ggtgggcact cgacacaaca tttgcgctct 900
gcagagagaa gattgggagt tactcggatc tgggaggaga gaaggtgtcc gagctgaggg 960
agtggacagt ttggcctttg gggtcggctt aggtcagggg cagggtcctc ccggatatgg 1020
cttttggcag gtctgagcca agcacctgcc cctgtgtgtg aagggcctgg ggtaggggca 1080
cccagcctgt gcctgcctgg agcctggtgg aaaaagccag aagaccctct ccctgagcat 1140
gagtggggcg ggcagaggcc tccgggtgag gagacagatg gggcctgcct tgctgccctg 1200
ggctggggct gcacagccgg ggtgcgtcca ggcaggaggg ctgagcctgg cttccagcag 1260
acaccctccc tccctgagct ggcctctcac caactgtctt gtccaccttg gtgttgctgg 1320
gcttgtgatc tacgttgcag gtgtaggtct gggtgccgaa gttgctggag ggcacggtca 1380
ccacgctgct gagggagtag agtcctgagg actgtaggac agctgggaag gtgtgcacgc 1440
cgctggtcag agcgcctgag ttccacgaca ccgtcaccgg ttcggggaag tagtccttga 1500
ctagggagcc cagggcggct gtgctctcgg aggtgcttct agagcagggc gccaggggga 1560
agaccgatgg gcccttggtg gaggctgagg agacggtgac cagggttcct tggccccagt 1620
agtccccgat ccccagtcgc tctctcgcac agtaatacac ggccgtgtcc gcagcggtca 1680
cagacctcag cttcagggag aactggttct tggacgtgtc tactgatatg gtgactcgac 1740
tcttgaggga ggggttgtag ttggtgctcc cactgtaata gatataccca atccactcca 1800
gtcccttccc tgggggctgc cggatccagc tccagtagta actactgatg gaggcaccag 1860
agacagtgca ggtgagggac agggtctccg aaggcttcac cagtcctggg cccgactcct 1920
gcagctgcac ctgggacagg acccatctgg gagctgccac taggagaagg aagaaccaca 1980
gatgcttcat 1990
<210> 71
<211> 241
<212> PRT
<213> Homo sapiens
<400> 71
Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Leu Ala Ala Pro
1 5 10 15
Arg Trp Val Leu Ser Gin Val Gln Leu Gln Glu Ser Gly Pro Gly Leu
20 25 30
Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ala
CA 02501984 2005-04-11
WO 2004/035603 PCT/US2003/032243
22/44
35 40 45
Ser Ile Ser Ser Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys
50 55 60
Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr
65 70 75 80
Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Ala Ser Pro Thr
85 90 95
Ser Lys Asn Gln Phe Ser Leu Lys Leu Arg Ser Val Thr Ala Ala Asp
100 105 110
Thr Ala Val Tyr Tyr Cys Ala Arg Glu Arg Leu Gly Ile Gly Asp Tyr
115 120 125
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
130 135 140
Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser
145 150 155 160
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
165 170 175
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
180 185 190
Pro Ala Val Leu Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
195 200 205
Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val
210 215 220
Ala Ser Pro His Lys Pro Ser Asn Thr Lys Val Ala Ser Pro Lys Thr
225 230 235 240
Val
<210> 72
<211> 12
<212> PRT
<213> Homo sapiens
<400> 72
Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro
1 5 10
<210> 73
<211> 115
<212> PRT
<213> Homo sapiens
<400> 73
Ala Pro Pro Val Ala Leu Ala Gly Pro Ser Val Phe Leu Phe Pro Pro
1 5 10 15
Lys Pro Lys Asp Thr Leu Net Ile Ser Arg Thr Pro Glu Val Thr Cys
20 25 30
Val Val Val Ala Ser Pro Val Ser His Glu Asp Pro Glu Val Gln Phe
35 40 45
Asn Trp Tyr Val Ala Ser Pro Gly Val Glu Val His Asn Ala Lys Thr
50 55 60
Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val
65 70 75 80
Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
85 90 95
Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
100 105 110
Lys Thr Lys
115
CA 02501984 2005-04-11
WO 2004/035603 PCT/US2003/032243
23/44
<210> 74
<211> 107
<212> PRT
<213> Homo sapiens
<400> 74
Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Glu
1 5 10 15
Glu Met Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe
20 25 30
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu
35 40 45
Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe
50 55 60
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly
65 70 75 80
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
85 90 95
Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys
100 105
<210> 75
<211> 310
<212> PRT
<213> Homo sapiens
<400> 75
Ser Thr Sex Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
1 5 10 15
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
20 25 30
Gly Val His Thr Phe Pro Ala Val Leu Gin Ser Ser Gly Leu Tyr Ser
35 40 45
Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gin Thr
50 55 60
Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
65 70 75 80
Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro
85 90 95
Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
100 105 110
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
115 120 125
Val Ser His Glu Asp Pro Glu Val Gin Phe Asn Trp Tyr Val Asp Giy
130 135 140
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Phe Asn
145 150 155 160
Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gin Asp Trp
165 170 175
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
180 185 190
Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gin Pro Arg Glu
195 200 205
Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
210 215 220
Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
225 230 235 240
Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr
CA 02501984 2005-04-11
WO 2004/035603 PCT/US2003/032243
24/44
245 250 255
Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
260 265 270
Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys
275 280 285
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu
290 295 300
Ser Leu Ser Pro Gly Lys
305 310
<210> 76
<211> 552
<212> DNA
<213> Homo sapiens
<400> 76
atgagggtcc ccgctcagct cctggggctc ctgctgctct ggttcccagg tgccaggtgt 60
aagcttgaca tccagctgac ccaatctcca tcctccctgt ctgcatctgt aggagacaga 120
gtcaccatca cttgccgggc aagtcagggc attagaaatg atttaggctg gtatcagcag 180
aaaccaggga aagcccctaa gcgcctgatc tatgctgcat ccagtttgca aagtggggtc 240
ccatcaaggt tcagcggcag tggatctggg acagaattca ctctcacaat cagcagcctg 300
cagcctgaag attttgcaac ttattactgt ctacagcata atacttaccc tccgacgttc 360
ggccaaggga ccaaggtgga aatcaaacga actgtggctg caccatctgt cttcatcttc 420
ccgccatctg atgagcagtt gaaatctgga actgctagcg ttgtgtgcct gctgaataac 480
ttctatccca gagaggccaa agtacagtgg aaggtggata acgccctcca atcgggtaac 540
tcccaggaga gt 552
<210> 77
<211> 552
<212> DNA
<213> Homo sapiens
<400> 77
actctcctgg gagttacccg attggagggc gttatccacc ttccactgta ctttggcctc 60
tctgggatag aagttattca gcaggcacac aacgctagca gttccagatt tcaactgctc 120
atcagatggc gggaagatga agacagatgg tgcagccaca gttcgtttga tttccacctt 180
ggtcccttgg ccgaacgtcg gagggtaagt attatgctgt agacagtaat aagttgcaaa 240
atcttcaggc tgcaggctgc tgattgtgag agtgaattct gtcccagatc cactgccgct 300
gaaccttgat gggaccccac tttgcaaact ggatgcagca tagatcaggc gcttaggggc 360
tttccctggt ttctgctgat accagcctaa atcatttcta atgccctgac ttgcccggca 420
agtgatggtg actctgtctc ctacagatgc agacagggag gatggagatt gggtcagctg 480
gatgtcaagc ttacacctgg cacctgggaa ccagagcagc aggagcccca ggagctgagc 540
ggggaccctc at 552
<210> 78
<211> 184
<212> PRT
<213> Homo sapiens
<400> 78
Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp Phe Pro
1 5 10 15
Gly Ala Arg Cys Lys Leu Asp Ile Gin Leu Thr Gin Ser Pro Ser Ser
20 25 30
Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
35 40 45
Gin Gly Ile Arg Asn Asp Leu Gly Trp Tyr Gin Gin Lys Pro Gly Lys
50 55 60
Ala Pro Lys Arg Leu Ile Tyr Ala Ala Ser Ser Leu Gin Ser G1y Val
65 70 75 80
CA 02501984 2005-04-11
WO 2004/035603 PCT/US2003/032243
25/44
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr
85 90 95
Ile Ser Ser Leu Gin Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gin
100 105 110
His Asn Thr Tyr Pro Pro Thr Phe Gly Gin Gly Thr Lys Val Glu Ile
115 120 125
Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
130 135 140
Glu Gin Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
145 150 155 160
Phe Tyr Pro Arg Glu Ala Lys Val Gin Trp Lys Val Asp Asn Ala Leu
165 170 175
Gin Ser Gly Asn Ser Gln Glu Ser
180
<210> 79
<211> 31
<212> PRT
<213> Homo sapiens
<400> 79
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gin
1 5 10 15
Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin Glu Ser
20 25 30
<210> 80
<211> 2011
<212> DNA
<213> Homo sapiens
<400> 80
atggaattgg ggctccgttg ggttttcctc gttgctcttt taagaggtgt ccagtgtcag 60
gtgcagctgg tggagtctgg gggaggcgtg gtccagcctg ggaggtccct gagactctcc 120
tgtgtagcct ctggattcac cttcagtagc tatggcatgc actgggtccg ccaggctcca 180
ggcaaggggc tggagtgggt ggcagttata tcatatgatg gaagtaataa atactatgca 240
gactccgtga agggccgatt caccatctcc agagacaatt ccaagaacac gctgtatctg 300
caaatgaaca gcctgagagt tgaggacacg gctgtgtatt actgtgcgag agatcacggt 360
gggaggtacg tctacgacta cggtatggac gtctggggcc aagggaccac ggtcaccgtc 420
tcctcagcct ccaccaaggg cccatcggtc ttccccctgg cgccctgctc tagaagcacc 480
tccgagagca cagccgcoct gggctgcctg gtcaaggact acttccccga accggtgacg 540
gtgtcgtgga actcaggcgc tctgaccagc ggcgtgcaca ccttcccagc tgtcctacag 600
tcctcaggac tctactccct cagcagcgtg gtgaccgtgc cctccagcaa cttcggcacc 660
cagacctaca cctgcaacgt agatcacaag cccagcaaca ccaaggtgga caagacagtt 720
ggtgagaggc cagctcaggg agggagggtg tctgctggaa gccaggctca gccctcctgc 780
ctggacgcac cccggctgtg cagccccagc ccagggcagc aaggcaggcc ccatctgtct 840
cctcacccgg aggcctctgc ccgccccagt catgctcagg gagagggtct tctggctttt 900
tccaccaggc tccaggcagg cacaggctgg gtgcccctac cccaggccct tcacacacag 960
gggcaggtgc ttggctcaga cctgccaaaa gccatatccg ggaggaccct gcccctgacc 1020
taagccgacc ccaaaggcca aactgtccac tccctcagct cggacacctt ctctcctccc 1080
agatccgagt aactcccaat cttctctctg cagagcgcaa atgttgtgtc gagtgcccac 1140
cgtgcccagg taagccagcc caggcctcgc cctccagctc aaggcgggac aggtgcccta 1200
gagtagcctg catccaggga caggccccag ctgggtgctg acacgtccac ctccatctct 1260
tcctcagccc cacctgtggc aggaccgtca gtcttcctct tccccccaaa acccaaggac 1320
accctcatga tctcccggac ccctgaggtc acgtgcgtgg tggtggacgt gagccacgaa 1380
gaccccgagg tccagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca 1440
aagccaaggg aggagcagtt caacagcacg ttccgtgtgg tcagcgtcct caccgttgtg 1500
caccaggact ggctgaacgg caaggagtac aagtgcaagg tctccaacaa aggcctccca 1560
gcccccatcg agaaaaccat ctccaaaacc aaaggtggga cccgcggggt atgagggcca 1620
CA 02501984 2005-04-11
WO 2004/035603 PCT/US2003/032243
26/44
catggacaga ggccggctcg gcccaccctc tgccctggga gtgaccgctg tgccaacctc 1680
tgtccctaca gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggagga 1740
gatgaccaag aaccaggtca gcctgccctg cctggtcaaa ggcttctacc ccagcgacat 1800
cgccgtggag tgggagagca atgggcagcc ggagaacaac tacaagacca cacctcccat 1860
gctggactcc gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg 1920
gcagcagggg aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac 1980
gcagaagagc ctctccctgt ctccgggtaa a 2011
<210> 81
<211> 2011
<212> DNA
<213> Homo sapiens
<400> 81
tttacccgga gacagggaga ggctcttctg cgtgtagtgg ttgtgcagag cctcatgcat 60
cacggagcat gagaagacgt tcccctgctg ccacctgctc ttgtccacgg tgagcttgct 120
gtagaggaag aaggagccgt cggagtccag catgggaggt gtggtcttgt agttgttctc 180
cggctgccca ttgctctccc actccacggc gatgtcgctg gggtagaagc ctttgaccag 240
gcaggtcagg ctgacctggt tcttggtcat ctcctcccgg gatgggggca gggtgtacac 300
ctgtggttct cggggctgcc ctgtagggac agaggttggc acagcggtca ctcccagggc 360
agagggtggg ccgagccggc ctctgtccat gtggccctca taccccgcgg gtcccacctt 420
tggttttgga gatggttttc tcgatggggg ctgggaggcc tttgttggag accttgcact 480
tgtactcctt gccgttcagc cagtcctggt gcacaacggt gaggacgctg accacacgga 540
acgtgctgtt gaactgctcc tcccgtggct ttgtcttggc attatgcacc tccacgccgt 600
ccacgtacca gttgaactgg acctcggggt cttcgtggct cacgtccacc accacgcacg 660
tgacctcagg ggtccgggag atcatgaggg tgtccttggg ttttgggggg aagaggaaga 720
ctgacggtcc tgccacaggt ggtgctgagg aagagatgga ggtggacgtg tcagcaccca 780
gctggggcct gtccctggat gcaggctact ctagggcacc tgtcccgcct tgagctggag 840
ggcgaggcct gggctggctt acctgggcac ggtgggcact cgacacaaca tttgcgctct 900
gcagagagaa gattgggagt tactcggatc tgggaggaga gaaggtgtcc gagctgaggg 960
agtggacagt ttggcctttg gggtcggctt aggtcagggg cagggtcctc ccggatatgg 1020
cttttggcag gtctgagcca agcacctgcc cctgtgtgtg aagggcctgg ggtaggggca 1080
cccagcctgt gcctgcctgg agcctggtgg aaaaagccag aagaccctct ccctgagcat 1140
gagtggggcg ggcagaggcc tccgggtgag gagacagatg gggcctgcct tgctgccctg 1200
ggctggggct gcacagccgg ggtgcgtcca ggcaggaggg ctgagcctgg cttccagcag 1260
acaccctccc tccctgagct ggcctctcac caactgtctt gtccaccttg gtgttgctgg 1320
gcttgtgatc tacgttgcag gtgtaggtct gggtgccgaa gttgctggag ggcacggtca 1380
ccacgctgct gagggagtag agtcctgagg actgtaggac agctgggaag gtgtgcacgc 1440
cgctggtcag agcgcctgag ttccacgaca ccgtcaccgg ttcggggaag tagtccttga 1500
ccaggcagcc cagggcggct gtgctctcgg aggtgcttct agagcagggc gccaggggga 1560
agaccgatgg gcccttggtg gaggctgagg agacggtgac cgtggtccct tggccccaga 1620
cgtccatacc gtagtcgtag acgtacctcc caccgtgatc tctcgcacag taatacacag 1680
ccgtgtcctc aactctcagg ctgttcattt gcagatacag cgtgttcttg gaattgtctc 1740
tggagatggt gaatcggccc ttcacggagt ctgcatagta tttattactt ccatcatatg 1800
atataactgc cacccactcc agccccttgc ctggagcctg gcggacccag tgcatgccat 1860
agctactgaa ggtgaatcca gaggctacac aggagagtct cagggacctc ccaggctgga 1920
ccacgcctcc cccagactcc accagctgca cctgacactg gacacctctt aaaagagcaa 1980
cgaggaaaac ccagcggagc cccaattcca t 2011
<210> 82
<211> 252
<212> PRT
<213> Homo sapiens
<400> 82
Met Glu Leu Gly Leu Arg Trp Val Phe Leu Val Ala Leu Ala Leu Leu
1 5 10 15
Arg Gly Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val
20 25 30
Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Val Ala Leu Ala Ser
35 40 45
CA 02501984 2005-04-11
WO 2004/035603 PCT/US2003/032243
27/44
Gly Phe Thr Phe Ser Ser Tyr Gly Met His Trp Val Ala Arg Gly Gln
50 55 60
Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Leu Ala Val Ile Ser Tyr
65 70 75 80
Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
85 90 95
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser
100 105 110
Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp His Gly
115 120 125
Gly Arg Tyr Val Tyr Asp Tyr Gly Met Asp Val Trp Gly Gln Gly Thr
130 135 140
Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
145 150 155 160
Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly
165 170 175
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
180 185 190
Ser Gly Ala Leu Thr Her Gly Val His Thr Phe Pro Ala Val Leu Gln
195 200 205
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
210 215 220
Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Ala Ser Pro His Lys
225 230 235 240
Pro Ser Asn Thr Lys Val Ala Ser Pro Lys Thr Val
245 250
<210> 83
<211> 752
<212> DNA
<213> Homo sapiens
<400> 83
atgagggtcc ccgctcagct cctggggctc ctgctgctct ggttcccagg ttccagatgc 60
gacatccaga tgacccaatc tccatcttcc gtgtctgcat ctataggaga cagagtctcc 120
atcacttgtc gggcgagtca gggtattagc agctggttag cctggtatca gcagaaacca 180
gggaaagccc ctacgctcct tatctatgct gcatccactt tgcaacgtgg ggtcccatca 240
aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 300
gaagattttg caacttactt ttgtcaacag gctaacagtt tcccattcac tttcggccct 360
gggaccaaag tggatatcaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcca 420
tctgatgagc agttgaaatc tggaactgct agcgttgtgt gcctgctgaa taacttctat 480
cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 540
gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 600
ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 660
ctgagctcgc ccgtcacaaa gagcttcaac aggggaagtg ggtagtcccg gactcgagcg 720
ggcagtgttt ctcgaagttg tcccctgagt gt 752
<210> 84
<211> 752
<212> DNA
<213> Homo sapiens
<400> 84
acactcaggg gacaacttcg agaaacactg cccgctcgag tccgggacta cccacttccc 60
ctgttgaagc tctttgtgac gggcgagctc aggccctgat gggtgacttc gcaggcgtag 120
actttgtgtt tctcgtagtc tgctttgctc agcgtcaggg tgctgctgag gctgtaggtg 180
ctgtccttgc tgtcctgctc tgtgacactc tcctgggagt tacccgattg gagggcgtta 240
tccaccttcc actgtacttt ggcctctctg ggatagaagt tattcagcag gcacacaacg 300
ctagcagttc cagatttcaa ctgctcatca gatggcggga agatgaagac agatggtgca 360
gccacagttc gtttgatatc cactttggtc ccagggccga aagtgaatgg gaaactgtta 420
CA 02501984 2005-04-11
WO 2004/035603 PCT/US2003/032243
28/44
gcctgttgac aaaagtaagt tgcaaaatct tcaggctgca ggctgctgat ggtgagagtg 480
aaatctgtcc cagatccact gccgctgaac cttgatggga ccccacgttg caaagtggat 540
gcagcataga taaggagcgt aggggctttc cctggtttct gctgatacca ggctaaccag 600
ctgctaatac cctgactcgc ccgacaagtg atggagactc tgtctcctat agatgcagac 660
acggaagatg gagattgggt catctggatg tcgcatctgg aacctgggaa ccagagcagc 720
aggagcccca ggagctgagc ggggaccctc at 752
<210> 85
<211> 234
<212> PRT
<213> Homo sapiens
<400> 85
Met Arg Val Pro Ala Gin Leu Leu Gly Leu Leu Leu Leu Trp Phe Pro
1 5 10 15
Gly Ser Arg Cys Asp Ile Gin Met Thr Gin Ser Pro Ser Ser Val Ser
20 25 30
Ala Ser Ile Gly Asp Arg Val Ser Ile Thr Cys Arg Ala Ser Gin Gly
35 40 45
Ile Ser Ser Trp Leu Ala Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro
50 55 60
Thr Leu Leu Ile Tyr Ala Ala Ser Thr Leu Gin Arg Gly Val Pro Ser
65 70 75 80
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
85 90 95
Ser Leu Gin Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gin Gin Ala Asn
100 105 110
Ser Phe Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg
115 120 125
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Giu Gin
130 135 140
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
145 150 155 160
Pro Arg Glu Ala Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser
165 k170 175
Gly Asn Ser Gin Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
180 185 190
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
195 200 205
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
210 215 220
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
225 230
<210> 86
<211> 1990
<212> DNA
<213> Homo sapiens
<400> 86
atgaagcatc tgtggttctt ccttctcctg gtggcagctc ccagatgggt cctttcccag 60
gtgcagctgc aggagtcggg cccaggactg gtgaagcctt cggagaccct gtccctcacc 120
tgcactgtct ctggtgcctc catcagtaat tactactgga gctggatccg gcagccccca 180
gggaagggac tggagtggat tgggtatgtc tcttacagtg ggagtacgta ctaCaacccc 240
tccctcaagg gtcgagtcac catgtcagta gacacgtcca agaaccagtt ctccctgaag 300
ctgagctctg tgaccgctgc ggacacggcc gtgtattact gtgcgagaga aaaactgggg 360
attggagact actggggcca gggaaccctg gtcaccgtct cctcagcctc caccaagggc 420
ccatcggtct tccccctggc gccctgctct agaagcacct ccgagagcac agccgccctg 480
ggctgcctgg tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgct 540
ctgaccagcg gcgtgcacac cttcccagct gtcctacagt cctcaggact ctactccctc 600
CA 02501984 2005-04-11
WO 2004/035603 PCT/US2003/032243
29/44
agcagcgtgg tgaccgtgcc ctccagcaac ttcggcaccc agacciacac ctgcaacgta 660
gatcacaagc ccagcaacac caaggtggac aagacagttg gtgagaggcc agctcaggga 720
gggagggtgt ctgctggaag ccaggctcag ccctcctgcc tggacgcacc ccggctgtgc 780
agccccagcc cagggcagca aggcaggccc catctgtctc ctcacccgga ggcctctgcc 840
cgccccactc atgctcaggg agagggtctt ctggcttttt ccaccaggct ccaggcaggc 900
acaggctggg tgcccctacc ccaggccctt cacacacagg ggcaggtgct tggctcagac 960
ctgccaaaag ccatatccgg gaggaccctg cccctgacct aagccgaccc caaaggccaa 1020
actgtccact ccctcagctc ggacaccttc tctcctccca gatccgagta actcccaatc 1080
ttctctctgc agagcgcaaa tgttgtgtcg agtgcccacc gtgcccaggt aagccagccc 1140
aggcctcgcc ctccagctca aggcgggaca ggtgccctag agtagcctgc atccagggac 1200
aggccccagc tgggtgctga cacgtccacc tccatctctt cctcagcacc acctgtggca 1260
ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 1320
cctgaggtca cgtgcgtggt ggtggacgtg agccacgaag accccgaggt ccagttcaac 1380
tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccacggga ggagcagttc 1440
aacagcacgt tccgtgtggt cagcgtcctc accgttgtgc accaggactg gctgaacggc 1500
aaggagtaca agtgcaaggt ctccaacaaa ggcctcccag cccccatcga gaaaaccatc 1560
tccaaaacca aaggtgggac ccgcggggta tgagggccac atggacagag gccggctcgg 1620
cccaccctct gccctgggag tgaccgctgt gccaacctct gtccctacag ggcagccccg 1680
agaaccacag gtgtacaccc tgcccccatc ccgggaggag atgaccaaga accaggtcag 1740
cctgacctgc ctggtcaaag gcttctaccc cagcgacatc gccgtggagt gggagagcaa 1800
tgggcagccg gagaacaact acaagaccac acctcccatg ctggactccg acggctcctt 1860
cttcctctac agcaagctca ccgtggacaa gagcaggtgg cagcagggga acgtcttctc 1920
atgctccgtg atgcatgagg ctctgcacaa ccactacacg cagaagagcc tctccctgtc 1980
tccgggtaaa 1990
<210> 87
<211> 1990
<212> DNA
<213> Homo sapiens
<400> 87
tttacccgga gacagggaga ggctcttctg cgtgtagtgg ttgtgcagag cctcatgcat 60
cacggagcat gagaagacgt tcccctgctg ccacctgctc ttgtccacgg tgagcttgct 120
gtagaggaag aaggagccgt cggagtccag catgggaggt gtggtcttgt agttgttctc 180
cggctgccca ttgctctccc actccacggc gatgtcgctg gggtagaagc ctttgaccag 240
gcaggtcagg ctgacctggt tcttggtcat ctcctcccgg gatgggggca gggtgtacac 300
ctgtggttct cggggctgcc ctgtagggac agaggttggc acagcggtca ctcccagggc 360
agagggtggg ccgagccggc ctctgtccat gtggccctca taccccgcgg gtcccacctt 420
tggttttgga gatggttttc tcgatggggg ctgggaggcc tttgttggag accttgcact 480
tgtactcctt gccgttcagc cagtcctggt gcacaacggt gaggacgctg accacacgga 540
acgtgctgtt gaactgctcc tcccgtggct ttgtcttggc attatgcacc tccacgccgt 600
ccacgtacca gttgaactgg acctcggggt cttcgtggct cacgtccacc accacgcacg 660
tgacctcagg ggtccgggag atcatgaggg tgtccttggg ttttgggggg aagaggaaga 720
ctgacggtcc tgccacaggt ggtgctgagg aagagatgga ggtggacgtg tcagcaccca 780
gctggggcct gtccctggat gcaggctact ctagggcacc tgtcccgcct tgagctggag 840
ggcgaggcct gggctggctt acctgggcac ggtgggcact cgacacaaca tttgcgctct 900
gcagagagaa gattgggagt tactcggatc tgggaggaga gaaggtgtcc gagctgaggg 960
agtggacagt ttggcctttg gggtcggctt aggtcagggg cagggtcctc ccggatatgg 1020
cttttggcag gtctgagcca agcacctgcc cctgtgtgtg aagggcctgg ggtaggggca 1080
cccagcctgt gcctgcctgg agcctggtgg aaaaagccag aagaccctct ccctgagcat 1140
gagtggggcg ggcagaggcc tccgggtgag gagacagatg gggcctgcct tgctgccctg 1200
ggctggggct gcacagccgg ggtgcgtcca ggcaggaggg ctgagcctgg cttccagcag 1260
acaccctccc tccctgagct ggcctctcac caactgtctt gtccaccttg gtgttgctgg 1320
gcttgtgatc tacgttgcag gtgtaggtct gggtgccgaa gttgctggag ggcacggtca 1380
ccacgctgct gagggagtag agtcctgagg actgtaggac agctgggaag gtgtgcacgc 1440
cgctggtcag agcgcctgag ttccacgaca ccgtcaccgg ttcggggaag tagtccttga 1500
ccaggcagcc cagggcggct gtgctctcgg aggtgcttct agagcagggc gccaggggga 1560
agaccgatgg gcccttggtg gaggctgagg agacggtgac cagggttccc tggccccagt 1620
agtctccaat ccccagtttt tctctcgcac agtaatacac ggccgtgtcc gcagcggtca 1680
cagagctcag cttcagggag aactggttct tggacgtgtc tactgacatg gtgactcgac 1740
ccttgaggga ggggttgtag tacgtactcc cactgtaaga gacataccca atccactcca 1800
CA 02501984 2005-04-11
WO 2004/035603 PCT/US2003/032243
30/44
gtcccttccc tgggggctgc cggatccagc tccagtagta attactgatg gaggcaccag 1860
agacagtgca ggtgagggac agggtctccg aaggcttcac cagtcctggg cccgactcct 1920
gcagctgcac ctgggacagg acccatctgg gagctgccac caggagaagg aagaaccaca 1980
gatgcttcat 1990
<210> 88
<211> 241
<212> PRT
<213> Homo sapiens
<400> 88
Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Leu Ala Ala Pro
1 5 10 15
Arg Trp Val Leu Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu
20 25 30
Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ala
35 40 45
Ser Ile Ser Asn Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys
50 55 60
Gly Leu Glu Trp Ile Gly Tyr Val Ser Tyr Ser Gly Ser Thr Tyr Tyr
65 70 75 80
Asn Pro Ser Leu Lys Gly Arg Val Thr Met Ser Val Ala Ser Pro Thr
85 90 95
Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp
100 105 110
Thr Ala Val Tyr Tyr Cys Ala Arg Glu Lys Leu Gly Ile Gly Asp Tyr
115 120 125
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
130 135 140
Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser
145 150 155 160
Thr Ala Ala Leu G1y Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
165 170 175
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
180 185 190
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
195 200 205
Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val
210 215 220
Ala Ser Pro His Lys Pro Ser Asn Thr Lys Val Ala Ser Pro Lys Thr
225 230 235 240
Val
<210> 89
<211> 702
<212> DNA
<213> Homo sapiens
<400> 89
atgaggctcc ccgctcagct cctggggctc ctgctgctct ggttcccagg tgccaggtgt 60
gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 120
atcacttgcc gggcaagtca gggcattaaa aatgatttag gctggtatca gcagaaacca 180
gggaaagccc ctaagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 240
aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 300
gaagattttg caacttatta ctgtctacag cataatagtt atccgtgcag ttttggccag 360
gggaccaagc tggagatcaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcca 420
tctgatgagc agttgaaatc tggaactgct agcgttgtgt gcctgctgaa taacttctat 480
cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 540
gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 600
CA 02501984 2005-04-11
WO 2004/035603 PCT/US2003/032243
31/44
ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 660
ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gt 702
<210> 90
<211> 702
<212> DNA
<213> Homo sapiens
<400> 90
acactctccc ctgttgaagc tctttgtgac gggcgagctc aggccctgat gggtgacttc 60
gcaggcgtag actttgtgtt tctcgtagtc tgctttgctc agcgtcaggg tgctgctgag 120
gctgtaggtg ctgtccttgc tgtcctgctc tgtgacactc tcctgggagt tacccgattg 180
gagggcgtta tccaccttcc actgtacttt ggcctctctg ggatagaagt tattcagcag 240
gcacacaacg ctagcagttc cagatttcaa ctgctcatca gatggcggga agatgaagac 300
agatggtgca gccacagttc gtttgatctc cagcttggtc ccctggccaa aactgcacgg 360
ataactatta tgctgtagac agtaataagt tgcaaaatct tcaggctgca ggctgctgat 420
tgtgagagtg aattctgtcc cagatccact gccgctgaac cttgatggga ccccactttg 480
caaactggat gcagcataga tcaggcgctt aggggctttc cctggtttct gctgatacca 540
gcctaaatca tttttaatgc cctgacttgc ccggcaagtg atggtgactc tgtctcctac 600
agatgcagac agggaggatg gagactgggt catctggatg tcacacctgg cacctgggaa 660
ccagagcagc aggagcccca ggagctgagc ggggagcctc at 702
<210> 91
<211> 234
<212> PRT
<213> Homo sapiens
<400> 91
Met Arg Leu Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp Phe Pro
1 5 10 15
Gly Ala Arg Cys Asp Ile Gin Met Thr Gin Ser Pro Ser Ser Leu Ser
20 25 30
Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gin Gly
35 40 45
Ile Lys Asn Asp Leu Gly Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro
50 55 60
Lys Arg Leu Ile Tyr Ala Ala Ser Ser Leu Gin Ser Gly Val Pro Ser
65 70 75 80
Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser
85 90 95
Ser Leu Gin Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gin His Asn
100 105 110
Ser Tyr Pro Cys Ser Phe Gly Gin Gly Thr Lys Leu Glu Ile Lys Arg
115 120 125
Thr Val Ala Ala Pro Ser Val Phe Ile She Pro Pro Ser Asp Glu Gin
130 135 140
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn She Tyr
145 150 155 160
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gin Ser
165 170 175
Gly Asn Ser Gin Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr
180 185 190
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
195 200 205
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Giy Leu Ser Ser Pro
210 215 220
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
225 230
<210> 92
CA 02501984 2005-04-11
WO 2004/035603 PCT/US2003/032243
32/44
<211> 1996
<212> DNA
<213> Homo sapiens
<400> 92
atgaaacatc tgtggttctt cctcctgctg gtggcagctc ccagatgggt cctgtcccag 60
gtgcagctgc aggagtcggg cccaggactg gtgaagcctt cacagaccct gtccctcacc 120
tgcactgtct ctggtgcctc catcagcagt ggtgcttaot actggagttg gatccgccag 180
cacccaggga agggcctgga gtggattggg tacatctata agagtgagac ctcctactac 240
aacccgtccc tcaagagtcg acttacccta tcagtagaca cgtctaagaa ccagttctcc 300
ctgaacctga tctctgtgac tgccgcggac acggccgtgt attattgtgc gagagataaa 360
ctggggatcg cggactactg gggccaggga accctggtca ccgtctcctc agcctccacc 420
aagggcccat cggtcttccc cctggcgccc tgctctagaa gcacctccga gagcacagcc 480
gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 540
ggcgctctga ccaccggcgt gcacaccttc ccagttgtcc tacagtcctc aggactctac 600
tccctcagca gcgtggtgac cgtgccctcc agcaacttcg gcacccagac ctacacctgc 660
aacgtagatc acaagcccag caacaccaag gtggacaaga cagttggtga gaggccagct 720
cagggaggga gggtgtctgc tggaagccag gctcagccct cctgcctgga cgcaccccgg 780
ctgtgcagcc ccagcccagg gcagcaaggc aggccccatc tgtctcctca cccggaggcc 840
tctgcccgcc ccactcatgc tcagggagag ggtcttctgg ctttttccac caggctccag 900
gcaggcacag gctgggtgcc cctaccccag gcccttcaca cacaggggca ggtgcttggc 960
tcagacctgc caaaagccat atccgggagg accctgcccc tgacctaagc cgaccccaaa 1020
ggccaaactg tccactccct cagctcggac accttctctc ctcccagatc cgagtaactc 1080
ccaatcttct ctctgcagag cgcaaatgtt gtgtcgagtg cccaccgtgc ccaggtaagc 1140
cagcccaggc ctcgccctcc agctcaaggc gggacaggtg ccctagagta gcctgcatcc 1200
agggacaggc cccagctggg tgctgacacg tccacctcca tctcttcctc agcaccacct 1260
gtggcaggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 1320
cggacccctg aggtcacgtg cgtggtggtg gacgtgagcc acgaagaccc cgaggtccag 1380
ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc acgggaggag 1440
cagttcaaca gcacgttccg tgtggtcagc gtcctcaccg ttgtgcacca ggactggctg 1500
aacggcaagg agtacaagtg caaggtctcc aacaaaggcc tcccagcccc catcgagaaa 1560
accatctcca aaaccaaagg tgggacccgc ggggtatgag ggccacatgg acagaggccg 1620
gctcggccca ccctctgccc tgggagtgac cgctgtgcca acctctgtcc ctacagggca 1680
gccccgagaa ccacaggtgt acaccctgcc cccatcccgg gaggagatga ccaagaacca 1740
ggtcagcctg acctgcctgg tcaaaggctt ctaccccagc gacatcgccg tggagtggga 1800
gagcaatggg cagccggaga acaactacaa gaccacacct cccatgctgg actccgacgg 1860
ctccttcttc ctctacagca agctcaccgt ggacaagagc aggtggcagc aggggaacgt 1920
cttctcatgc tccgtgatgc atgaggctct gcacaaccac tacacgcaga agagcctctc 1980
cctgtctccg ggtaaa 1996
<210> 93
<211> 1996
<212> DNA
<213> Homo sapiens
<400> 93
tttacccgga gacagggaga ggctcttctg cgtgtagtgg ttgtgcagag cctcatgcat 60
cacggagcat gagaagacgt tcccctgctg ccacctgctc ttgtccacgg tgagcttgct 120
gtagaggaag aaggagccgt cggagtccag catgggaggt gtggtcttgt agttgttctc 180
cggctgccca ttgctctccc actccacggc gatgtcgctg gggtagaagc ctttgaccag 240
gcaggtcagg ctgacctggt tcttggtcat ctcctcccgg gatgggggca gggtgtacac 300
ctgtggttct cggggctgcc ctgtagtgac agaggttggc acagcggtca ctcccagggc 360
agagggtggg ccgagccggc ctctgtccat gtggccctca taccccgcgg gtcccacctt 420
tggttttgga gatggttttc tcgatggggg ctgggaggcc tttgttggag accttgcact 480
tgtactcctt gccgttcagc cagtcctggt gcacaacggt gaggacgctg accacacgga 540
acgtgctgtt gaactgctcc tcccgtggct ttgtcttggc attatgcacc tccacgccgt 600
ccacgtacca gttgaactgg acctcggggt cttcgtggct cacgtccacc accacgcacg 660
tgacctcagg ggtccgggag atcatgaggg tgtccttggg ttttgggggg aagaggaaga 720
ctgacggtcc tgccacaggt ggtgctgagg aagagatgga ggtggacgtg tcagcaccca 780
gctggggcct gtccctggat gcaggctact ctagggcacc tgtcccgcct tgagctggag 840
ggcgaggcct gggctggctt acctgggcac ggtgggcact cgacacaaca tttgcgctct 900
CA 02501984 2005-04-11
WO 2004/035603 PCT/US2003/032243
33/44
gcagagagaa gattgggagt tactcggatc tgggaggaga gaaggtgtcc gagctgaggg 960
agtggacagt ttggcctttg gggtcggctt agatcagggg cagggtcctc ccggatatgg 1020
cttttggcag gtctgagcca agcacctgcc cctgtgtgtg aagggcctgg ggtaggggca 1080
cccagcctgt gcctgcctgg agcctggtgg aaaaagccag aagaccctct ccctgagcat 1140
gagtggggcg ggcagaggcc tccgggtgag gagacagatg gggcctgcct tgctgccctg 1200
ggttggggct gcacagccgg ggtgcgtcca ggcaggaggg ctgagcctgg cttccagcag 1260
acaccctccc tccctgagct ggcctctcac caactgtctt gtccaccttg gtgttgctgg 1320
gcttgtgatc tacgttgcag gtgtaggtct gggtgccgaa gttgctggag ggcacggtca 1380
ccacgctgct gagggagtag agtcctgagg actgtaggac agctgggaag gtgtgcacgc 1440
cgctggtcag agcgcctgag ttccacgaca ccgtcaccgg ttcggggaag tagtccttga 1500
ccaggcagcc cagggcggct gtgctctcgg aggtgcttct agagcagggc gccaggggga 1560
agaccgatgg gcccttggtg gaggctgagg agacggtgac cagggttccc tggccccagt 1620
agtccgcgat ccccagttta tctctcgcac aataatacac ggccgtgtcc gcggcagtca 1680
cagagatcag gttcagggag aactggttct tagacgtgtc tactgatagg gtaagtcgac 1740
tcttgaggga cgggttgtag taggaggtct cactcttata gatgtaccca atccactcca 1800
ggcccttccc tgggtgctgg cggatccaac tccagtagta agcaccactg ctgatggagg 1860
caccagagac agtgcaggtg agggacaggg tctgtgaagg cttcaccagt cctgggcccg 1920
actcctgcag ctgcacctgg gacaggaccc atctgggagc tgccaccagc aggaggaaga 1980
accacagatg tttcat 1996
<210> 94
<211> 243
<212> PRT
<213> Homo sapiens
<400> 94
Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Leu Ala Ala Pro
1 5 10 15
Arg Trp Val Leu Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu
20 25 30
Val Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ala
35 40 45
Ser Ile Ser Ser Gly Ala Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro
50 55 60
Gly Lys Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Lys Ser Glu Thr Ser
65 70 75 80
Tyr Tyr, Asn Pro Ser Leu Lys Ser Arg Leu Thr Leu Ser Val Ala Ser
85 90 95
Pro Thr Sex Lys Asn Gln Phe Ser Leu Asn Leu Ile Ser Val Thr Ala
100 105 110
Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Lys Leu Gly Ile Ala
115 120 125
Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
130 135 140
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser
145 150 155 160
Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
165 170 175
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
180 185 190
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
195 200 205
Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys
210 215 220
Asn Val Ala Ser Pro His Lys Pro Ser Asn Thr Lys Val Ala Ser Pro
225 230 235 240
Lys Thr Val
<210> 95
CA 02501984 2005-04-11
WO 2004/035603 PCT/US2003/032243
34/44
<211> 702
<212> DNA
<213> Homo sapiens
<400> 95
atgagggtcc ccgctcagct cctggggctc ctgctgctct ggttcccagg cgccaggtgt 60
gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 120
atcacttgcc gggcaagtca ggacattaga aatgatttag gctggtatca gcagaaacca 180
gggaaagccc ctaagcgcct gatctatgct gcatccaatt tgcaaagtgg ggtcccatca 240
aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 300
gaagattttg caacttatta ctgtctacag cataatagct accctcccac tttcggcgga 360'
gggaccaagg tggaaatcaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcca 420
tctgatgagc agttgaaatc tggaactgct agcgttgtgt gcctgctgaa taacttctat 480
cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 540
gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 600
ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 660
ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gt 702
<210> 96
<211> 702
<212> DNA
<213> Homo sapiens
<400> 96
acactctccc ctgttgaagc tctttgtgac gggcgagctc aggccctgat gggtgacttc 60
gcaggcgtag actttgtgtt tctcgtagtc tgctttgctc agcgtcaggg tgctgctgag 120
gctgtaggtg ctgtccttgc tgtcctgctc tgtgacactc tcctgggagt tacccgattg 180
gagggcgtta tccaccttcc actgtacttt ggcctctctg ggatagaagt tattcagcag 240
gcacacaacg ctagcagttc cagatttcaa ctgctcatca gatggcggga agatgaagac 300
agatggtgca gccacagttc gtttgatttc caccttggtc cctccgccga aagtgggagg 360,
gtagctatta tgctgtagac agtaataagt tgcaaaatct tcaggctgca ggctgctgat 420
tgtgagagtg aattctgtcc cagatccact gccgctgaac cttgatggga ccccactttg 480
caaattggat gcagcataga tcaggcgctt aggggctttc cctggtttct gctgatacca 540
gcctaaatca tttctaatgt cctgacttgc ccggcaagtg atggtgactc tgtctcctac 600
agatgcagac agggaggatg gagactgggt catctggatg tcacacctgg cgcctgggaa 660
ccagagcagc aggagcccca ggagctgagc ggggaccctc at 702
<210> 97
<211> 234
<212> PRT
<213> Homo sapiens
<400> 97
Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp Phe Pro
1 5 10 15
Gly Ala Arg Cys Asp Ile Gln Met Thr Gin Ser Pro Ser Sex Leu Ser
20 25 30
Ala'Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp
35 40 45
Ile Arg Asn Asp Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
50 55 60
Lys Arg Leu Ile Tyr Ala Ala Ser Asn Leu Gln Ser Gly Val Pro Ser
65 70 75 80
Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser
85 90 95
Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn
100 105 110
Ser Tyr Pro Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg
115 120 125
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
130 135 140
CA 02501984 2005-04-11
WO 2004/035603 PCT/US2003/032243
35/44
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
145 150 155 160
Pro Arg Glu Ala Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gln Ser
165 170 175
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
180 185 190
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
195 200 205
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
210 215 220
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
225 230
<210> 98
<211> 1990
<212> DNA
<213> Homo sapiens
<400> 98
atgaaacacc tgtggttctt ccttctcctg gtggcagctc ccagatgggt cctgtcccag 60
gtgcagctgc aggagtcggg cccaggactg gtgaagcctt cggagaccct gtccctcacc 120
tgcactgtct ctggtgtctc catcagtaat tactactgga gctggatccg gcagtcccca 180
gggaagggac tggagtggat tggatatatc tattacagtg ggagtcccta ttacaacccc 240
tccctcaaga gtcgagtcac tatatctgca gacacgtcca agaaccaatt ctccctgaag 300
ctgagctctg tgaccgctgc ggacacggcc atttattact gtgcgagaga aaaactgggg 360
attggagact actggggcca gggaaccctg gtcaccgtct cctcagcctc caccaagggc 420
ccatcggtct tccccctggc gccctgctct agaagcacct ccgagagcac agccgccctg 480
ggctgcctgg tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgct 540
ctgaccagcg gcgtgcacac cttcccagct gtcctacagt cctcaggact ctactccctc 600
agcagcgtgg tgaccgtgcc ctccagcaac ttcggcaccc agacctacac ctgcaacgta 660
gatcacaagc ccagcaacac caaggtggac aagacagttg gtgagaggcc agctcaggga 720
gggagggtgt ctgctggaag ccaggctcag ccctcctgcc tggacgcacc ccggctgtgc 780
agccccagcc cagggcagca aggcaggccc catctgtctc ctcacccgga ggcctctgcc 840
cgccccactc atgctcaggg agagggtctt ctggcttttt ccaccaggct ccaggcaggc 900
acaggctggg tgcccctacc ccaggccctt cacacacagg ggcaggtgct tggctcagac 960
ctgccaaaag ccatatccgg gaggaccctg cccctgacct aagccgaccc caaaggccaa 1020
actgtccact ccctcagctc ggacaccttc tctcctccca gatccgagta actcccaatc 1080
ttctctctgc agagcgcaaa tgttgtgtcg agtgcccacc gtgcccaggt aagccagccc 1140
aggcctcgcc ctccagctca aggcgggaca ggttccctag agtagcctgc atccagggac 1200
aggccccagc tgggtgctga cacgtccacc tccatctctt cctcagcatC acctgtggca 1260
ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 1320
cctgaggtca cgtgcgtggt ggtggacgtg agccacgaag accccgaggt ccagttcaac 1380
tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccacggga ggagcagttc 1440
aacagcacgt tccgtgtggt cagcgtcctc accgttgtgc accaggactg gctgaacggc 1500
aaggagtaca agtgcaaggt ctccaacaaa ggcctcccag cccccatcga gaaaaccatc 1560
tccaaaacca aaggtgggac ccgcggggta tgagggccac atggacagag gccggctcag 1620
cccaccctct gccctgggag tgaccgctgt gccaacctct gtccctacag ggcagccccg 1680
agaaccacag gtgtacaccc tgcccccatc ccgggaggag atgaccaaga accaggtcag 1740
cctgacctgc ctgctcaaag gcttctaccc cagcgacatc gccgtggagt gggagagcaa 1800
tgggcagccg gagaacaact acaagaccac acctcccatg ctggactccg acggctcctt 1860
cttcctctac agcaagctca ccgtggacaa gagcaggtgg cagcagggga acgtcttctc 1920
atgctccgtg atgcatgagg ctctgcacaa ccactacacg cagaagagcc tctccctgtc 1980
tccgggtaaa 1990
<210> 99
<211> 1990
<212> DNA
<213> Homo sapiens
<400> 99
CA 02501984 2005-04-11
WO 2004/035603 PCT/US2003/032243
36/44
tttacccgga gacagggaga ggctcttctg cgtgtagtgg ttgtgcagag cctcatgcat 60
cacggagcat gagaagacgt tcccctgctg ccacctgctc ttgtccacgg tgagcttgct 120
gtagaggaag aaggagccgt cggagtccag catgggaggt gtggtcttgt agttgttctc 180
cggctgccca ttgctctccc actccacggc gatgtcgctg gggtagaagc ctttgaccag 240
gcaggtcagg ctgacctggt tcttggtcat ctcctcccgg gatgggggca gggtgtacac 300
ctgtggttct cggggctgcc ctgtagggac agaggttggc acagcggtca ctcccagggc 360
agagggtggg ccgagccggc ctctgtccat gtggccctca taccccgcgg gtcccacctt 420
tggttttgga gatggttttc tcgatggggg ctgggaggcc tttgttggag accttgcact 480
tgtactcctt gccgttcagc cagtcctggt gcacaacggt gaggacgctg accacacgga 540
acgtgctgtt gaactgctcc tcccgtagct ttgtcttggc attatgcacc tccacgCcgt 600
ccacgtacca gttgaactgg acctcggggt cttcgtggct cacgtccacc accacgcacg 660
tgacctcagg ggtccgggag atcatgaggg tgtccttggg ttttgggggg aagaggaaga 720
ctgacggtcc tgccacaggt ggtgctgagg aagagatgga ggtggacgtg tcagcaccca 780
gctggggcct gtccctggat gcaggctact ctagggcacc tgtcccgcct tgagctggag 840
ggcgaggcct gggctggctt acctgggcac ggtgggcact cgacacaaca tttgcgctct 900
gcagagagaa gattgggagt tactcggatc tgggaggaga gaaggtgtcc gagctgaggg 960
agtggacagt ttggcctttg gggtcggctt aggtcagggg cagggtcctc ccggatatgg 1020
cttttggcag gtctgagcca agcacctgcc cctgtgtgtg aagggcctgg ggtaggggca 1080
cccagcctgt gcctgcctgg agcctggtgg aaaaagccag aagaccctct ccctgagcat 1140
gagtggggcg ggcagaggcc tccgggggag gagacagatg gggcctgcct tgctgccctg 1200
ggctggggct gcacagccgg ggtgcgtcca ggcaggaggg ctgagcctgg cttccagcag 1260
acaccctccc tccctgagct ggcctctcac caactgtctt gtccaccttg gtgttgctgg 1320
gcttgtgatc tacgttgcag gtgtaggtct gggtgccgaa gttgctggag ggcacggtca 1380
ccacgctgct gagggagtag agtcctgagg actgtaggac agctgggaag gtgtgcacgc 1440
cgctggtcag agcgcctgag ttccacgaca ccgtcaccgg ttcggggaag tagtccttga 1500
ccaggcagcc cagggcggct gtgctctcgg aggtgcttct agagcagggc gccaggggga 1560,
agaccgatgg gcccttggtg gaggctgagg agacggtgac cagggttccc tggccccagt 1620
agtctccaat ccccagtttt tctctcgcac agtaataaat ggccgtgtcc gcagcggtca 1680
cagagctcag cttcagggag aattggttct tggacgtgtc tgcagatata gtgactcgac 1740
tcttgaggga ggggttgtaa tagggactcc cactgtaata gatatatcca atccactcca 1800
gtcccttccc tggggactgc cggatccagc tccagtagta attactgatg gagacaccag 1860
agacagtgca ggtgagggac agggtctccg aaggcttcac cagtcctggg cccgactcct 1920
gcagctgcac ctgggacagg acccatctgg gagctgccac caggagaagg aagaaccaca 1980
ggtgtttcat 1990
<210> 100
<211> 239
<212> PRT
<213> Homo sapiens
<400> 100
Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Leu Ala Ala Pro
1 5 10 15
Arg Trp Val Leu Ser Gin Val Gln Leu Gln Glu Ser Gly Pro Gly Leu
20 25 30
Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val
35 40 45
Ser Ile Ser Asn Tyr Tyr Trp Ser Trp Ile Arg Gln Ser Pro Gly Lys
50 55 60
Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Pro Tyr Tyr
65 70 75 80
Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Ala Asp Thr Ser Lys
85 90 95
Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala
100 105 110
Ile Tyr Tyr Cys Ala Arg Glu Lys Leu Gly Ile Gly Asp Tyr Trp Gly
115 120 125
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
130 135 140
Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala
145 150 155 160
CA 02501984 2005-04-11
WO 2004/035603 PCT/US2003/032243
37/44
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
165 170 175
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
180 185 190
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
195 200 205
Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Ala Ser
210 215 220
Pro His Lys Pro Ser Asn Thr Lys Val Ala Ser Pro Lys Thr Val
225 230 235
<210> 101
<211> 702
<212> DNA
<213> Homo sapiens
<400> 101
atgagggtcc ccgctcagct cctggggctc ctgctgctct ggttcccagg tgccaggtgt 60
gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtcggaga cagagtcacc 120
atcacttgcc gggcaagtca gggcattaga aatgatttag gctggtatca gcagaaacca 180
gggaaagccc ctaagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 240
aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 300
gaagattttg caacttatta ctgtctacag cataatagtt accctcccac tttcggccct 360
gggaccaagg tggatatcaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcca 420
tctgatgagc agttgaaatc tggaactgct agcgttgtgt gcctgctgaa taacttctat 480
cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 540
gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 600
ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 660
ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gt 702
<210> 102
<211> 702
<212> DNA
<213> Homo sapiens
<400> 102
acactctccc ctgttgaagc tctttgtgac gggcgagctc aggccctgat gggtgacttc 60
gcaggcgtag actttgtgtt tctcgtagtc tgctttgctc agcgtcaggg tgctgctgag 120
gctgtaggtg ctgtccttgc tgtcctgctc tgtgacactc tcctgggagt tacccgattg 180
gagggcgtta tccaccttcc actgtacttt ggcctctctg ggatagaagt tattcagcag 240
gcacacaacg ctagcagttc cagatttcaa ctgctcatca gatggcggga agatgaagac 300
agatggtgca gccacagttc gtttgatatc caccttggtc ccagggccga aagtgggagg 360
gtaactatta tgctgtagac agtaataagt tgcaaaatct tcaggctgca ggctgctgat 420
tgtgagagtg aattctgtcc cagatccact gccgctgaac cttgatggga ccccactttg 480
caaactggat gcagcataga tcaggcgctt aggggctttc cctggtttct gctgatacca 540
gcctaaatca tttctaatgc cctgacttgc ccggcaagtg atggtgactc tgtctccgac 600
agatgcagac agggaggatg gagactgggt catctggatg tcacacctgg cacctgggaa 660
ccagagcagc aggagcccca ggagctgagc ggggaccctc at 702
<210> 103
<211> 234
<212> PRT
<213> Homo sapiens
<400> 103
Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp Phe Pro
1 5 10 15
Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
20 25 30
Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly
CA 02501984 2005-04-11
WO 2004/035603 PCT/US2003/032243
38/44
35 40 45
Ile Arg Asn Asp Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
50 55 60
Lys Arg Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser
65 70 75 80
Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser
85 90 95
Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn
100 105 110
Ser Tyr Pro Pro Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg
115 120 125
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
130 135 140
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
145 150 155 160
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
165 170 175
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
180 185 190
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
195 200 205
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
210 215 220
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
225 230
<210> 104
<211> 1990
<212> DNA
<213> Homo sapiens
<400> 104
atgaaacatc tgtggttctt ccttctcctg gtggcagctc ccagatgggt cctgtcccag 60
gtgcagctgc aggagtcggg cccaggactg gtgaagcctt cggagaccct gtccctcacc 120
tgcactgtct ctggtggctc catcagtcgt tactactgga gctggatccg gcagccccca 180
gggaagggac tggagtggat tgggtatgtc tcttacagtg ggagcaccta ctacaacccc 240
tccctcaaga gtcgagtcac catatcagta gacacgtcca agaaccagtt ctccctgaag 300
ctgagctctg tgaccgctgc ggacacggcc gtgtattact gtgcgagaga taaactgggg 360
attggagact actggggcca gggaaccctg gtcaccgtct cctcagcctc caccaagggc 420
ccatcggtct tccccctggc gccctgctct agaagcacct ccgagagcac agccgccctg 480
ggctgcctgg tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgct 540
ctgaccagcg gcgtgcacac cttcccagct gtcctacagt cctcaggact ctactccctc 600
agcagcgtgg tgaccgtgcc ctccagcaac ttcggcaccc agacctacac ctgcaacgta 660
gatcacaagc ccagcaacac caaggtggac aagacagttg gtgagaggcc agctcaggga 720
gggagggtgt ctgctggaag ccaggctcag ccctcctgcc tggacgcacc ccggctgtgc 780
agccccagcc cagggcagca aggcaggccc catctgtctc ctcacccgga ggcctctgcc 840
cgccccactc atgctcaggg agagggtctt ctggcttttt ccaccaggct ccaggcaggc 900
acaggctggg tgcccctacc ccaggccctt cacacacagg ggcaggtgct tggctcagac 960
ctgccaaaag ccatatccgg gaggaccctg cccctgacct aagccgaccc caaaggccaa 1020
actgtccact ccctcagctc ggacaccttc tctcctccca gatccgagta actcccaatc 1080
ttctctctgc agagcgcaaa tgttgtgtcg agtgcccacc gtgcccaggt aagccagccc 1140
aggcctcgcc ctccagctca aggcgggaca ggtgccctag agtagcctgc atccagggac 1200
aggccccagc tgggtgctga cacgtccacc tccatctctt cctcagcacc acctgtggca 1260
ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 1320
cctgaggtca cgtgcgtggt ggtggacgtg agccacgaag accccgaggt ccagttcaac 1380
tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccacggga ggagcagttc 1440
aacagcacgt tccgtgtggt cagcgtcctc accgttgtgc accaggactg gctgaacggc 1500
aaggagtaca agtgcaaggt ctccaacaaa ggcctcccag cccccatcga gaaaaccatc 1560
tccaaaacca aaggtgggac ccgcggggta tgagggccac atggacagag gccggctcgg 1620
cccaccctct gccctgggag tgaccgctgt gccaacctct gtccctacag ggcagccccg 1680
CA 02501984 2005-04-11
WO 2004/035603 PCT/US2003/032243
39/44
agaaccacag gtgtacaccc tgcccccatc ccgggaggag atgaccaaga accaggtcag 1740
cctgacctgc ctggtcaaag gcttctaccc cagcgacatc gccgtggagt gggagagcaa 1800
tgggcagccg gagaacaact acaagaccac acctcccatg ctggactccg acggctcctt 1860
cttcctctac agcaagctca ccgtggacaa gagcaggtgg cagcagggga acgtcttctc 1920
atgctccgtg atgcatgagg ctctgcacaa ccactacacg cagaagagcc tctccctgtc 1980
tccgggtaaa 1990
<210> 105
<211> 1990
<212> DNA
<213> Homo sapiens
<400> 105
tttacccgga gacagggaga ggctcttctg cgtgtagtgg ttgtgcagag cctcatgcat 60
cacggagcat gagaagacgt tcccctgctg ccacctgctc ttgtccacgg tgagcttgct 120
gtagaggaag aaggagccgt cggagtccag catgggaggt gtggtcttgt agttgttctc 180
cggctgccca ttgctctccc actccacggc gatgtcgctg gggtggaaac ctttgaccag 240
gcaggtcagg ctgacctggt tcttggtcat ctcctcccgg gatgggggca gggtgtacac 300
ctgtggttct cggggctgcc ctgtagggac agaggttggc acagcggtca ctcccacggc 360
agagggtggg ccgagccggc ctctgtccat gtggccctca taccccgcgg gtcccacctt 420
tggttttgga gatggttttc tcgatggggg ctgggaggcc tttgttggag accttgcact 480
tgtactcctt gccgttcagc cagtcctggt gcacaacggt gaggacgctg accacacgga 540
acgtgctgtt gaactgctcc tcccgtggct ttgtcttggc attatgcacc tccacgccgt 600
ccacgtacca gttgaactgg acctcggggt cttcgtggct cacgtccacc accacgcacg 660
tgacctcagg ggtccgggag atcatgaggg tgtccttggg ttttgggggg aagaggaaga 720
ctgacggtcc tgccacaggt ggtgctgagg aagagatgga ggtggacgtg tcagcaccca 780
gctggggcct gtccctggat gcaggctact ctagggcacc tgtcccgcct tgagctggag 840
ggcgaggcct gggctggctt acctgggcac ggtgggcact cgacacaaca tttgcgctct 900
gcagagagaa gattgggagt tactcggatc tgggaggaga gaaggtgtcc gagctgaggg 960
agtggacagt ttggcctttg gggtcggctt aggtcagggg cagggtcctc ccggatatgg 1020
cttttggcag gtctgagcca agcacctgcc cctgtgtgtg aagggcctgg ggtaggggca 1080
cccagcctgt gcctgcctgg agcctggtgg aaaaagccag aagaccctct ccctgagcat 1140
gagtggggcg ggcagaggcc tccgggtgag gagacagatg gggcctgcct tgctgccctg 1200
ggctggggct gcacagccgg ggtgcgtcca ggcaggaggg ctgagcctgg cttccagcag 1260
acaccctccc tccctgagct ggcctctcac caactgtctt gtccaccttg gtgttgctgg 1320
gcttgtgatc tacgttgcag gtgtaggtct gggtgccgaa gttgctggag ggcacggtca 1380
ccacgctgct gagggagtag agtcctgagg actgtaggac agctgggaag gtgtgcacgc 1440
cgctggtcag agcgcctgag ttccacgaca ccgtcaccgg ttcggggaag tagtccttga 1500
ccaggcagcc cagggcggct gtgctctcgg aggtgcttct agagcagggc gccaggggga 1560
agaccgatgg gcccttggtg gaggctgagg agacggtgac cagggttccc tggccccagt 1620
agtctccaat ccccagttta tctctcgcac agtaatacac ggccgtgtcc gcagcggtca 1680
cagagctcag cttcagggag aactggttct tggacgtgtc tactgatatg gtgactcgac 1740
tcttgaggga ggggttgtag taggtgctcc cactgtaaga gacataccca atccactcca 1800
gtcccttccc tgggggctgc cggatccagc tccagtagta acgactgatg gagccaccag 1860
agacagtgca ggtgagggac agggtctccg aaggcttcac cagtcctggg cccgactcct 1920
gcagctgcac ctgggacagg acccatctgg gagctgccac caggagaagg aagaaccaca 1980
gatgtttcat 1990
<210> 106
<211> 241
<212> PRT
<213> Homo sapiens
<400> 106
Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Leu Ala Ala Pro
1 5 10 15
Arg Trp Val Leu Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu
20 25 30
Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly
35 40 45
Ser Ile Ser Arg Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys
CA 02501984 2005-04-11
WO 2004/035603 PCT/US2003/032243
40/44
50 55 60
Gly Leu Glu Trp Ile Gly Tyr Val Ser Tyr Ser Gly Ser Thr Tyr Tyr
65 70 75 80
Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Ala Ser Pro Thr
85 90 95
Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp
100 105 110
Thr Ala Val Tyr Tyr Cys Ala Arg Asp Lys Leu Gly Ile Gly Asp Tyr
115 120 125
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
130 135 140
Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser
145 150 155 160
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
165 170 175
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
180 185 190
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
195 200 205
Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val
210 215 220
Ala Ser Pro His Lys Pro Ser Asn Thr Lys Val Ala Ser Pro Lys Thr
225 230 235 240
Val
<210> 107
<211> 702
<212> DNA
<213> Homo sapiens
<400> 107
atgaggctcc ctgctcagct cctggggctc ctgctgctct ggttcccagg tgccaggtgt 60
gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 120
atcacttgcc gggcaagtca gggcattaga aatgatttag gctggtatca gcagaaaccg 180
gggaaagccc ctaagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 240
aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 300
gaagattttg caacttatta ctgtctacag cataatagtt acccgtgcag ttttggccag 360
gggaccaagc tggagatcaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcca 420
tctgatgagc agttgaaatc tggaactgct agcgttgtgt gcctgctgaa taacttctat 480
cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 540
gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 600
ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 660
ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gt 702
<210> 108
<211> 702
<212> DNA
<213> Homo sapiens
<400> 108
acactctccc ctgttgaagc tctttgtgac gggcgagctc aggccctgat gggtgacttc 60
gcaggcgtag actttgtgtt tctcgtagtc tgctttgctc agcgtcaggg tgctgctgag 120
gctgtaggtg ctgtccttgc tgtcctgctc tgtgacactc tcctgggagt tacccgattg 180
gagggcgtta tccaccttcc actgtacttt ggcctctctg ggatagaagt tattcagcag 240
gcacacaacg ctagcagttc cagatttcaa ctgctcatca gatggcggga agatgaagac 300
agatggtgca gccacagttc gtttgatctc cagcttggtc ccctggccaa aactgcacgg 360
gtaactatta tgctgtagac agtaataagt tgcaaaatct tcaggctgca ggctgctgat 420
tgtgagagtg aattctgtcc cagatccact gccgctgaac cttgatggga ccccactttg 480
caaactggat gcagcataga tcaggcgctt aggggctttc cccggtttct gctgatacca 540
CA 02501984 2005-04-11
WO 2004/035603 PCT/US2003/032243
41/44
gcctaaatca tttctaatgc cctgacttgc ccggcaagtg atggtgactc tgtctcctac 600
agatgcagac agggaggatg gagactgggt catctggatg tcacacctgg cacctgggaa 660
ccagagcagc aggagcccca ggagctgagc agggagcctc at 702
<210> 109
<211> 234
<212> PRT
<213> Homo sapiens
<400> 109
Met Arg Leu Pro Ala Gin Leu Leu Gly Leu Leu Leu Leu Trp Phe Pro
1 5 10 15
Gly Ala Arg Cys Asp Ile Gin Met Thr Gin Ser Pro Ser Ser Leu Ser
20 25 30
Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gin Gly
35 40 45
Ile Arg Asn Asp Leu Gly Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro
50 55 60
Lys Arg Leu Ile Tyr Ala Ala Ser Ser Leu Gin Ser Gly Val Pro Ser
65 70 75 80
Arg Phe Her Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser
85 90 95
Ser Leu Gin Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gin His Asn
100 105 110
Ser Tyr Pro Cys Ser Phe Gly Gin Gly Thr Lys Leu Glu Ile Lys Arg
115 120 125
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gin
130 135 140
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
145 150 155 160
Pro Arg Glu Ala Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser
165 170 175
Gly Asn Ser Gin Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr
180 185 190
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
195 200 205
His Lys Val Tyr Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro
210 215 220
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
225 230
<210> 110
<211> 1996
<212> DNA
<213> Homo sapiens
<400> 110
atgaagcatc tgtggttctt cctcctgctg gtggcagctc ccagatgggt cctgtcccag 60
gtgcagctgc aggagtcggg cccaggactg gtgaagcctt tacagaccct gtccctcacc 120
tgcactgtct ctggtggctc catcagcagt ggtgtttact actggagctg gatccgccag 180
cacccgggga agggcctgga gtggattggg tacatctata acagtaagac ctcctattat 240
aatccgtccc tcaagagtcg acttacccta tcagtagaca cgtctaagaa ccagttctcc 300
ctgaacctga tctctgtgac tgccgcggac acggccgtgt attactgtgc gagagataaa 360
ttggggatcg cggactactg gggccaggga accctggtca ccgtctcctc agcctccacc 420
aagggcccat cggtcttccc cctggcgccc tgctctagaa gcacctccga gagcacagcc 480
gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 540
ggcgctctga ccagcggcgt gcacaccttc ccagctgtcc tacagtcctc aggactctac 600
tccctcagca gcgtggtgac cgtgccctcc agcaacttcg gcacccagac ctacacctgc 660
aacgtagatc acaagcccag caacaccaag gtggacaaga cagttggtga gaggccagct 720
cagggaggga gggtgtctgc tggaagccag gctcagccct cctgcctgga cgcaccccgg 780
CA 02501984 2005-04-11
WO 2004/035603 PCT/US2003/032243
42/44
ctgtgcagcc ccagcccagg gcagcaaggc aggccccatc tgtctcctca cccggaggcc 840
tctgcccgcc ccactcatgc tcagggagag ggtcttctgg ctttttccac caggctccag 900
gcaggcacag gctgggtgcc cctaccccag gcccttcaga cacaggggca ggtgcttggc 960
tcagacctgc caaaagccat atccgggagg accctgcccc tgacctaagc cgaccccaaa 1020
ggccaaactg tccactccct cagctcggac accttctctc ctcccagatc cgagtaactc 1080
ccaatcttct ctctgcagag cgcaaatgtt gtgtcgagtg ccaaccgtgc ccaggtaagc 1140
cagcccaggc ctcgccctcc agctcaaggc gggacaggtg ccctagagta gcctgcatcc 1200
agggacaggc cccagctggg tgctgccacg tccacctcca tctcttcctc agcaccacct 1260
gtggcaggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 1320
cggacccctg aggtcacgtg cgtggtggtg gacgtgagcc acgaagaccc cgaggtccag 1380
ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc acgggaggag 1440
cagttcaaca gcacgttccg tgtggtcagc gtcctcaccg ttgtgcacca ggactggctg 1500
aacggcaagg agtacaagtg caaggtctcc aacaaaggcc tcccagcccc catcgagaaa 1560
accatctcca aaaccaaagg tgggacccgc ggggtatgag ggccacatgg acagaggccg 1620
gctcggccca ccctctgccc tgggagtgac cgctgtgcca acctctgtcc ctacagggca 1680
gccccgagaa ccacaggtgt acaccctgcc cccatcccgg gaggagatga ccaagaacca 1740
ggtcaccctg acctgcctgg tcaaaggctt ctaccccagc gacatcgccg tggagtggga 1800
gagcaatggg cagccggaga acaactacaa gaccacacct cccatgctgg actccgacgg 1860
ctccttcttc ctctacagca agctcaccgt ggacaagagc aggtggcagc aggggaacgt 1920
cttctcatgc tccgtgatgc atgaggctct gcacaaccac tacacgcaga agagcctctc 1980
cctgtctccg ggtaaa 1996
<210> 111
<211> 1996
<212> DNA
<213> Homo sapiens
<400> 111
tttacccgga gacagggaga ggctcttctg cgtgtagtgg ttgtgcagag cctcatgcat 60
cacggagcat gagaagacgt tcccctgctg ccacctgctc ttgtccacgg tgagcttgct 120
gtagaggaag aaggagccgt cggagtccag catgggaggt gtggtcttgt agttgttctc 180
cggctgccca ttgctctccc actccacggc gatgtcgctg gggtagaagc ctttgaccag 240
gcaggtcagg ctgacctggt tcttggtcat ctcctcccgg gatgggggca gggtgtacac 300
ctgtggttct cggggctgcc ctgtagggac agaggttggc acagcggtca ctcccagggc 360
agagggtggg ccgagccggc ctctgtccat gtggccctca taccccgcgg gtcccacctt 420
tggttttgga gatggttttc tcgatggggg ctgggaggcc tttgttggag accttgcact 480
tgtactcctt gccgttcagc cagtcctggt gcacaacggt gaggacgctg accacacgga 540
acgtgctgtt gaactgctcc tcccgtggct ttgtcttggc attatgcacc tccacgccgt 600
ccacgtacca gttgaactgg acctcggggt cttcgtggct cacgtccacc accacgcacg 660
tgacctcagg ggtccgggag atcatgaggg tgtccttggg ttttgggggg aagaggaaga 720
ctgacggtcc tgccacaggt ggtgctgagg aagagatgga ggtggacgtg tcagcaccca 780
gctggggcct gtccctggat gcaggctact ctagggcacc tgtcccgcct tgagctggag 840
ggcgaggcct gggctggctt acctgggcac ggtgggcact cgacacaaca tttgcgctct 900
gcagagagaa gattgggagt tactcggatc tgggaggaga gaaggtgtcc gagctgaggg 960
agtggacagt ttggcctttg gggtcggctt aggtcagggg cagggtcctc ccggatatgg 1020
cttttggcag gtctgagcca agcacctgcc cctgtgtgtg aagggcctgg ggtaggggca 1080
cccagcctgt gcctgcctgg agcctggtgg aaaaagccag aagaccctct ccctgagcat 1140
gagtggggcg ggcagaggcc tccgggtgag gagacagatg gggcctgcct tgctgccctg 1200
ggctggggct gcacagccgg ggtgcgtcca ggcaggaggg ctgagcctgg cttccagcag 1260
acaccctccc tccctgagct ggcctctcac caactgtctt gtccaccttg gtgttgctgg 1320
gcttgtgatc tacgttgcag gtgtaggtct gggtgccgaa gttgctggag ggcacggtca 1380
ccacgctgct gagggagtag agtcctgagg actgtaggac agctgggaag gtgtgcacgc 1440
cgctggtcag agcgcctgag ttccacgaca ccgtcaccgg ttcggggaag tagtccttga 1500
ccaggcagcc cagggcggct gtgctctcgg aggtgcttct agagcagggc gccaggggga 1560
agaccgatgg gcccttggtg gaggctgagg agacggtgac cagggttccc tggccccagt 1620
agtccgcgat ccccaattta tctctcgcac agtaatacac ggccgtgtcc gcggcagtca 1680
cagagatcag gttcagggag aactggttct tagacgtgtc tactgatagg gtaagtcgac 1740
tcttgaggga cggattataa taggaggtct tactgttata gatgtaccca atccactcca 1800
ggcccttccc tgggtgctgg cggatccagc tccagtagta aacaccactg ctgatggagc 1860
caccagagac agtgcaggtg agggacaggg tctgtaaagg cttcaccagt cctgggcccg 1920
actcctgcag ctgcacctgg gacaggaccc atctgggagc tgccaccagc aggaggaaga 1980
CA 02501984 2005-04-11
WO 2004/035603 PCT/US2003/032243
43/44
accacagatg cttcat 1996
<210> 112
<211> 235
<212> PRT
<213> homo sapiens
<400> 112
Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp
1 5 10 15
Val Leu Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys
20 25 30
Pro Leu Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile
35 40 45
Ser Ser Gly Val Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys
50 55 60
G1y Leu Glu Trp Ile Gly Tyr Ile Tyr Asn Ser Lys Thr Ser Tyr Tyr
65 70 75 80
Asn Pro Ser Leu Lys Ser Arg Leu Thr Leu Ser Val Asp Thr Ser Lys
85 90 95
Asn Gln Phe Ser Leu Asn Leu Ile Ser Val Thr Ala Ala Asp Thr Ala
100 105 110
Val Tyr Tyr Cys Ala Arg Asp Lys Leu Gly Ile Ala Asp Tyr Trp Gly
115 120 125
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
130 135 140
Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala
145 150 155 160
,Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
165 170 175
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
180 185 190
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
195 200 205
Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His
210 215 220
Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val
225 230 235
<210> 113
<211> 702
<212> DNA
<213> Homo sapiens
<400> 113
atgagggtcc ctgctcagct cctggggctc ctgctgctct ggttcccagg tgccaggtgt 60
gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 120
atcacttgcc ggacaagtca gggcattaga aatgatttag gctggtatca gcagaaacca 180
gggaaagccc ctaagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 240
aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 300
gaagattttg caacttatta ctgtctacag cataatagct accctcccac tttcggcgga 360
gggaccaagg tggagatcaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcca 420
tctgatgagc agttgaaatc tggaactgct agcgttgtgt gcctgctgaa taacttctat 480
cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 540
gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 600
ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 660
ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gt 702
<210> 114
<211> 702
CA 02501984 2005-04-11
WO 2004/035603 PCT/US2003/032243
44/44
<212> DNA
<213> Homo sapiens
<400> 114
acactctccc ctgttgaagc tctttgtgac gggcgagctc aggccctgat gggtgacttc 60
gcaggcgtag actttgtgtt tctcgtagtc tgctttgctc agcgtcaggg tgctgctgag 120
gctgtaggtg ctgtccttgc tgtcctgctc tgtgacactc tcctgggagt,tacccgattg 180
gagggcgtta tccaccttcc actgtacttt ggcctctctg ggatagaagt tattcagcag 240
gcacacaacg ctagcagttc cagatttcaa ctgctcatca gatggcggga agatgaagac 300
agatggtgca gccacagttc gtttgatctc caccttggtc cctccgccga aagtgggagg 360
gtagctatta tgctgtagac agtaataagt tgcaaaatct tcaggctgca ggctgctgat 420
tgtgagagtg aattctgtcc cagatccact gccgctgaac cttgatggga ccccactttg 480
caaactggat gcagcataga tcaggcgctt aggggctttc cctggtttct gctgatacca 540
gcctaaatca tttctaatgc cctgacttgt ccggcaagtg atggtgactc tgtctcctac 600
agatgcagac agggaggatg gagactgggt catctggatg tcacacctgg cacctgggaa 660
ccagagcagc aggagcccca ggagctgagc agggaccctc at 702
<210> 115
<211> 234
<212> PRT
<213> Homo sapiens
<400> 115
Met Arg Val Pro Ala Gin Leu Leu Gly Leu Leu Leu Leu Trp Phe Pro
1 5 10 15
Gly Ala Arg Cys Asp Ile Gln Met Thr Gin Ser Pro Ser Ser Leu Ser
20 25 30
Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Gly
35 40 45
Ile Arg Asn Asp Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
50 55 60
Lys Arg Leu Ile Tyr Ala Ala Ser Ser Leu Gin Ser Gly Val Pro Ser
65 70 75 80
Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser
85 90 95
Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn
100 105 110
Ser Tyr Pro Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg
115 120 125
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
130 135 140
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
145 150 155 160
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gin Ser
165 170 175
Gly Asn Ser Gin Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr
180 185 190
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
195 200 205
His Lys Val Tyr Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro
210 215 220
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
225 230
