Note: Descriptions are shown in the official language in which they were submitted.
DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
CECI EST LE TOME 2 DE 2
~~ TTENANT LES PAGES 284 A 436
NOTE : Pour les tomes additionels, veuillez contacter 1e Bureau canadien des
brevets
JUMBO APPLICATIONS/PATENTS
THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME
THIS IS VOLUME 2 OF 2
CONTAINING PAGES 284 TO 436
NOTE: For additional volumes, please contact the Canadian Patent Office
NOM DU FICHIER / FILE NAME
NOTE POUR LE TOME / VOLUME NOTE:
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284
Also preferred is a composition of matter comprising isolated nucleic acid
molecules wherein the nucleotide sequences of said nucleic acid molecules
comprise
a panel of at least two nucleotide sequences, wherein at least one sequence in
said
panel is at least 95% identical to a sequence of at least 50 contiguous
nucleotides in a
sequence selected from the group consisting of: a nucleotide sequence of SEQ
ID
NO:X wherein X is any integer as defined in Table 1; and a nucleotide sequence
encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1
and contained in the deposit with the ATCC Deposit Number shown for said cDNA
clone in Table 1. The nucleic acid molecules can comprise DNA molecules or RNA
molecules.
Also preferred is an isolated polypeptide comprising an amino acid sequence
at least 90% identical to a sequence of at least about 10 contiguous amino
acids in the
amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in
Table 1.
Also preferred is a polypeptide, wherein said sequence of contiguous amino
acids is included in the amino acid sequence of SEQ ID NO:Y in the range of
positions beginning with the residue at about the position of the First Amino
Acid of
the Secreted Portion and ending with the residue at about the Last Amino Acid
of the
Open Reading Frame as set forth for SEQ ID NO:Y in Table 1-.
Also preferred is an isolated polypeptide comprising an amino acid sequence
at least 95% identical to a sequence of at least about 30 contiguous amino
acids in the
amino acid sequence of SEQ ID NO:Y.
Further preferred is an isolated polypeptide comprising an amino acid
sequence at least 95% identical to a sequence of at least about 100 contiguous
amino
acids in the amino acid sequence of SEQ ID NO:Y.
Further preferred is an isolated polypeptide comprising an amino acid
sequence at least 95% identical to the complete amino acid sequence of SEQ ID
NO:Y.
Further preferred is an isolated polypeptide comprising an amino acid
sequence at least 90% identical to a sequence of at least about 10 contiguous
amino
acids in the complete amino acid sequence of a secreted protein encoded by a
human
cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in
the
deposit. with the ATCC Deposit Number shown for said cDNA clone in Table 1.
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Also preferred is a polypeptide wherein said sequence of contiguous amino
acids is included in the amino acid sequence of a secreted portion of the
secreted
protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in
Table 1 and contained in the deposit with the ATCC Deposit Number shown for
said
cDNA clone in Table 1.
Also preferred is an isolated polypeptide comprising an amino acid sequence
at least 95% identical to a sequence of at least about 30 contiguous amino
acids in the
amino acid sequence of the secreted portion of the protein encoded by a human
cDNA
clone identified by a cDNA Clone Identifier in Table 1 and contained in the
deposit
with the ATCC Deposit Number shown for said cDNA clone in Table 1.
Also preferred is an isolated polypeptide comprising an amino acid sequence
at least 95% identical to a sequence of at least about 100 contiguous amino
acids in
the amino acid sequence of the secreted portion of the protein encoded by a
human
cDNA clone identified by a cDNA Clone Identifier in Table l and contained in
the
deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.
Also preferred is an isolated polypeptide comprising an amino acid sequence
at least 95% identical to the amino acid sequence of the secreted portion of
the protein
encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1
and contained in the deposit with the ATCC Deposit Number shown for said cDNA
clone in Table 1.
Further preferred is an isolated antibody which binds specifically to a
polypeptide comprising an amino acid sequence that is at least 90% identical
to a
sequence of at least 10 contiguous amino acids in a sequence selected from the
group
consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer
as
defined in Table 1; and a complete amino acid sequence of a protein encoded by
a
human cDNA clone identified by a cDNA Clone Identifier in Table 1 and
contained
in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table
1.
Further preferred is a method for detecting in a biological sample a
polypeptide comprising an amino acid sequence which is at least 90% identical
to a
sequence of at-least 10 contiguous amino acids in a sequence selected from the
group
consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer
as _.
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defined in Table 1; and a complete amino acid sequence of a protein encoded by
a
human cDNA clone identified by a cDNA Clone Identifier in Table 1 and
contained
in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table
.
1; which method comprises a step of comparing an amino acid sequence of at
least
one polypeptide molecule in said sample with a sequence selected from said
group
and determining whether the sequence of said polypeptide molecule in said
sample is
at least 90% identical to said sequence of at least 10 contiguous amino acids.
Also preferred is the above method wherein said step of comparing an amino
acid sequence of at least one polypeptide molecule in said sample with a
sequence
selected from said group comprises determining the extent of specific binding
of
polypeptides in said sample to an antibody which binds specifically to a
polypeptide
comprising an amino acid sequence that is at least 90% identical to a sequence
of at
least 10 contiguous amino acids in a sequence selected from the group
consisting of:
an amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in
Table 1; and a complete amino acid sequence of a protein encoded by a human
cDNA
clone identified by a cDNA Clone Identifier in Table 1 and contained in the
deposit
with the ATCC Deposit Number shown for said cDNA clone in Table 1.
Also preferred is the above method wherein said step of comparing sequences
is performed by comparing the amino acid sequence determined from a
polypeptide
molecule in said sample with said sequence selected from said group.
Also preferred is a method for identifying the species, tissue or cell type of
a
biological sample which method comprises a step of detecting polypeptide
molecules
in said sample, if any, comprising an amino acid sequence that is at least 90%
identical to a sequence of at least 10 contiguous amino acids in a sequence
selected
from the group consisting of an amino acid sequence of SEQ >D NO:Y wherein Y
is
any integer as defined in Table 1; and a complete amino acid sequence of a
secreted
protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in
Table 1 and contained in the deposit with the ATCC Deposit Number shown for
said
cDNA clone in Table 1.
Also preferred is the above method for identifying the species, tissue or cell
.
type of a biological sample, which method comprises a step of detecting
polypeptide
molecules comprising an amino acid sequence in a panel of at least two amino
acid
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sequences, wherein at least one sequence in said panel is at least 90%
identical to a
sequence of at least 10 contiguous amino acids in a sequence selected from the
above
group.
Also preferred is a method for diagnosing in a subject a pathological
condition
associated with abnormal structure or expression of a gene encoding a secreted
protein identified in Table 1, which method comprises a step of detecting in a
biological sample obtained from said subject polypeptide molecules comprising
an
amino acid sequence in a panel of at least two amino acid sequences, wherein
at least
one sequence in said panel is at least 90% identical to a sequence of at least
10
contiguous amino acids in a sequence selected from the group consisting of: an
amino
acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table 1;
and a
complete amino acid sequence of a secreted protein encoded by a human cDNA
clone
identified by a cDNA Clone Identifier in Table l and contained in the deposit
with the
ATCC Deposit Number shown for said cDNA clone in Table 1.
In any of these methods, the step of detecting said polypeptide molecules
includes using an antibody.
Also preferred is an isolated nucleic acid molecule comprising a nucleotide
sequence which is at least 95% identical to a nucleotide sequence encoding a
polypeptide wherein said polypeptide comprises an amino acid sequence that is
at
least 90% identical to a sequence of at least 10 contiguous amino acids in a
sequence
selected from the group consisting of: an amino acid sequence of SEQ m NO:Y
wherein Y is any integer as defined in Table 1; and a complete amino acid
sequence
of a secreted protein encoded by a human cDNA clone identified by a cDNA Clone
Identifier in Table l and contained in the deposit with the ATCC Deposit
Number
shown for said cDNA clone in Table 1.
Also preferred is an isolated nucleic acid molecule, wherein said nucleotide
sequence encoding a polypeptide has been optimized for expression of said
polypeptide in a prokaryotic host.
Also preferred is an isolated nucleic acid molecule, wherein said polypeptide
comprises an amino acid sequence selected from the group consisting of an
amino
acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table 1;
and a
complete amino acid sequence of a secreted protein encoded by a human cDNA
clone
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identified by a cDNA Clone Identifier in Table 1 and contained in the deposit
with the
ATCC Deposit Number shown for said cDNA clone in Table 1.
Further preferred is a method of making a recombinant vector comprising
inserting any of the above isolated nucleic acid molecule into a vector. Also
preferred
is the recombinant vector produced by this method. Also preferred is a method
of
making a recombinant host cell comprising introducing the vector into a host
cell, as
well as the recombinant host cell produced by this method.
Also preferred is a method of making an isolated polypeptide comprising
culturing this recombinant host cell under conditions such that said
polypeptide is
expressed and recovering said polypeptide. Also preferred is this method of
making
an isolated polypeptide, wherein said recombinant host cell is a eukaryotic
cell and
said polypeptide is a secreted portion of a human secreted protein comprising
an
amino acid sequence selected from the group consisting of: an amino acid
sequence of
SEQ ID NO:Y beginning with the residue at the position of the First Amino Acid
of
the Secreted Portion of SEQ ID NO:Y wherein Y is an integer set forth in Table
1 and
said position of the First Amino Acid of the Secreted Portion of SEQ ID NO:Y
is
defined in Table 1; and an amino acid sequence of a secreted portion of a
protein
encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1
and contained in,the deposit with the ATCC Deposit Number shown for said cDNA
clone in Table 1. The isolated polypeptide produced by this method is also
preferred.
Also preferred is a method of treatment of an individual in need of an
increased level of a secreted protein activity, which method comprises
administering
to such an individual a pharmaceutical composition comprising an amount of an
isolated polypeptide, polynucleotide, or antibody of the claimed invention
effective to
increase the level of said protein activity in said individual.
The above-recited applications have uses in a wide variety of hosts. Such
hosts include, but are not limited to, human, murine, rabbit, goat, guinea
pig, camel,
horse, mouse, rat, hamster, pig, micro-pig, chicken, goat, cow, sheep, dog,
cat, non-
human primate, and human. In specific embodiments, the host is a mouse,
rabbit,
goat, guinea pig, chicken, rat, hamster, pig, sheep, dog or cat. In preferred
embodiments, the host is a mammal. In most preferred embodiments, the host is
a
human.
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In specific embodiments of the invention, for each "Contig m" listed in the
fourth column of Table 5, preferably excluded are one or more polynucleotides
comprising, or alternatively consisting of, a nucleotide sequence referenced
in the
fifth column of Table.5 and described by the general formula of a-b, whereas a
and b
are uniquely determined for the corresponding SEQ ID NO:X referred to in
column 3
of Table 5. Further specific embodiments are directed to polynucleotide
sequences
excluding one, two, three, four, or more of the specific polynucleotide
sequences
referred to in the fifth column of Table 5. In'no way is this listing meant to
encompass all of the sequences which may be excluded by the general formula,
it is
just a representative example. All references available through these
accessions are
hereby incorporated by reference in their entirety.
TABLE 5
Gene cDNA CloneNT Contig Public Accession Numbers
No. ID SEQ ID
ID
NO:
X
1 HTPFX16 33 1051320AA585439, N99013, AA585440,
AL040768,
AL044015, AL046147, AL042245,
AL049007,
AL043950, AL036500, AL043468,
AL042700,
AL044771, AL134123, AL046994,
AL042712,
AI535639, AL040414, AL040571,
AL037341,
AL046097, AI525556, AL045671,
AI525316,
AA585453, AL046150, AL044258,
228355,
AL040856, AA585434, AI541510,
AI546855,
AL041577, AL043848, AL046914,
AL044029,
AL046850, AL037435, AL043570,
AL079876,
AI525328, AI541374, N99040,
AL043201,
AL040252, AL041459, AL045991,
AL044201,
AI556967, AI541514, C15189,
AL043814,
AI541523, AI526180, AL047593,
AL044064,
' AL039316, 230131, AI546999,
AL043923,
AL043537, AL048647, AL043128,
AI541534,
AI525306, AL037335, AA585101,
AL045994,
AI535660, AI546945, AL03737.1,
AI557731,
AL043604, AI541365, AI557807,
AI526140,
AI541509, AI546828, AI541017,
AI525431,
AL037323, AL038532, AA585356,
AI526194,
AL043845, C16300, AI541317,
AL044583,
AIS47039, AL040329, AI526196,
AI541535,
AL041374, AL040082, AI557799,
AI540967,
AI54i508, AL040263, AI541307,
AL039338,
T11028, AI536138, AI557082,
AI557262,
AI525653, AI546899, AI526144,
AI541205,
D61254; 829445, AI535813,
AI5S7787,
AL040148, AL043627, AL041523,_.
AL044377,
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AL041730, 828735, AL038983,
AL046392,
AL044272, AL040090, AI546875,
AI526184,
AL040510, AL040625, AL045817,
AL041142,
AL041238, AL041133, AL041131,
AL046330,
AL040322, AL047183, AL041051,
AL041292,
AL047036, AL040119, AL047170,
AL047057,
AL041227, AL047219, AL040463,
AL039915,
AL043612, AL041197, AL040155,
AL041346,
AL040529, AL041096, AL047012,
AL041358,
AL041277, AL041163, AL041098,
AL040621,
AL043538, AL041324, AL040464,
AL044162,
AL041086, AL043496, AL041296,
AL041233,
AL043467, AL041159, AL045725,
AL044186,
AL041140, AL040193, AL044037,
AL040168,
AL040091, AL040128, AL040255,
AL040342,
AL040285, AL040332, AL040617,
AL040553,
AL045684, AL040745, AL043677,
AL040370,
AA585438, AL046442, AL040839,
AL041752,
AL040149, AL043775, D57491,
AL044165,
AI557084, AL043492, AL041602,
AL045920,
AL041278, AL038838, AL040253,
AL044074,
AL041635, AL045990, AL040458,
AL044199,
AL044187, AI525320, AJ239433,
AL040294,
AL041186, AL040052, AL042135,
AL037727,
T23985, AL040576, AI142134,
AI546891,
AL045753, AL044274, AI557796,
AL079878,
AL049018, AL040444, AL039744,
AL045857,
AL038822, AI525321, AL046327,
AI541013,
AL041168, AA585476, AL049069,
AI557238,
AL043444, AL041246, AL040472,
AL040238,
AL041955, AL041347, AI540920,
C16305,
AL038761, AL040075, T41289,
T23957,
AI541506, AL080031, AI557155,
AI541345,.
AL045989, 829177, AL080075,
AF221069,
AF221068, AR017907, I13349,
A91965,
I66498, I66495, I66494, 166487,
I66497,
I66496, I66486, I66488, I66489,
I66490,
I66491, I66492, I66493, I66481,
A83642,
A83643,I66485,I66482,I66483,I66484,
A83151, X81969, I05488, I61310,
A60977,
A91752, I08196, A32110, A58524,
A58523,
A68112, A68104, AR028564,
AR067731,
A06419, A21892, A23997, A84772,
AR067732, A68114, A20702,
A86792,
A58522, AR062872, AR027319,
AR037157,
AR054109, A21895, A84776,
A25909,
A60985, A64973, A84773, A84775,
A91751,
AR062873, A47368, A43189,
AR027318;
A84774, A43188, A60990, I19516,
A05160,
A08030, A20502, I19517, A89633,
A89634,
A60987, I63560, I63561, I63563,
AR062871,
I08776, AR009152, AR009151,
E14304,
AR002333, A81878, I25027,
I26929, I44515,
I26928, I26930, I26927, I44516,
E 16678,
I25041, A95096, A95106, A95105,
A29109,
A32111, A98767, A93963, A93964,
I15353,
A91750, A20700, AF082186,
AR035975,
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AR035974, AR035977; AR035976,
AR035978, I08051, A60961,
A92133, I07249,
AR068508, AR068510, AR068509,
A63954,
I91969, AR038762, I58322,
I58323,
AR003585, A76773, A22413,
A38214, I56772,
I95540, AR038855, A58521,
A91754,
AR031374, AR031375, AR020969,
E12584,
AR036903, A90655, AR008429,
I01987,
AJ244004, A85395, A85476,
A49700, I44681,
I18895, AJ244005, AJ244003,
A92666,
A92668, A9266'7, A92665,
A98420, A98423,
A98432, A98436, A98417, A98427,
I63120,
I19525, Y16359, D78345, X83865,
A93016,
A18053, M28262, AJ244007,
I15717, I15718,
E03627, I49890, I48927, A02712,
A77094,
A77095, I84553, A95051, I84554,
A18050,
A23334, A75888, 170384, A60111,
A23633,
AR007512, I08396, A60212,
A60209, A60210,
A60211, I00682, A11623, E00609,
A11624,
E13740, A11178, E01007, A10361,
A62298,
A35536, A35537, A02135, A04663,
A02136,
A04664, I08395, I06859, U94592,
AR043601,
A11245, I62368, X12660, A02710,
E12615,
AR035193, A07700, A13392,
A13393,
AR031488, I13521, I52048,
A27396,
AR027100, I44531, I28266,
I21869, A70040,
A82653, E16636, A24783, A24782,
I03331,
A95117, AF149828, I01995,
AR031566,
I60241, I60242, AR038066,
A20699, E00696,
E00697, E03813, AR027099,
Y09813,
AR051652, AR051651, A62300,
and A25246.
2 HPJCP79 12 1097166AI131158, AI143233, AI084060,
AI796995,
AW024300, AI690136, AW068636,
AA187976, AI990865, AA188104,
AA702670,
AI248604, AW241327, AI796740,
AI638567,
AA526922, AI147820, AA728795,
AI955736,
AI125679, AA036781, AI298768,
AI653728,
W58025, AI042104, AA903790,
W57793,
AI681520, N41467, AI148201,
AI825715,
AI982785, AL121341, AW068147,
AA449957,
AI917005, AA036838, AI125117,
AW139372,
AA536124, AI342363, 863264,
H03741,
AA451767, AI242453, AI928172,
AI884809,
AI536994, AI917991, AA833643,
AW236630,
AI769003, AI654929, AA364665,
T88911,
H06496, AI672523, AA343825,
845487,
866598, T79474, AA356796,
AA135794,
807310, AA961256, AW 118743,
W95716,
AI339456, AI863802, AA135750,
W95688,
AA046714, 866752, 863216,
AA046595,
T84216, AW027460, AI203114,
AI685956,
AA112706, D20929, AI557697,
AI525221,
D42044, AL035413, AI637994,
AI990544,
AW071667, and AW235783.
2 HPJCP79 34 1035470W55896, W55856, AI033991,
AI581098,
AW068238, 876895, 886326,
AW362820,
877060, AI028412, AI220844,
AA862718,
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AA728770, AA040365, AA040364,
T94138,
D42044, AL035413, AI219616,
and
AW515266.
2 HPJCP79 35 1018167AI131158, AI143233, AI084060,
AI796995,
AW024300, AI690136, AW068636,
AA 187976, AI990865, AA 188104,
AA702670,
AI248604, AW241327, AI796740,
AI638567,
AA526922, AI147820, AI955736,
AA728795,
AI125679, AI298768, AI653728,
AA036781,
W58025, AI042104, AA903790,
W57793,
AI681520, N41467, AI148201,
AI825715,
AI982785, AL121341, AW068147,
AA449957,
AI917005, AI125117, AA036838,
AW 139372,
AI342363, AA536124, AA451767,
863264,
H03741, AI242453, AI928172,
AI884809,
AI536994, AI917991, AA833643,
AW236630,
AI769003, T08888, H06553,
AI654929,
AA364665, T88911, H06496,
AA112706,
AI672523, AA343825, T79565,
845487,
AW068635, 866598, T79474,
AA356796,
AA135794, 807310, AA961256,
AW118743,
824756, W95716, AA112705,
AI339456,
AI863802, AA135750, W95688,
807309,
AA046714, 866752, 863216,
AA046595,
T84216, AW027460, AI203114,
AA303951,
AI685956, AI652260, D20929,
AI557697,
AI525221, D42044, AL035413,
AI637994,
AI990544, AW071667, and AW235783.
3 HWHS013 13 1115032AI628384, AI951331, AA625266,
AI985375,
AI247256, and AA41931 I.
_3 HWHS013 36 1045505T83059, AA419311, AI247256,
AI628384,
AI951331, and AI985375.
4 HSYAB05 14 1188478AA190860, AA355750, AA368604,
AI674193, AL119522, AW384394,
AW363220, AL119396, AW392670,
AL134902, AL119443, AW372827,
AL 119497, AL 119319, 299396,
AL 119457,
AL119399, AL119324, U46341,
U46347,
AL119484, AL119363, AL119341,
AL119391,
AL119355, U46346, U46350,
U46351,
U46349, AL119444, AL119483,
AL119335,
AL 119439, AL 119401, AL
119496, AL 134524,
AL037205, U46345, AL042614,
AL042896,
AL134527, AI142132, AL134538,
AL119418,
AL043037, AL042980, AL042975,
AI142137,
AL042450, AL042965, AL042970,
AL043019,
AL134526, AL042542, AL042544,
AI142139,
AL042984, AL043029, AL042551,
AL043003,
AL119488, AL119464, AB026436,
AR069079,
AR054110, A81671, AR060234,
AR066494,
and AR043113.
4 HSYABOS 37 1030176AA355750, AL119522, AW384394,
AW363220, AL119396, AW392670,
AL134902, AL119443, AW372827,
AL 119497, AL 119319, 299396,
AL 119457,
AL119399, AL119324, U46341,
U46347,
AL119484, AL119363, AL119341,
AL119391,
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AL119355, U46346, U46350,
U46351,
U46349, AL119444, AL119483,
AL119335,
AL 119439, AL 119401, AL
119496, AL 134524,
AL037205, U46345, AL042614,
AL042896,
AL134527, AI142132, AL134538,
AL119418,
AL043037, AL042980, AL042975,
AI142137,
AL042450, AL042965, AL042970,
AL043019,
AL134526, AL042542, AL042544,
AI142139,
AL042984, AL043029, AL042551,
AL043003,
AL119488, AL119464, AB026436,
AR069079,
AR054110, A81671, AR060234,
AR066494,
and AR043113.
HKIYI48 15 1212672AA421803, AA767372, AA746191,
W90802,
AA629842, AA723382, AA768200,
AI379904,
AI248375, W90746, 820049,
AI651629,
860506, AI338678, AI470342,
AI221377,
AA325442, T09446, AW170290,
and 245731.
5 HKIYI48 38 1042106820049, W90802, AA421803,
and 245731.
5 HKIYI48 39 1039455AA629842, AA767372, AA746191,
AI248375, AA723382, AA768200,
AI379904,
AA325442, W90746, AI470342,
AI338678,
and AA421803.
5 HKIYI48 40 1039454AA421803, AA767372, W90802,
AA746191,
AA629842, AA723382, AA768200,
AI379904,
W90746, AI248375, 820049,
860506,
AI338678, AI470342, AA325442,
245731,
and AI651629.
6 HARM138 16 1164812AW410706, AI748798, AA725637,
AA402147, AI419313, AL049126,
AI147283, _
. AI990726, AW410705, AI478369,
AA020743,
AI148856, AA284946, AA393789,
AW236603, AA393884, AI889197,
856450,
T27037, AI921312, AA394113,
AI189667,
AI948651, AA284766, AW172788,
AA180305, AA402989, AI948636,
AI866673,
AW071781, H19479, AA284767,
T27036,
AW237208, AI500384, AI678354,
239780,
AI826066, AI470278, H20936,
AI270491,
H84411, AA292332, H19561,
AA285099,
H20935, AI336345, 245818,
243720,
AI541240, AW004032, AA020742,
815049,
AI801457, F04685, AI290375,
AW 135154, .
AI480181, AI611126, AI982513,
AA179952,
F21516, AA634385, 841485,
and AI699619.
6 HARM138 41 1135023AA020742, 299396, AW392670,
AL119497,
AL119496, AW372827, U46341,
AW384394,
AW363220, AL119319, AL119457,
AL119443, AL119324, AL119484,
AL119363,
AL119341, AL119391, AL119355,
U46346,
U46350, AI826066, U46347,
U46351, U46349,
AL134524, AL119483, AL119396,
AL119335,
AL119444, AL134920, AL134533,
AL119439,
AL042450, AL119522, AL134526,
AL119399,
AL042975, AL037205, U46345,
AL042551,
AL042614, AL134525, AL134538,
AL042965,
AI142132, AL134531, AL119418,
AL042544,
AL042970, AL043029, AL042542,
AL043019,
CA 02396719 2002-07-24
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AL042984, AL043003, AL119464,
ALI 19488,
I25027, AR054109, I44515,
I26928, I26930,
I26927, I25041, I44516, AR035224,
I85513,
AR009152, AR009151, AR027099,
I05393,
A10617, AR028792, A94046,
A94054,
A01324, A01323, AR034783,
I63120,
AR067733, AR064322, AR064323,
AR064320, AR064321, A32110,
A94048,
A94061, AR038307, AR038321,
A49045,
A83642, A83643, A70359, AR019094,
I05430,
A92666, A92668, I49890, A92667,
A92665,
AR028791, AR028793, AR019098,
AR020199, AR020200, AR001287,
AR020198, AR020197, AR029418,
AR067734, AR067731, AR067732,
AR029417, 189986, AR018924,
AR018923,
A48774, A48775, AR000006,
AR015960,
AR000007, AR015961, AR051652,
A92081,
A92080, A92077, A92078, A92079,
A91752,
A91751, I09121, AR068508,
AR068510,
AR068509, I91969, AR003585,
I58322,
I58323, A91754, A63067, A51047,
A63064,
A63072, A85308, AR031375,
AR068507,
AR068506, A60213, AR062871,
AR051957,
A44171, A29109, A32111, AR068550,
A23373, AR068551, A49700,
A60207,
A60208, I58669, A58521, Y14971,
AB026436,
AR055065, A93444, A46342,
A46343,
A30600, AR055066, AR054110,
A81671,
AR032878, AR060234, AR066494,
and
AR069079.
6 HARMJ38 42 1038066AA020742.
6 HARMJ38 43 1031878AW410706, AI748798, AA402147,
AA725637, AI419313, AL049126,
AI147283,
AI478369, AA020743, AI148856,
AA284946,
AA393884, AA393789, AW236603,
AW410705, AI889197, T27037,
AI921312,
AA394113, AI189667, AA284766,
AA180305,
AW 172788, AA402989, AI990726,
AI948636,
AI866673, AW071781, H19479,
AA284767,
856450, T27036, AW237208,
AI500384,
AI678354, 239780, AI470278,
H20936,
AI270491, H84411, AA292332,
AI826066,
AA285099, H20935, AI336345,
H19561,
243720, AI541240, AW004032,
815049,
AI801457, AI948651, F04685,
AW135154,
AI290375, AI480181, AI611126,
AI982513,
AA179952, F21516,-AA634385,
841485,
245818, AI699619, AI791349,
Y17793,
AF064854, and AI673327.
7 HTTIJ31 17 1165265AL046746, AL041706, AA488903,
AW102955, AL120058, AI110760,
AI471543,
AI445582, AI631119, AI961232,
AW024544,
F28204, N23097, AA838140,
AI888852,
AA525409, AA568314, AI125167,
AI925321,
AL120343, AW410402, AA640305,
AW410844, AA736713, AA679538,
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
295
AW167374, AW265468, AA427470,
AA449661, AA604333, AI890283,
AA084439,
AA573024, AL121039, AI702049,
AL040921,
AA515677, AI547110, AI538404,
AI754257,
AI572680, AF034176, AW021674,
AI564209,
AI792586, AI792498, AI908381,
AI312267,
AI445768, AI345166, AW148821,
AI252611,
AI431434, AW411147, AW021608,
AA064983, AA533660, AA584360,
AW 162288, AA502104, AW069110,
AA610644, AC009516, AP000553,
AC007227,
AL022322, AL133448, 297630,
AC006449,
AC005529, AL034423, AC005399,
AL031662,
AC005484, AL031311, AL022165,
299716,
AL008719, AP000692, AC003043,
AC006285,
L44140, AC004812, AC016025,
AC006088,
AC004263, AC005015, AC006006,
AC005971, AP000501, AC005531,
283844,
AC005102, AP000503, AL0224'76,
AC005288,
284469, AC005695, AC005231,
AC003688,
AC004890, AC005527, AC004895,
298048,
AC004703, AL034420, AC005500,
Y14768,
AL022326, AC004491, AL008582,
AC000025,
M63796, AL096791, 299128,
AL121653,
AC004560, U62317, AF207550,
AC006312,
AC004821, AC004033, AC006538,
298051,
AC004851, AF196779, 295113,
AC007676,
AL022316, AL109628, AJ246003,
AC005755,
AC007055, AL049869, AC002314,
AL009179,
AC004253, AC005049, AC005081,
AC004019, AC004922, AF111168,
AL050318,
AL133163, AP000505, AP000555,
AL021154,
AL022320, AC004150, AC005808,
AL096701,
U52112, AC004531, AC002418,
AP000359,
297054, AC005331, AC002312,
AC004000,
AC006077, AC004999, AC007537,
AC005913, AC006064, AC002126,
AF053356,
AC005280, AL035072, AC005031,
AF001550,
AL035405, AC004167, AC005412,
AL023879,
AC004099, U91326, AC003006,
AC005785,
U80017, AC007421, AL133243,
AF109907,
AC00211-5, AP000557, AF045555,
AC007731,
AC002492, AL035079, AL109627,
AL021391,
298941, AL033521, AC004386,
AC004972,
AL022163, AC005821, AL035455,
AC005071,
AC006084, AL049759, AC004883,
AC004876,
AL031846, AC002~96, AC005519,
AL109952,
AC004858, AL050321, AC006509,
AC006057,
AC004659, AL049761, AC005225,
U62292,
AC005914, AC002404, AC002039,
AC005209, AC007546, AC005619,
AC005180, AL008710, AL031767,
AC002553,
AC005200, AC005358, U91327,
AC005300,
AL031281, AL135744, AC004887,
AC005228,
AC000353, AL133245, AC002477,
AC007384,
AF003626, AL035086, U85195,
AP000691,
AC003689, AL035422, AB023048,
293017,
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
296
AL031255, AC006261, AC005562,
AC007666,
AF205588, AC004584, AC005736,
AL022336,
AC007308, AL022313, 285986,
AC005523,
' AL035659, AC002563, AL021453,
AC007435,
AL022315, AL008729, AL035249,
AE0006S8,
AC006001, AL022323, AC004638,
AC004477,
AL117258, AL117330, AP000356,
AC007993,
AC005089, U78027, AC003109,
D87675,
AL049776, 297056, AC007919,
AC005839,
281364, AL031848, AL031670,
AF134726,
AC005694, AC002310, AD000092,
282244,
AL035587, AC004832, AC005250,
AC010205,
AL031427, AL049636, AC004878,
AB023049,
AL008716, AF111167, AL121915,
AL049694,
AP000116, AC004973, 293241,
AL031657,
AC004134, AC007277, AC005086,
AC005911, AL121655, AC002395,
AL080243,
and U91321.
7 HTTIJ31 44 977169 AC009516 and AP000553.
8 HHTLB76 18 1138860AA829036, AI061313, AA449997,
AI733856,
AW328331, AI609972, AA535216,
AI612142,
AI755214, AI610941, AL079734,
AI279417,
AI7S4S67, AI5832S2, AA704393,
AA410788,
AI754105, AI912401, AI249365,
T74524,
AI962030, AL041375, AA584862,
AA483606,
AI926033, AI380617, AW237905,
AA225406,
AA570740, AW272294, AW327624,
AA838091, AA169245, AA420546,
T57767,
AI884383, H07953, AI491765,
AI635028,
AA502991, AI446623, AI114557,
AA349193,
AA568204, AW265359, AI792521,
AL037714,
AI859438, AA469327, AA829065,
AI251576,
AA862312, AW301771, AA228778,
AA828592, AA492584, AA297666,
AIS81193,
AI628859, AI249688, AI457596,
AI457389,
AA534064, AA812684, AA552989,
AJ229041,
AL050317, AC008179, AC005874,
AF134471,
297181, AJ003147, U82695,
AC004526,
AC005529, AL022162, AP000245,
AL022098,
AF031078; AF030876, AC006263,
AL031295,
AC007228, AC003037, AL049839,
AC004S34,
AC007401, AC006427, 285987,
AP000014,
AC002369, AC002123, AP000036,
AC002378,
AC004970, AL133238, AF031076,
AC006080,
AC005538, AL022336; AC001231,
AP000206,
AP000128, AC005527, AL023807,
AP000098,
AL031289, AC005520, AL078638,
AC.003035, .
AC005060, AC005533, AC003684,
AL022337,
AJ229042, AL031121, AL008729,
AL031681,
AL009031, AC002326, AP000501,
AC002302,
AF001550, AC004554, AC005284,
293930,
AC005192, AC004887, AF011889,
M63544,
AC004890, 284466, AL080243,
AC007073,
AC007376, AL034402, M63543,
AP000330,
AL031597, AL096703, U91321,
298941,
AC005914, AL049869, AC005531,
AC007919,
AC005799, AF111169, AC006928,
AC003657,
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
297
AL031123, 297353, AP000032,
D86995,
AC002347, AL078477, 298751,
AL022163,
285994, AP000260, AC004966,
AC002070,
AP000696, AL009181, AC004778,
AC005871,
AC007450, AL031737, AL034429,
AC007298,
AC005291, AC006961, AC007263,
AL031664,
AC004125, AP000099, AC005859,
AL034384,
AC005722, AC004967, AC006101,
AC003663, AP000212, AP000134,
AP000030,
AC004551, AP000466, AC004685,
AP000689,
AC005694, AC016831, L44140,
AC005703,
AL133244, AC008372, AC005036,
AC007731,
AC007057, AC004020, AC006211,
AP000013,
AC004865, AC007129, AC006992,
AC004674, AL121653, AP000295,
AC007917,
AC004787, AF003529, U95739,
298304,
AC002549, AC007051, 283826,
AL022326,
AJ251973, AL031733, AC004816,
AC006257,
AL049795, AC008041, AC005822,
AC005500,
AC004895, AL021939, AC005295,
282195,
AC004216, AC005699, AC005081,
AC004453, AC006252, AC002067,
AF196969,
AC002351, AC005377, AC007011,
AC007207, AC007276, AL 122003,
AP000103,
AP000109, AP000041, AC005702,
AL033525,
AL049712, 295331, 285996,
AC005274,
AL133445, AP000247, AL035420,
AC007993,
AP000121, AP000053, AP000168,
AL022320,
AC005697, AL008719, AC005255,
AC002288,
AP000111, AP000043, AC007151,
AL133163,
AC002365, 284572, AL008730,
AC002492,
AL031577, AL031297, AL132642,
283836,
AP000500, AF196970, AP000263,
AL031848,
AC004682, AC002045, AP000688,
AF 121782,
AC008115, AF015262, AC012384,
AC005245,
AB003151, AF003626, AL078593,
AC006203,
AC007684, AJ229043, AC002456,
AC004687;
AP000056, AC006011, AC004143,
298050,
AC003003, AL033397, AC006130,
AC005823,
AL034549, 282198, 298044,
AC005399,
AF091512, AC006505, and AC006160.
8 HHTLB76 45 1136207AL134524, AI525316, AA829036,
AI733856,
AI061313, AA535216, AI609972,
AI612142,
AI279417, AW328331, AI610941,
AL079734,
AI583252, AI912401, AA483606,
AI380617,
AI249365, AW237905, AA570740,
AI962030,
T74524, AA169245, AA704393,
AI755214,
AA838091, AA225406, AI251576,
AI792521,
AI754567, AI884383, AA568204,
AW272294,
AI754105, AA584862, AL037714,
AI635028,
AA502991, H07953, AI583466,
AW265359,
AA862312, AW301771, AA829065,
AA828047, AA828592, AL041375,
AI306232,
AI890971, AW274191, AA631517,
AA013168, AI114557; AI281431,
AI961983,
AI417469, AI053696, AI628859,
AA558404,
AI280266, AA629894, AA629902,
H53284,
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
298
AW021116, AW439703, AA019973,
AI310464; AI793172, AI793209,
AJ229041,
AL050317, AR038762, AF134471,
AC008179,
U82695, AP000245, AF031078,
AF030876,
AJ003147, AL022098, AC005874,
297181,
AP000036, M63544, AF031076,
AL031295,
AC002369, AC006263, M63543,
AL049839,
AC004534, AC002123, AC005913,
AC001231, 285987, AC006427,
AC007228,
AC004526, AL132642, AL133238,
AP000206,
AP000128, AC005538, AL009031,
AC002326,
AC006080, AC003684, AC003037,
AL031289,
AF011889, AL022337, AL078638,
AC007263,
AC005192, AL031737, AC004890,
AC005060,
AL031597, AC005036, 283826,
AC003035,
AC007073, AC007401, AL008729,
AC005533,
AF111169, AC005529, AP000330,
AC007130,
AJ229042, AL023807, AL049869,
AC005699,
285994, AC004966, AC006211,
AC002302,
AC004685, AC004554, 293020,
284466,
AL031733, AL022336, AC004967,
AP000260,
AL049712, AC007376, AC005859,
AL009181,
AL034384, D86995, AC006101,
AL096703,
AC005284, AL080243, U95739,
AC007917,
AJ229043, AC006515, AF003529,
295331,
AC007057, 285996, AC005799,
AC004887,
AC005694, AC007919, AP000099,
AL049795,
AP000030, AC006961, AC005527,
AF015262,
AC007298, AP000240, AC004778,
AC002378,
AL034429, AC007051, 298941,
AC002347,
AL022162, AC004787, AC005871,
AC008115,
AP000466, AP000696, AL035461,
AP000212,
AP000134, AF003626, AC005377,
AL022163,
AF001550, AF196970, AC007878,
AP000032,
AC005971, AP000689, AC007276,
AC007684,
AC005386, AL078593, AC008372,
U91321,
AC006928, AC007151, AL031681,
AP000694,
AC007129, 295113, AC003657,
AB003151,
AC004970, AC004551, AC005531,
AC005291, AL031123, AP000295,
AC005295,
AP000103, AC004020, AP000109,
AP000041,
AL008719, AC002351, AC005722,
AF196969,
268287, AC006257, AC016831,
AC002549,
AP000697, AC005245, AC009044,
AL133244,
AP000098, AC005255, 297353,
298044,
AC005822, AC005914, AL031121,
AC006974,
AC006160, AL049636, AL022326,
AF099810,
AL034402, AC002045, AC002067,
AP000501,
AC004816, AF091512, U07000,
AC005703;
AC007011, AC008041, AP000155,
AC003003,
AL122003, AC007731, AC006011,
AC007157,
AL121757, AC004674, AC006075,
AC005399,
AL022316, 282195, AP000263,
AC006992,
AL021977, AL031120, AC007450,
298304,
AC002070, AC005071, AL035089,
L44140,
AC006953, AP000247, AP000688,
AL121653,
AC004865, AC004963, AC005500,
AL078584,
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
299
AC003098, AP000201, AP000013,
AC007262,
AP000143, AC004682, AL049553,
AP000097,
AL133445, AC004453, AC005786,
AP000493,
298200, AC003663, AC004143,
AC005520,
AC005081, AL078477, AC006238,
AP000111,
AP000043, AL033525, and AP000555.
9 HTEGF16 19 1160908AA203365, AI813708, AW300554,
AW026370, AI654923, AA252931,
AW299408, N70033, AI859872,
AA977420,
AA629258, N46021, 877192,
AI659888,
AA252888, H11937, AA149537,
AI024609,
N69894, AI768599, AI420563,
AI472810,
AI810949, AA489009, N98886,
AI698854,
AA364127, 238327, AA488535,
AI187721,
AA488402, AI803900, AA309914,
and
AW612414.
9 HTEGF16 47 1049385877192, H11937, N46021, N69894,
N70033,
N 98886, AA203365, AA252888,
AA252931,
AA977420, AA488402, AA488535,
AA629258, AI024609, 238327,
AI420563,
AI654923, AI698854, AI768599,
AI803900,
AI810949, AI813708, AW026370,
AW299408,
AW300554, and AW612414.
9 HTEGF16 48 952104D51799, D80439, AA305409,
D80212,
D59859, C14331, D59619, D80210,
D80240,
D80166, D80157, D81030, C14014,
C14389,
D51060, D80219, D80164, D51423,
D80133,
D80253, D51759, AA514186,
D80268,
D80195, D59610, D80022, D80366,
D58283,
D80188, D80247, D80391, D80248,
D45260,
C06015, D59787, D59502, D59889,
D59467,
D59275, D80038, D80043, D80227,
D80045,
D81026, D57483, D80196, D80269,
D80024,
D59653, D51022, D50979, D80522,
D50995,
AA305578, C05695, D59927,
C15076,
C14429, D80241, D80193, D80302,
C03092,
AA514188, D80251, D80378,
D51103,
D81111, AW 177440, AW 178893,
D80064,
AW360811, D59373, D80949,
AA809122,
D58246, H67854, T11417, C14344,
F13647,
C75259, D51221, AW377671,
AW375405,
AI525920, AI525917, H67866,
AI525923,
T03269, T03116, AA514184,
AW360817,
AW360834, D80258, C14227,
D59503,
D59317, D80014, D59474, D59695,
AW366296, C14973, D51079,
AW178906,
AI535686, AW360844, D80228,
C14407,
AW 179328, T48593, D59551,
D58101,
AW375406, AW378534, AW179332,
AW377672, AW 179023, AW 178905,
AW177731, C14957, AW378528,
AWi78762,
AW179019, D60010, AW378533,
AW378532,
C14046, AW177501, AW177511,
AI525235,
221582, D60214, AW 179020,
AI525215,
AI525227, AW377676, AI557774,
AW352171,
AW360841, AW352170, AW178907,
AW 177733, AW 178908, AW 179024,
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
300
AW352120, AI525912, AI525925,
D51250,
D59627, AW 178914, AW 178774,
AW 176467,
AI525242, AW178986, AA285331,
AW 177505, AI557751, AI525222,
AW 178775,
D51053, AW367950, AW 178909,
AW 177456,
AW 179004, AW 179329, AW
178980, D45273,
C 14298, AW 178754, 230160,
AW 179018,
D51213, AW352158, C16955,
D80168,
AW378542, AW352117, C05763,
233452,
T02974, 217814, AW178781,
AW378539,
AW 178911, AW378543, AW378525,
AW378540, AW352163, AW360855,
AW 177734, AI525237, H67858,
AW 179009,
AW 179012, C04682, AW 177728,
AW 177722,
AI525238, T02868, AI910186,
AW369651,
C139S8, AI525928, N66429,
C14077, D80314,
D31458, AI525216, D50981,
F13796,
AW 177508, AI525228, AW 177497,
T03048,
AI525239, AR060385, A62298,
AR018138,
AR008278, A82595, A84916,
A62300,
AB028859, AJ132110, AF058696,
AB002449,
I50126, I50132, I50128, I50133,
I14842,
AR054175, AR008277, AR008281,
AR016514, A63261, X67155,
Y17187,
AR060138, A45456, Y17188,
A94995,
D26022, A26615, AR052274,
A43192,
. Y12724, A43190, AR038669,
A25909,
AR066488, Y09669, AR066487,
A70867,
A67220, D89785, A78862, D34614,
A30438,
AR062872, AR008443, I79511,
AR016691,
AR016690, U46128, A64136,
A68321,
D88547, 182448, D50010, X68127,
X82626,
X72378, AR008408, AF123263,
AR032065,
AR060133, and AR025207.
HISET33 20 1102795AL134524, AI142134, AL045327,
AL134110,
AL045328, AL038983, AL038878,
AL047163,
AL119324, AL042898, AL037727,
U46344,
AL039432, AW372827, AL135012,
AL037295, AL038838, AL048677,
AL037343,
AL039643, AI318479, AL038651,
AIS47295,
AL119457, D29033, AL037436,
AL037335,
AL037323, AL049018, AL119399,
AL037443,
AL038532, AW392670, AL038822,
AL042420, AL047611, AL042655,
AI431323,
AL045494, AL045891, AL042523,
AL038761,
AL037435, AL042741, AL040472,
299396,
AL043941, AL043321, AL044125,
AL043923,
AL043814, AL047012, AL041238,
AL044186,
AL040617, AL043845, AL041347,
AL042931,
AL040576, AL040193, AL045753,
AL041955,
AL040463, AL047170, AL044037,
AL041635,
AL040294, AL044064, AL041459,
AL041577,
AL038024, AL043089, AL044162,
AL047219,
AL040625, AL048657, AL045684,
AL041752,
AL046850, AL040768, AL045671,
AL046994,
AL046914, AL048714, AL039360,
AL038745,
AL119443, AL042519, AL043496,
AL040052,
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
301
AL042802, AL042508, AL043538,
AL040444,
AL040621, AL040464, AL042488,
AL046356,
AL040510, AL043467, AW363350,
AL043677, AL040839, AL043492,
AL041602,
AL044074, AL041730, AL041523,
AL043627,
AL041374, AL043848, AL043570,
AL047183,
AI431307, AL042135, AI623302,
AI432644,
AI431316, AI431238, AL042515,
AL046442,
AL041324, AL041133, AL042533,
AL039316,
AI432666, AL041098, AL040322,
AW384394,
AL042853, AL046392, AL042832,
AI431235,
AI431315, AL040119, AW363220,
AL044272,
AL044258, AL043091, AL037341,
AL042468,
AL134920, AL041096, AL119396,
AL042729,
AL042096, AI431246, AL043166,
AL042842,
AL042787, AL041168, AL119497,
AL038040,
AW081103, AI432653, AI431230,
AI431321,
U46350, AL041163, AL041.1~59,
AL119483,
AL045817, AI431257, AL045920,
AL040148,
AL119418, AL079852, AL047057,
AL040207,
AI432654, AL045326, AI432650,
AI432677,
AL040458, AL044187, U46349,
AL043278,
U46351, AL043295, AL038041,
AL119335,
AL041296, AW084068, U46341,
AI431328,
AL041358, AL041292, AL119319,
AL040571,
AL045990, AL047675, AL119341,
U46346,
AI431353, AL119522, AI431312,
AI432656,
AI431231, AI432655, AR066494,
AR064707,
AR023813, AJ238010, A93923,
A93916,
D 17247, A93931, AL 133053,
AR060234,
AL122101, AL133074, AF019249,
AL133049,
Y17793, A85203, A81671, AL133082,
AL133076, AL133068, AR069079,
AR054110,
and AB026436.
HISET33 49 977186AW392670, 299396, AL119396,
AL042965,
AW372827, AW384394, AL119497,
AL119401, AL119443, AL119483,
AW363220, AL119418, AL134524,
U46350,
AL134533, U46346, AL119335,
AL119522,
AL134902, AL134920, AL119341,
U46349,
U46341, U46351, AL119496,
AL042896,
AI142132, AL119399, AL134531,
AL134538,
AI142139, LJ46345, A81671,
AR060234,
AR066494, AR069079, AR054.110,
and
AB026436.
1 I HTFMX90 21 1055914AL134524, AL042420, AA515728,
AA829036, AI279417, AW237905,
AI887235,
AI358712, AI380617, AI912401,
AW419389,
AI926728, AA706495, AI244127,
AA225406,
AI755214, AI754105, AA572813,
AI754567,
AW026305, AI421950, AI904840,
AI419337,
AA704393, AI090377, AI224583,
AA573067,
AA640430, AI859438, AA502991,
AI612142,
AL118925, AA640410, AL031428,
AP000245,
AP000128, AP000206, AL109627,
AC005274,
AF139813, AC005740, AC004228,
AL034549,
282206, AL050331, AC005670,
AL109801,
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
302
AL079303, AP000503, AF134726,
AC005921,
AP000696, AC005520, AC003669,
AC005071,
AC006480, AC007371, AC005228,
AC006530, AC0072,16, AC002477,
AC006449, AL031589, AC007052,
AC007363,
AP000694, AC002301, AC002425,
AP000113,
AP000045, AL021154, AC004139,
AC003982,
AL049780; AL021155, AC005668,
AC005412,
AC003101, AP000211, AP000133,
AC006064,
AC004827, AC005399, AL121652,
AL031311,
AB014078, 273417, AC004020,
AC008179,
AP000697, AC005899, AL049697,
AL133244,
AL031664, AL022069, U80017,
AL096791,
AC004821, AC004859, AL022334,
AL137100,
AC004797, AC002549, AC007421,
AC006388, AC002365, L44140,
AC006501,
AL023803, AC002429, AC003663,
AC004805,
AP000514, AL021394, AC007707,
284469,
AC004181, AC002565, AC002073,
AC004765, AB020868, AL034420,
AJ003147,
U95742, AC006312, AC005031,
AC004774,
AC004841, AC007731, AC005011,
AF042484,
AP000497, AC005500, AL133396,
AC005039;
AL121655, AL035587, AC000353,
U91321,
AL049831, 282195, AC006441,
AL049839,
AC005920, AL033521, Y14768,
AC002045,
AL022399, AC004905, AC006211,
AL049759,
AL031602, AL133448, AC006061,
AC004079,
293241, AL031228, AC006487,
AC007773,
282194, AL035423, AC005701,
AL024507,
AP000505, AC007842, AP000240,
AC005696,
AL078477, 282172, AC004891,
AC006581,
AC005295, AL034554, AF165926,
AC002115,
AF064861, AL034548, AC005799,
AC004975,
AC005536, AC005585, 284486,
AL031737,
AL031670, AL035410, AC007298,
AF111168,
AL049871, AP000098, AC002302,
AC007917,
AB023048, AC005913, AC006597,
AC002401, AL078583, AC002558,
AL133353,
AP000563, AC007041, AC008125,
AL031666,
AL079304, AL133245, AP000114,
AP000046,
AL009181, 282215, AC005231,
285986,
AB000876, AC007686, AC005049,
AL035683,
M89651, AL035089, AL031283,
AC005901,
AP000557, AL133243, AC005409,
AC002314,
AC005778, AC004383, AC006115,
AC007243, AL096775, AC008372,
293017,
AC004972, U91326, AF124523,
AL109798,
AC006111, AC005939, AP000022,
AL035413,
AC005089, AC010205, AF064857,
AC005411,
AC005592, AC005225, AP000501,
AC002316,
AF196779, AC007151, AC005940,
AL035088,
AC006023, 297056, AC002080,
AC002449,
U95743, AC007386, AL031774,
AL121748,
AC004491, AC007226, AL021393,
AL109623,
AC005058, AC004125, 293244,
AL022316,
AC007868, AC005086, AC000063,
and
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
303
AL035407.
11 HTFMX90 51 1052131AA515728, AA829036, AI279417,
AW237905, AI887235, AI358712,
AI380617,
AI912401, AI244127, AI926728,
AI755214,
AA225406, AW419389, AI754105,
AA572813, AI754567, AA706495,
AW026305, AI421950, AI904840,
AI419337,
AA704393, AI090377, AI224583,
AA640430,
AI859438, AA573067, AA502991,
AA640410,
AL118925, AI311647, AI361090,
AC007216,
AC005274, U91321, AC007363,
AP000128,
AP000206, AC000353, AC006449,
AP000245,
Y10196, AC007052, AF111169,
AL031428,
AC006480, AC005740, AP000503,
AC004228,
AF139813, AL109801, 282206,
AC003669,
AP000696, AC003101, AL079303,
AC005670,
AL031589, AL109627, AC005412,
AC006530,
AC005921, AL049697, AP000694,
AC006501,
AC005520, AF134726, AL049780,
AC004139,
AC002301, AC003982, AC006061,
AC006064, AC003663, AC004765,
AL035410,
AC002477, AP000211, AP000133,
AC004827,
AL031311, AC002425, AC005668,
AC004020,
AC005039, AP000113, AP000045,
AL133244,
AC005228, U80017, AL034549,
AL022334,
AP000697, AC007277, AC004821,
AC002365,
AF042484, AL031664, AC004805,
AC006388,
U95742, AC006211, AL021155,
AL022069,
AL034420, AC005399, AC004905,
AL021154,
AC005778, AC004797, AB020868,
AJ003147,
293241, AC002395, AL121652,
AL096791,
AC008179, AB014078, AC005071,
AP000514,
284469, AP000497, AC007421,
AC007707,
AL049831, AL121655, AL035407,
293244,
AL078477, AC002073, AC002045,
AF111168,
AC006312, AL031737, AC002565,
AC005031,
AC004774, AB023048, AL133396,
AC004181,
AL050331, AL049759, AC004859,
AL031602,
AC005913, AC006581, AC004841,
AC002558, 273417, AL031228,
AL035587,
AC005011, AC005701, AC006115,
282195,
AC005920, AC005899, AC007842,
AL023803,
AC007773, Y14768, L44140,
AC005696,
AP000240, AL022399, AC002316,
282194,
282172, AL033521, AF165926,
AL133448,
AC007055, AL024507, AC005536,
AC005585,
AC004975, AF196779, AC002429,
AL035423,
AC006441, AF064861, AL049871~,
298946,
AC005295, AP000505, AC007371,
AC004125,
AC007731, AC002115, AL133353,
AC008372,
AL031666, AC005500, AP000022,
AL009181,
AC005005, AC007298, 285986,
AL133243,
AC004891, AC006088, AL031283,
AC005821,
282215, AC005049, AC005409,
AC004079,
AL137100, AP000098, AR036572,
U91328,
AC002302, AL021394, AC005088,
AC008125,
AC005690, AC004491, AL031670,
AF124523,
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
304
AL033392, AC010205, AC005411,
AL109798,
AC006487, AP000365, AP000114,
AP000046,
AC007243, AC002401, AE000658,
AL049757,
AC005231, AC004383, U91326,
AC004972,
AL079304, AC006111, AP000501,
AB000876,
AC002314, AC007227, AL121748,
AC007041,
AC005086, AF064857, AL049839,
293017,
AL035683, AL022238, AC006511,
AC005901,
AC004938, AC007677, AL078583,
AC003041,
AC008101, 298742, AL139054,
AC007917,
AC003035, AC007386, AC002544,
AC007066, AL0313.68, AC007686,
AL022328,
U85195, AC007221, AC004929,
and
AC002080.
12 HE8FD93 22 1091113AC007350.
12 HE8FD93 53 1046606AA142989, AA649871, AA150979,
AA730301, AI423201, and AC007350.
12 HE8FD93 54 948832 AC007350.
13 HSUMA53 23 1183531AW375619, AW375590, AI954621,
AW362044, H98087, AW205215,
AW205991,
AI361588, AW193731, AA588837,
AA534307, AI631442, AW291638,
AW408128, AI097266, AA731997,
H89823,
898300, W01922, AI656446,
898059,
AI432644, AI623302, AI431255,
AI431337,
AI431351, AI432654, AW128900,
AI432658,
AI432674, AI432661, AI431346,
AI791349,
AI432675, AI431353, AI431347,
AI432653,
AI431230, AI431328, AI431354,
AI432655,
AI431310, AI431312, AI431241,
AI431345,
AW081103, AI432677, AI431254,
AI432651,
AI432647, AI432665, AI431357,
AI432673,
AI432649, AI431243, AI492519,
AI431330,
AI432672, AI431248, AI432662,
AI432676,
AW 128846, AI431340, AW 128897,
AI432664,
AI432650, AI431307, AI431316,
AI492520,
AI432643, AI492509, AI431751,
AW128884,
AI432657, AI431352, AI431247,
AI431356,
AI492510, AI431308, AI432645,
AW 129223,
AB006624, Y17793, AF064854,
AF019249,
and AF048686.
13 HSUMA53 55 951341 AA380314, AA374807, and AB006624.
13 HSUMA53 56 898943 AW375619, AW375590, AI954621,
AW362044, H98087, AW205215,
AW205991,
AI361588, AW193731, AA588837,
AA534307, AI631442, AW291638,
AW408128, AI097266, AA731997,
H89823,
898300, W01922, AI656446,
898059,
. AI432644, AI623302, AI431255,
AI431337,
AI431351, AI432654, AW128900,
AI432658,
AI432674, AI432661, AI431346,
AI791349, .
AI432675, AI431353, AI431347,
AI432653,
AI431230, AI431328, AI431354,
AI432655,
AI431310, AI431312, AI431241,
AI431345,
AW081103, AI432677, AI431254,
AI432651,
AI432647, AI432665, AI431357,
AI432673,
AI432649, AI431243, AI492519,
AI431330,
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
305
AI432672, AI431248, AI432662,
AI432676,
AW 128846, AI431340, AW 128897,
AI432664,
AI432650, AI431307, AI431316,
AI492520,
AI432643, AI492509, AI431751,
AW128884,
AI432657, AI431352, AI431247,
AI431356,
AI492510, AI431308, AI432645,
AW 129223,
AB006624, Y17793, AF064854,
AF019249,
and AF048686.
14 HEOQX60 24 1035205AA614014, AW084519, AA903338,
AI244442, AI342240, AI962752,
AI717991,
AW139714, AA354504, AI660761,
AW207518, AW402742, D20168,
AA380395,
T29020, AW138271, AI219797,
AI041118,
F19235, AI798637, F16699,
AI201892,
AL047399, AI808637, AI564067,
AW393736,
AW393737, AA748165, AA918804,
AL041067, AA282129, AL041068,
AA282128, AA811883, AI921541,
AW393733; 299396, AL119443,
AW392670,
AW372827, AL119497, AL119418,
AL119457, AW384394, AL119496,
AL119483, U46341, AW363220,
U46349,
AL119324, U46346, U46351,
AL119391,
AL036418, AL038837, AL134524,
AA631969,
AL119335, AL119399, AL119484,
AL037051,
AL036725, U46350, AL119319,
AL042544,
AL134902, AL119363, AL119341,
AL119444,
AL119396, AL119439, U46347,
AL134518,
AL119355,.AI142132, AL119522,
AL037205,
AL042614, AL038509, AL036858,
AL036924,
AL039074, AL119401, AL134528,
U46345,
AL042965, AL042975, AL134525,
AL134538,
AL119464, AL042970,.AL042450,
AL042984,
AI142137, AL042551, AL037082,
AL043019,
AL043029, AL042542, AL037639,
AL037094,
AL036196, AL037526, AL043003,
AL119488,
AL036767, AL038851, AL037085,
AL037077,
AL036268, AL036190, AL038520,
AL037615,
AL036998, D30758, AR060234,
AR066494,
A81671, AR023813, AR064707,
AR054110,
AR069079, and AB026436.
14 HEOQX60 57 918920 AI253192, AI913253, AI597595,
AA713495,
AA972065, AA135967, AI928486,
AA621901,
AI261821, N87925, AA767100,
AA427998,.
AW375405, C14331, AA514186,
D80188,
D58283, D59275, AW366296,
D57483,
D80253, D80166, C14014, D51060,
D80024,
AA305409, C14389, D59859,
D80043,
D80366, D51799, D80248, D51423,
D59619,
D80210, D80240, D50979, D81030,
C14429,
D80212, D80022, D80219, D80195,
D59467,
D80391, D80164, D59610, D59787,
D80227,
AA305578, D59502, D80133,
D80522,
D81026, D80269, D5.9889,
D80196, D51022,
D80268, D50995, D59927, D80251,
C15076,
D80038, D80439, D80193, D80045,
D80241,
AA514188, AW360811, D80378,
AW177440,
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
306
D80247, D80302, AW 178893,
D59373,
AW377671, AA809122, C75259,
AI557751,
AI905856, T03269, T48593,
C05695,
AW 178906, AW 179328, D51103,
AW360844,
D51759, AW360817, D80157,
AW375406,
AW378534, AW179332, AW377672,
AW 179023, AW 178905, AW378532,
AW177501, AW177511, AW352171,
AW377676, AW178983, AW352170,
AW 177731, AW 178907, AW378528,
AW 178762, AW 179019, AW
179024,
AW352117, AW176467, D51250,
D80134,
AW360841, AW177505, D80132,
C14407,
D58253, AW 179020, AW 178775,
AW 178909,
AW 177456, AW 179329, AI535686,
AW 178980, AW 178914, AW
177733,
AW 178908, AW 178754, AW
179018,
AW352158, D52291, AW367967,
AW369651,
D59653, C06015, AW360834,
AW 178774,
AW352120, AW179004, D45260,
AW179012,
AW352163, AW378525, D51079,
F13647,
D80949, AW378543, D59627,
C14344,
AW 177728, D80258, H67854,
AW 179009,
AW 178911, AW 177722, AW367950,
AI910186, AW378540, D80168,
AW352174,
C03092, D80064, H67866, AW178781,
C14298, C14227, D59503, T11417,
AI525923,
C14957, D59317, D58101, D81111,
AW177734, AI525917, D58246,
AW178986,
D59474, 221582, D51221, AA514184,
D80228, T03116, AW 177508,
AW 177723,
D80014, AI525920, C14973,
AI525912,
AW378533, AW177497, D60010,
D51213,
D45273, AI525235, D59551,
AI525227,
D51097, C14046, D60214, AI557774,
N66429,
T03048, AI525215, C16955,
AI525242,
AA285331, AW378542, AI535959,
AI525925,
AI525222, AW378539, AI525237,
C05763,
233452, T02974, AW360855,
I25810, X99477,
A62298, A84916, A62300, A82595,
AR018138, Y17188, AJ132110,
AB028859,
AR008278, AF058696, AR016808,
A30438,
Y17187, A94995, AR060385,
X82626,
AB002449, X67155, D26022,
Y12724,
A25909, AR016514, A67220,
D89785,
A78862, D34614, AR008443,
I50126, I50132,
I50128, I50133, D88547, AR066488,
AR060138, A45456, A26615,
AR052274,
A43190, AR038669, Y09669,
X68127,
A43192, AR025207, AR066487,
AR008277,
AR008281, D50010, I14842,
AR054175,
U46128, AR016691, AR016690,
AR066490,
A63261, U79457, AR008408,
I18367,
AR062872, A70867, 282022,
D13509,
AR060133, AB012117, A64136,
A68321,
I79511, A44171, A85396, D88507,
AR066482,
AF123263, A85477; I19525,
A86792,
CA 02396719 2002-07-24
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AR032065, X93549, and AR008382.
15 HFXDI56 25 1218288T85435, T85337, AA904610,
AI220608,
AL117340, AP000080, and X55499.
15 HFXDI56 58 1218744T85435, T85337, AA904610,
AI220608,
AL117340, and AP000080.
15 HFXDI56 59 1035197AL117340.
15 HFXDI56 60 609313 T85435, T85337, AA904610,
AI220608,
AL117340, AP000080, and X55499.
16 HCLTHQ40 26 1179279AI826538, AL038664, AI051237,
AI267318,
AI688542, AI052104, AW 163010,
AI376453,
AW192514, AI818589, AW029328,
AI678648,
AI566340, AI972077, AI811155,
AI936746,
AI089502, AI372947, AI004230,
AI354532,
AL119666, AW074007, AI084362,
AI027083,
AI691080, AA862706, AA621070,
AI744332,
AI149953, AW242075, AI149949,
AI150745,
AI199180, AI625208, AI003733,
W20002,
AI627187, AW 130451, AW270647,
AI014764,
AI091649, AA041468, W55944,
AW149580,
AI445868, AW151070, AA148318,
AI092273,
AI005484, AI372493, AA040575,
AA595861,
AI689545, AI524423, AI521587,
AA342697,
AA908191, AI689268, AI270577,
AI952557,
AI372494, AI619883, AI538583,
AW263138,
AI026832, AA040673, W25901,
AI368864,
AW316596, AI539834, AA349447,
AA721376, AW292131, 819495,
AA662403,
AW085967, T75472, AA808860,
AA199620,
AI125767, AW090571, H20652,
N78681,
AA176087, N32970, AI074758,
AA740389,
AA814692, AA300365, AA329440,
AI547225,
AW275741, AA894651, AA302328,
AI680268, T48533, AA719848,
F13229,
AA383093, AA386145, AA970611,
AI536066,
D31244, 244196, H20558, AI350433,
AW243606, AI784415, AI676163,
AA063203,
D82747, W26208, AA471277,
AA903068,
AA664940, AA853050, AA897635,
AW021288, D31438, AI535982,
AI419708,
AI680414, AA090164, AA362084,
AA386197,
862151, AI250661, 862259,
W28043,
AI521566, AI611841, F10830,
AA355685,
AA343846, 843842, AA334321,
T69962,
AA093703, T24661, AA845417,
D56184,
AA332748, 240172, 839290,
T27330,
AI695489, D80027, 812857,
838429,
AI524545, AA095572, W 15187,
T28780,
F24108, AA176086, AW37.5368,
AA323934,
AA337850, D81428, AA373966,
AA344329,
AW293901, AA039822, AW375337,
T69912,
AI802643, F35697, AA848160,
AI523217,
AI241878, AA148317, H60591,
AI709179,
T25879, AA970902, N63253,
T79010,
AW 169467, T16724, AA093662,
N84238,
AW241154, AW270806, AI198549,
AW380896, AI630928, AL038665,
AI284509,
AI557186, AL041150, AI433157,
AI554821,
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AW 151136, AI539771, AI537677,
AI494201,
AI500659, AI815232, AI801325,
AI500523,
AI436429, AI582932, AI923989,
AI284517,
AI371228, AI500706, AI445237,
AI491776,
AW 151138, AI889189, AI521560,
AI500662,
AI889168, AI866573, AI633493,
AI434256,
AI805769, AI888661, AI284513,
AI888118,
AI889147, AI440252, AI610557,
AI366900,
AI538850, AI872423, AW 172723,
AI440263,
AI866469, AI434242, AI859991,
AI866786,
AI860003, AI242736, AI887499,
AL080007,
AW 151132, AL045500, AI866465,
D31885,
AE000658, U85195, AF223953,
AF172088,
AF133669, I89947, AL049938,
AL122101,
A58524, A58523, AF113019,
AL133072,
AL122049, AR011880, AL050138,
U35846,
AF111851, AF017437, AL049423,
Y09972,
AL133113, A77033, A77035,
AL110280,
AL050277, AL137459, AF124728,
E06743,
AR038854, AL137476, AL133640,
L31396,
L31397, AL137539, AL110221,
AL122110,
Y16645, AL133080, I33392,
AL133077,
AB029066, AL137550, E07361,
AF061943,
AC002471, AC005374, AF097996,
A65340,
AL050024, Y11587, AL133081,
AF185614,
AL137463, A21625, U91329,
AF076464,
AF113677, U67958, U58996,
U42766,
X72889, U30290, AF081195,
X00474,
AF028823, AL137526, AL117583,
AL117585,
AL122123, I09360, A18777,
AL049300,
AL117460, AF158248, AL049283,
X84990,
AL133075, U49434, AL133565,
S78214,
I00734, AF078844, AF113694,
AL133084,
AF118094, AL133070, AL110197,
AL133098,
AL049466, E00617, E00717,
E00778,
AL133015, A03736, AJ012755,
AL110269,
S61953, AL137298, E03671,
I42402, I46765,
AL117457, AL096744, AL050108,
AF102578,
AF104032, 566283, AL049447,
I89944,
AB031064, L19437, AL117649,
Y10823,
249216, X61399, AR034821,
AF100931,
AF113691, AL133558, A45787,
AL133608,
U78525, AL110222, X63574,
AF008439,
AW273036, AW473757, AW474360,
AW510479, AW514205, AW516993,
AW518951, AW630807, AW662059,
and
AW 664721. .
18 HKZCK47 28 1045503H95452, N30257, N40659, N41980,
W32409,
W32855, AA001776, AA747202,
AA876069,
D81852, D81929, D81992, AI373734,
AI299574, AI221043, AI910470,
and
AW 196676.
21 HRDEP41 31 1129342AI819066, AI096889, N57566,
AI652475,
AI762701, AI571414, AW072021,
AW136982,
AA770246, AI360284, AI185794,
AA653289,
AI632543, N39261, AI093864,
AI361124,
W78162, AW072658, AA434490,
AI092771,
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W79205, AL044476, AI126467,
AI377502,
AI371895, AA086103, AI768304,
N50074,
AI347630, W57608, AI004516,
AI684524,
AA702096, AI992245, AA434395,
AI360808,
AI359558, AA324953, AA781993,
AI933385,
AA026053, AI243341, AI654422,
H09521,
AA086102, AA789226, AA988193,
H09520,
245285, W28501, 240993, C04672,
AI623520,
AA969690, AI674810, AW138990,
AI214707,
AI368537, N46707, 249982,
U30172, and
249983.
22 HKGBJ74 32 1189265AW245735, AW245623, AW245881,
N57252,
AW409766, AW247020, AW250562,
AI885802, AW250222, AI829136,
H98166,
AW072012, AW409650, AA236737,
AA130880, AA081326, AA176211,
AA081347, AA618083, AA176210,
AA486434, AI092870, AI589540,
AW273066,
AI745511, AI953980, AW409675,
AA524975,
AW 173680, AW 149138, AW248461,
AA056429, T66277, AI686881,
AW246286,
AA633178, AI361334, AA313766,
AA081346,
AI972465, AA878148, AA828127,
AA135151,
AA490323, AA541297, AI208260,
AA448202,
N31322, AI283002, AW 102925,
AA132698,
AA024484, AA814452, AI278141,
AI338188,
AI802624, AA922736, N35048,
AI002040,
AA502359, AW079983, AA634343,
AA564581, AW251043, AI143056,
N21370,
AI087009, AL045365, AI038842,
AA081325,
AA235062, AA724588, AA234910,
AW272966, AA219233, AW245379,
AA219293, AA380625, AA135288,
AA147048, AW002314, AA627088,
244936,
T66190, AA604261, AA490427,
AA132809,
AW263614, AA845407, AI300044,
AA393562, AA939182, AA309709,
AA936143, AA298671, AA298672,
T90842,
802758, AA579652, AA025328,
AA297935,
AL135190, AI798307, AA054656,
AA296918,
F 12298, AI024248, AI480173,
N45060,
AW250136, AI952399, AA297613,
802757,
AA298705, AI469323, AA130935,
AW247374, AI766398, AA876843,
AA297025, AA346949, AW380104,
AA551740, AA285283, AA309007,
AI337489,
AA090542, T19272, AI799323,
T73385,
F09925, AA090691, AA598486,
AI627371,
AA102203, AA147239, AW246489,
AI358227, AW380065, AW409756,
AA759368, AI954095, AI434969,
AI241741,
AW 198112, AW 176261, AI612913,
AI554821,
AI680221, AI868204, AI613038,
AI580436,
AI345688, AI289791, AI961589,
AI698391,
AI440238, AI582932, AI583578,
AL120254,
AI669639, AI565031, AI537187,
AL039086,
AI973272, AW129929, AI433157,
AI702073,
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AI633125, AI432570; AW 172982,
AI539260,
AI690389; AL042745, AI345415,
AW084151,
AI610362, AA937574, AI687568,
AI43361 I,
AI590043, AI309306, AI584130,
AI251221,
AW022682, AW163834, AI638644,
AI927233,
AI559863, AI624938, AI699823,
AL042515,
AL037582, AI799183, AL037602,
AI582966,
AI583065, AI801793, AW 167146,
AW 167926,
_ AL040241, AI249389, AI161279,
N75779,
AL036954, AI475339, AI889189,
AA464646,
AL046466, AI623835, AI471909,
AI628325,
AI590134, 869938, AI524654,
AI318280,
AI886415, AA954134; AA595932,
AL118781,
AW 149069, AI933992, AI491710,
AI114703,
AI621341, AI254042, AW268080,
AW075669,
AI916419, AW089387, AI811840,
AA693331,
AI565172, AI345612, AW022636,
AI685517,
AL134830, AW020397, AI690748,
AW 150557, AI635016, AI679959,
AL043981,
AI954080, AW025279, AI524179,
AI446511,
AI280637, AF090385, AF161489,
AF046025,
AF110956, AB024303, AL137530,
U77594,
I89947, A65340, AF113699,
AL133619,
I33392, I09499, M92439, X99226,
AL049324,
AL117416, AL050138, AL137292,
AR034821,
X83508, I48978, AL137558,
S68736,
AB007812, AL136884, S36676,
AL133665,
I32738, AF200464, AL049938,
A65341,
AF079763,.AL137533, U73682,
AL117435,
A03736, AL133080, AF176651,
AF102578,
297214, E04233, AF038847,
237987,
AF177401, AF044323, AL096720,
AL137550,
U35846, E01614, E13364, AL080110,
A08913,
AL137480, AL080124, I26207,
AF026124,
A08912, AF139986, A08910,
AF106862,
AR020905, A08909, AL117587,
AR038854,
E02914, A08908, AF159615,
AF090900,
S77771, A12297, AL133049,
S76508, S78453,
AL049283, AL137459, AL110221,
AF113676,
AL137641, AF090934, AF183393,
AF090903,
AL110222, D83032, AF017437,
X80340,
S53987, X63574, I00734, S61953,
A18777,
AL122110, AF118070, AF107847,
AF069506,
U58996, AJ242859, X52128,
AF017790,
AF 106697, AL 133016, E00617,
E00717,
E00778, A21101, U72621, AF061573,
AL137476, A15345, I89931,
AF028823,
AL049339, A83556, A77033,
A77035,
AL117460, AF117657, AL080140,
AJ003118,
AL133568, AL117394, AL122093,
AL050092,
AL096751, AF032666, I49625,
AL137271,
U86379, AF000301, AL137529,
AF090901,
X65873, AL 122098, AF 1 I
1112, A08907,
AF100781, AL133558, A65336,
D44497,
I52013, AF047716, Y09972,
AL137548,
AL050366, A08911, I29004,
I89934, X98066,
AL133067, 282022, AL122100,
AL137711,
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AL080126, AL110280, X63410,.583456;
AL133104, AF126247, I48979,
AF141289,
582852, AB025103, U42031,
AL080127,
AL117578, M27260, AL110225,
AL110224,
X93495, AJ005690, X53587,
Y10655,
AL133637, AF118094, Y11254,
AL080159,
AR054987, U68233, I92592,
X92070,
M96857, AL023657, AF055917,
AF185576,
AF137367, AF008439, I68732,
AF013214,
AF017152, AF207750, AF113690,
L13297,
A07588, X66871, AF067728,
AL133640,
AF111849, AF125948, AF199027,
AL122121,
AF030513, AL137665, AL080163,
AF057300,
AF057299, AJ012755, S75997,
AL137526,
X99257, AF111851, U95114,
213966, E01812,
AF047443, U35146, AL137538,
AL133557,
AL050149, U57352, D16301,
AF081197,
AF081195, X72889, S78214,
AF113691.;
AF026816, AL137463, AL117648,
X82434,
_ ' AF113677, AL050277, A86558,
AF090943,
AL122050, AL137523, AL049382,
AL049314,
AF115392, X63162, AF153205,
AJ238278,
AL117585, AL080234, U96683,
S69510,
AL133075, U72620, A08916,
AR011880,
AF115410, and AW467171.
22 HKGBJ74 67 1025650AW245735, AW245623, AW245881,
N57252,
AW247020, AW250562, AI885802,
AW250222, AI829136, H98166,
AW072012,
AW409650, AA236737, AW409766,
AA130880, AA081326, AA176211,
AA081347, AA618083, AA 176210,
AA486434, AI092870, AI589540,
AW273066,
AI745511, AI953980, AW409675,
AA5249'7S,
AW 173680, AW 149138, AW248461,
T66277,
AI686881, AW246286, AA633178,
AAOS6429, AI361334, AA313766,
AA081346,
AI972465, AA878148, AA828127,
AA135151;
AA490323, AA541297, AI208260,
AA448202,
N31322, AI283002, AW102925,
AA132698,
AA024484, AA8144S2, AI278141,
AI338188,
AI802624, AA922736, N35048,
AI002040,
AA502359, AW079983, AA634343,
AA564581, AW251043, AI143056,
N21370,
AI038842, AA081325, AI087009,
AA235062,
AA724588, AA234910, AW272966,
AA219233, AW245379, AA219293,
AA380625, AA135288, AA147048,
AW002314, AA627088, 244936,
T66190,
AL04S365, AA490427, AA132809,
AW263614, AA845407, AI300044,
AA393562, AA939182, AA309709,
AA936143, AA298671, AA298672,
T90842,
802758, AA604261, AA579652,
AA025328,
AA297935, AL135190, AI798307,
AA054656,
AA296918, F12298, N45060,
AI024248,
AA297613, 802757, AA298705,
AI9S2399,
AI469323, AA130935, AI480173,
AW247374,
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AI766398, AA876843, AA297025,
AA346949,
AW250136, AW380104, AA551740,
AA285283, AA309007, AI337489,
AA090542,
T19272, AI799323, T73385,
F09925,
AA090691, AA598486, AI627371,
AA102203,
AW246489, AA147239, AI358227,
AW380065, AA759368, AW409756,
AI339364, AF090385, AF161489,
AF046025,
AF110956, AB024303, and AW467171.
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Having generally described the invention, the same will be more readily
understood by reference to the following examples, which are provided by way
of
illustration and are not intended as limiting.
Examples
Example 1: Isolation of a Selected cDl~lA Clone From the Deposited Sample
Each cDNA clone in a cited ATCC deposit is contained, in a plasmid vector.
Table 1 identifies the vectors used to construct the cDNA library from which-
each
clone was isolated. In many cases, the vector used to construct the library is
a phage
vector from which a plasmid has been excised. The table immediately below
correlates the related plasmid for each phage vector used in constructing the
cDNA
library. For example, where a particular clone is identified in Table 1 as
being
isolated in the vector "Lambda Zap," the corresponding deposited clone is in
"pBluescript."
Vector Used to Construct Library Corresponding Deposited Plasmid
Lambda Zap pBluescript (pBS)
Uni-Zap XR pBluescript (pBS)
Zap Express pBK
lafinid BA plafmid BA
pSportl pSportl
pCMVSport 2.0 pCMVSport 2.0
pCMVSport 3.0 pCMVSport 3.0
pCR~2.1 pCR2.1
Vectors Lambda Zap (U.S. Patent Nos. 5,128,256 and 5,286,636), Uni-Zap
XR (U.S. Patent Nos. 5,128, 256 and 5,286,636), Zap Express (U.S. Patent Nos.
5,128,256 and 5,286,636), pBluescript (pBS) (Short, J. M. et al., Nucleic
Acids Res.
16:7583-7600 (1988); Alting-Mees, M. A. and Short, J. M., Nucleic Acids Res.
17:9494 (1989)) and pBK (Aping-Mees, M. A. et al., Strategies 5:58-61 (1992))
are
commercially available from Stratagene Cloning Systems, Inc., 11011 N. Torrey
Pines Road, La Jolla, CA, 92037. pBS contains an ampicillin resistance gene
and -,
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314
pBK contains a neomycin resistance gene. Both can be transformed into E. coli
strain
XL-1 Blue, also available from Stratagene. pBS comes in 4 dorms SK+, SK-, KS+
and KS. The S and K refers to the orientation of the polylinker to the T7 and
T3
primer sequences which flank the polylinker region ("S" is for SacI and "K" is
for
KpnI which are the first sites on each respective end of the linker). "+" or "-
" refer to
the orientation of the fl origin of replication ("ori"), such that in one
orientation,
single stranded rescue initiated from the fl on generates sense strand DNA and
in the
other, antisense.
Vectors pSportl, pCMVSport 2.0 and pCMVSport 3.0, were obtained from
Life Technologies, Inc., P. O. Box 6009, Gaithersburg, MD 20897. All Sport
vectors
contain an ampicillin resistance gene and may be transformed into E. coli
strain
DH10B, also available from Life Technologies. (See, for instance, Gruber, C.
E., et
al., Focus 15:59 (1993).) Vector lafmid BA (Bento Soares, Columbia University,
NY) contains an ampicillin resistance gene and can be transformed into E. coli
strain
XL-1 Blue. Vector pCR~2.1, which is available from Invitrogen, 1600 Faraday
Avenue, Carlsbad, CA 92008, contains an ampicillin resistance gene and may be
transformed into E. coli strain DH10B, available from Life Technologies. (See,
for
instance, Clark, J. M., Nuc. Acids Res. 16:9677-9686 (1988) and Mead, D. et
al.,
Bio/Technology 9: (1991).) Preferably, a polynucleotide of the present
invention
does not comprise the phage vector sequences identified for the particular
clone in
Table 1, as well as the corresponding plasmid vector sequences designated
above.
The deposited material in the sample assigned the ATCC Deposit Number
cited in Table 1 for any given cDNA clone also may contain one or more
additional
plasmids, each comprising a cDNA clone different from that given clone. Thus,
deposits sharing the same ATCC Deposit Number contain at least a plasmid for
each
cDNA clone identified in Table 1. Typically, each ATCC deposit sample cited in
Table 1 comprises a mixture of approximately equal amounts (by weight) of
about 50
plasmid DNAs, each containing a different cDNA clone; but such a deposit
sample
may include plasmids for more or less than 50 cDNA clones, up to about 500
cDNA
clones.
Two approaches can be used to isolate a particular clone from the deposited
sample of plasmid DNAs cited for that clone in Table 1. First, a plasmid is
directly -.
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isolated by screening the clones using a polynucleotide probe corresponding to
SEQ
ID NO:X.
Particularly, a specific polynucleotide with 30-40 nucleotides is synthesized
using an Applied Biosystems DNA synthesizer according to the sequence
reported.
The oligonucleotide is labeled, for instance, with 32P-y-ATP using T4
polynucleotide
kinase and purified according to routine methods. (E.g., Maniatis et al.,
Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring, NY
(1982).)
The plasmid mixture is transformed into a suitable host, as indicated above
(such as
XL-1 Blue (Stratagene)) using techniques known to those of skill in the art,
such as
those provided by the vector supplier or in related publications or patents
cited above.
The transformants are plated on 1.5% agar plates (containing the appropriate
selection
agent, e.g., ampicillin) to a density of about 150 transformants (colonies)
per plate.
These plates are screened using Nylon membranes according to routine methods
for
bacterial colony screening (e.g., Sambrook et al., Molecular Cloning: A
Laboratory
Manual, 2nd Edit., (1989), Cold Spring Harbor Laboratory Press, pages 1.93 to
1.104), or other techniques known to those of skill in the art.
Alternatively, two primers of 17-20 nucleotides derived from both ends of the
SEQ ID NO:X (i.e., within the region of SEQ ID NO:X bounded~by the 5' NT and
the 3' NT of the clone defined in Table 1) are synthesized and used to amplify
the
desired cDNA using the deposited cDNA plasmid as a template. The polymerase
chain reaction is carried out under routine conditions, for instance, in 25 u1
of reaction
mixture with 0.5 ug of the above cDNA template. A convenient reaction mixture
is
1.5-5 mM MgClz, 0.01 % (w/v) gelatin, 20 uM each of dATP, dCTP, dGTP, dTTP, 25
pmol of each primer and 0.25 Unit of Taq polymerase. Thirty five cycles of PCR
(denaturation at 94 degree C for 1 min; annealing at 55 degree C for 1 min;
elongation
at 72 degree C for 1 min) are performed with a Perkirt-Elmer Cetus automated
thermal cycler. The amplified product is analyzed by agarose gel
electrophoresis and
the DNA band with expected molecular weight is excised and purified. The PCR
product is verified to be the selected sequence by subcloning and sequencing
the
DNA product.
Several methods are available for the identification of the 5' or 3' non-
coding
portions of a gene which may not be present in the deposited clone. These
methods
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include but are not limited to, filter probing, clone enrichment using
specific probes,
and protocols similar or identical to 5' and 3' "RACE" protocols which are
well
known in the art. For instance, a method similar to 5' RACE is available for
generating the missing 5' end of a desired full-length transcript. (Fromont-
Racine et
al., Nucleic Acids Res. 21(7):1683-1684 (1993).)
Briefly, a specific RNA oligonucleotide is ligated to the 5' ends of a
population of RNA presumably containing full-length gene RNA transcripts. A
primer set containing a primer specific to the ligated RNA oligonucleotide and
a
primer specific to a known sequence of the gene of interest is used to PCR
amplify
the 5' portion of the desired full-length gene. This amplified product may
then be
sequenced and used to generate the full length gene.
This above method starts with total RNA isolated from the desired source,
although poly-A+ RNA can be used. The RNA preparation, can then be treated
with
phosphatase if necessary to eliminate 5' phosphate groups on degraded or
damaged
RNA which may interfere with the later RNA lipase step. The phosphatase should
then be inactivated and the RNA treated with tobacco acid pyrophosphatase in
order
to remove the cap structure present at the 5' ends of messenger RNAs. This
reaction
leaves a 5' phosphate group at the S' end of the cap cleaved RNA which can
then be
ligated to an RNA oligonucleotide using T4 RNA lipase.
This modified RNA preparation is used as a template for first strand cDNA
synthesis using a gene specific oligonucleotide. The first strand synthesis
reaction is
used as a template for PCR amplification of the desired 5' end using a primer
specific
to the ligated RNA oligonucleotide and a primer specific to the known sequence
of
the gene of interest. The resultant product is then sequenced and analyzed to
confirm
that the 5' end sequence belongs to the desired gene.
Example 2: Isolation of Genomic Clones Corresponding to a Polynucleotide
A human genomic P1 library (Genomic Systems, Inc.) is screened by PCR
using primers selected for the cDNA sequence corresponding to SEQ 1D NO:X.,
according to the method described in Example 1. (See also, Sambrook.)
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Example 3: Tissue Distribution of Polypeptide
Tissue distribution of mRNA expression of polynucleotides of the present
invention is determined using protocols for Northern blot analysis, described
by,
among others, Sambrook et al. For example, a cDNA probe produced by the method
described in Example 1 is labeled with P32 using the rediprimeTM DNA labeling
system (Amersham Life. Science), according to manufacturer's instructions.
After
labeling, the probe is purified using CHROMA SPIN-100TM column (Clontech
Laboratories, Inc.), according to manufacturer's protocol number PT1200-1. The
purified labeled probe is then used to examine various human tissues for mRNA
expression.
Multiple Tissue Northern (MTN) blots containing various human tissues (H)
or human immune system tissues (IM) (Clontech) are examined with the labeled
probe using ExpressHybTM hybridization solution (Clontech) according to
manufacturer's protocol number PT1190-1. Following hybridization and washing,
the
blots are mounted and exposed to film at -70 degree C overnight, and the films
developed according to standard procedures.
Example 4: Chromosomal Mapping of the Polynucleotides
An oligonucleotide primer set is designed according to the sequence at the 5'
end of SEQ ID NO:X. This primer preferably spans about 100 nucleotides. This
primer set is then used in a polymerase chain reaction under the following set
of
conditions : 30 seconds,95 degree C; 1 minute, 56 degree C; 1 minute, 70
degree C.
This cycle is repeated 32 times followed by one 5 minute cycle at 70 degree C.
Human, mouse, and hamster DNA is used as template in addition to a somatic
cell
hybrid panel containing individual chromosomes or chromosome fragments (Bios,
Inc). The reactions is analyzed on either 8% polyacrylamide gels or 3.5 %
agarose
gels. Chromosome mapping is determined by the presence of an approximately 100
by PCR fragment in the particular somatic cell hybrid.
Example 5: Bacterial Expression of a Polypeptide
A polynucleotide encoding a polypeptide of the present invention is amplified
using PCR oligonucleotide primers corresponding to the 5' and 3' ends of the
DNA
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sequence, as outlined in Example 1, to synthesize insertion fragments. The
primers
used to amplify the cDNA insert should preferably contain restriction sites,
such as
BamHI and XbaI, at the 5' end of the primers in order to clone the amplified
product
into the expression vector. For example, BamHI and XbaI correspond to the
restriction enzyme sites on the bacterial expression vector pQE-9. (Qiagen,
Inc.,
Chatsworth, CA). This plasmid vector encodes antibiotic resistance (Amps, a
bacterial origin of replication (ori), an IPTG-regulatable promoter/operator
(P/0), a
ribosome binding site (RBS), a 6-histidine tag (6-His), and restriction enzyme
cloning
sites.
The pQE-9 vector is digested with BamHI and XbaI and the amplified
fragment is ligated into the pQE-9 vector maintaining the reading frame
initiated at
the bacterial RBS. The ligation mixture is then used to transform the E. coli
strain
M15/rep4 (Qiagen, Inc.) which contains multiple copies of the plasmid pREP4,
which
expresses the lacI repressor and also confers kanamycin resistance (Kan~.
Transformants are identified by their ability to grow on LB plates and
ampicillin/kanamycin resistant colonies are selected. Plasmid DNA is isolated
and
confirmed by restriction analysis.
Clones containing the desired constructs are grown overnight (0/N) in liquid
culture in LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml).
The O/N culture is used to inoculate a large culture at a ratio of 1:100 to
1:250. The
cells are grown to an optical density 600 (O.D.boo) of between 0.4 and 0.6.
IPTG
(Isopropyl-B-D-thiogalacto pyranoside) is then added to a final concentration
of 1
mM. IPTG induces by inactivating the lacI repressor, clearing the P/O leading
to
increased gene expression.
Cells are grown for an extra 3 to 4 hours. Cells are then harvested by
centrifugation (20 mins at 6000Xg). The cell pellet is solubilized in the
chaotropic
agent 6 Molar Guanidine HCl by stirring for 3-4 hours at 4 degree C. The cell
debris
is removed by centrifugation, and the supernatant containing the polypeptide
is loaded
onto a nickel-nitrilo-tri-acetic acid ("Ni-NTA") affinity resin column
(available from
QIAGEN, Inc., supra). Proteins with a 6 x His tag bind to the Ni-NTA resin
with
high affinity and can be purified in a simple one-step procedure (for details
see: The
QIAexpressionist (1995) QIAGEN, Inca, supra).
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Briefly, the supernatant is loaded onto the column in 6 M guanidine-HC1, pH
8, the column is first washed with 10 volumes of 6 M~ guanidine-HCI, pH 8,
then
washed with 10 volumes of 6 M guanidine-HCl pH 6, and finally the polypeptide
is
. eluted with 6 M guanidine-HCI, pH 5.
The purified protein is then renatured by dialyzing it against phosphate-
buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus 200 mM NaCI.
Alternatively, the protein can be successfully refolded while immobilized on
the Ni-
NTA column. The recommended conditions are as follows: renature using a linear
6M-1M urea gradient in 500 mM NaCI, 20% glycerol, 20 mM Tris/HCl pH 7.4,
containing protease inhibitors. The renaturation should be performed over a
period of
1.5 hours or more. After renaturation the proteins are eluted by the addition
of 250
mM immidazole. Immidazole is removed by a final dialyzing step against PBS or
50
mM sodium acetate pH 6 buffer plus 200 mM NaCI. The purified protein is stored
at
4 degree C or frozen at -80 degree C.
In addition to the above expression vector, the present invention further
includes an expression vector comprising phage operator and promoter elements
operatively linked to a polynucleotide of the present invention, called
pHE4a.. (ATCC
Accession Number 209645, deposited on February 25, 1998.) This vector
contains:
1 ) a neomycinphosphotransferase gene as a selection marker, 2) an E. coli
origin of
replication, 3) a T5 phage promoter sequence, 4) two lac operator sequences,
5) a
Shine-Delgarno sequence, and 6) the lactose operon repressor gene (lacIq). The
origin of replication (oriC) is derived from pUCl9 (LTI, Gaithersburg, MD).
The
promoter sequence and operator sequences are made synthetically. ,
DNA can be inserted into the pHEa by restricting the vector with NdeI and
XbaI, BamHI, XhoI, or Asp718, running the restricted product on a gel, and
isolating
the larger fragment (the stuffer fragment should be about 310 base pairs). The
DNA
insert is generated according to the PCR protocol described in Example l,
using PCR
primers having restriction sites for NdeI (5' primer) and XbaI, BamHI, XhoI,
or
Asp718 (3' primer). The PCR insert is gel purified and restricted with
compatible
enzymes. The insert and vector are ligated according to standard protocols.
The engineered vector could easily be substituted in the above protocol to
express protein in a bacterial system.
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Example 6: Purification of a Polypeptide from an Inclusion Body
The following alternative method can be used to purify a polypeptide
expressed in E coli when it is present in the form of inclusion bodies. Unless
otherwise specified, all of the following steps are conducted at 4-10 degree
C.
Upon completion of the production phase of the E. coli fermentation, the cell
culture is cooled to 4-10 degree C and the cells harvested by continuous
centrifugation at 15,000 rpm (Heraeus Sepatech). On the basis of the expected
yield
of protein per unit weight of cell paste and the amount of purified protein
required, an
appropriate amount of cell paste, by weight, is suspended in a buffer solution
containing 100 mM Tris, 50 mM EDTA, pH 7.4. The cells are dispersed to a
homogeneous suspension using a high shear mixer.
The cells are then lysed by passing the solution through a microfluidizer
(Microfuidics, Corp. or APV Gaulin, Inc.) twice at 4000-6000 psi. The
homogenate
15- is then mixed with NaCI solution to a final concentration of 0.5 M NaCI,
followed by
centrifugation at 7000 xg for 15 min. The resultant pellet is washed again
using O.SM
NaCI, 100 mM Tris, 50 mM EDTA, pH 7.4.
The resulting washed inclusion bodies are solubilized with 1.5 M guanidine
hydrochloride (GuHCI) for 2-4 hours. After 7000 xg centrifugation for 1 S
min., the
pellet is discarded and the polypeptide containing supernatant is incubated at
4 degree
C overnight to allow further GuHCI extraction. .
Following high speed centrifugation (30,000 xg) to remove insoluble particles,
the GuHCI solubilized protein is refolded by quickly mixing the GuHCI extract
with
20 volumes of buffer containing 50 mM sodium, pH 4.5, 150 mM NaCI; 2 mM EDTA
by vigorous stirnng. The refolded diluted protein solution is kept at 4 degree
C
without mixing for 12 hours prior to further purification steps.
To clarify the refolded polypeptide solution, a previously prepared tangential
filtration unit equipped with 0.16 um membrane filter with appropriate surface
area
(e.g., Filtron), equilibrated with 40 mM sodium acetate, pH 6.0 is employed.
The
filtered sample is loaded onto a canon exchange resin (e.g., Poros HS-50,
Perseptive
Biosystems). The column is washed with 40 mM sodium acetate, pH 6.0 and eluted
with 250 mM, 500 mM, 1000 mM, and 1500 mM NaCI in the same buffer, in a
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stepwise manner. The absorbance at 280 nm of the effluent is continuously
monitored. Fractions are collected and further analyzed by SDS-PAGE.
Fractions containing the polypeptide are then pooled and mixed with 4
volumes of water. The diluted sample is then loaded onto a previously prepared
set of
tandem columns of strong anion (Poros HQ-50, Perseptive Biosystems) and weak
anion (Poros CM-20, Perseptive Biosystems) exchange resins. The columns are
equilibrated with 40 mM sodium acetate, pH 6Ø Both columns are washed with
40
mM sodium acetate, pH 6.0, 200 mM NaCI. The CM-20 column is then eluted using
a 10 column volume linear gradient, ranging from 0.2 M NaCI, 50 mM sodium
acetate, pH 6.0 to 1.0 M NaCI, 50 mM sodium acetate, pH 6.5. Fractions are ,
collected under constant AZ$o monitoring of the effluent. Fractions containing
the
polypeptide (determined, for instance, by 16% SDS-PAGE) are then pooled.
The resultant polypeptide should exhibit greater than 95% purity after the
above refolding and purification steps. No major contaminant bands should be
observed from Commassie blue stained 16% SDS-PAGE gel when 5 ug of purified
protein is loaded. The purified protein can also be tested for endotoxin/LPS
contamination, and typically the LPS content is less than 0.1 ng/ml according
to LAL
assays. .
Example 7: Cloning and Expression of a Polvnentide in a Baculovirus
Expression System
In this example, the plasmid shuttle vector pA2 is used to insert a
polynucleotide into a baculovirus to express a polypeptide. This expression
vector
contains the strong polyhedrin promoter of the Autographa californica nuclear
polyhedrosis virus (AcMNPV) followed by convenient restriction sites such as
BamHI, Xba I and Asp718.~ The polyadenylation site of the simian virus 40
("SV40")
is used for efficient polyadenylation. For easy selection of recombinant
virus, the
plasmid contains the beta-galactosidase gene from E. coli under control of a
weak
Drosophila promoter in the same orientation, followed by the polyadenylation
signal
of the polyhedrin gene. The inserted genes are flanked on both sides by viral
sequences for cell-mediated homologous recombination with wild-type: viral DNA
to
generate a viable virus that express the cloned polynucleotide.
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Many other baculovirus vectors can be used in place of the vector above, such
as pAc373, pVL941, and pAcIMI, as one skilled in the art would readily
appreciate,
as long as the construct provides appropriately located signals for
transcription,
translation, secretion and the like, including a signal peptide and an in-
frame AUG as
required. Such vectors are described, for instance, in Luckow et al., Virology
170:31
39 (1989).
Specifically, the cDNA sequence contained in the deposited clone, including
the AUG initiation codon and the naturally associated leader sequence
identified in
Table 1, is amplified using the PCR protocol described in Example 1. If the
naturally '
occurnng signal sequence is used to produce the secreted protein, the pA2
vector does
not need a second signal peptide. Alternatively, the vector can be modified
(pA2 GP)
to include a baculovirus leader sequence, using the standard methods described
in
Summers et al., "A Manual of Methods for Baculovirus Vectors and Insect Cell
Culture Procedures," Texas Agricultural Experimental Station Bulletin No. 1555
(1987).
The amplified fragment is isolated from a 1 % agarose gel using a
commercially available kit ("Geneclean," BIO 101 Inc., La Jolla, Ca.). The
fragment
then is digested with appropriate restriction.enzymes and again purified on a
1%
agarose gel.
The plasmid is digested with the corresponding restriction enzymes and
optionally, can be dephosphorylated using calf intestinal phosphatase, using
routine
procedures known in the art. The DNA is then isolated from a 1 % agarose gel
using a
commercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Ca.).
The fragment and the dephosphorylated plasmid are ligated together with T4
DNA ligase. E. coli HB101 or other suitable E. coli hosts such as XL-1 Blue
(Stratagene Cloning Systems, La Jolla, CA) cells are transformed with the
ligation
mixture and spread on culture plates. Bacteria containing the plasmid are
identified
by digesting DNA from individual colonies and analyzing the digestion product
by
gel electrophoresis. The sequence of the cloned fragment is confirmed by DNA
sequencing.
Five ug of a plasmid containing the polynucleotide is co-transfected with 1.0
ug of a commercially available linearized baculovirus DNA ("BaculoGoldTM
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baculovirus DNA", Pharmingen, San Diego, CA), using the lipofection method
described by Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987).
One ug
of BaculoGoldTM virus DNA and 5 ug of the plasmid are mixed in a sterile well
of a
microtiter plate containing 50 u1 of serum-free Grace's medium (Life
Technologies
Inc., Gaithersburg, MD). Afterwards, 10 u1 Lipofectin plus 90 u1 Grace's
medium are
added, mixed and incubated for 15 minutes at room temperature. Then the
transfection mixture is added drop-wise to S~ insect cells (ATCC CRL 1711)
seeded
in a 35 mm tissue culture plate with 1 ml Grace's medium without serum. The
plate is
then incubated for 5 hours at 27 degrees C. The transfection solution is then
removed
from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal
calf
serum is added. Cultivation is then continued at 27 degrees C for four days.
After four days the supernatant is collected and a plaque assay is performed,
as described by Summers and Smith, supra. An agarose gel with "Blue Gal" (Life
Technologies Inc., Gaithersburg) is used to allow easy identification and
isolation of
gal-expressing clones, which produce blue-stained plaques. (A detailed
description of
a "plaque assay" of this type can also be found in the user's guide for insect
cell
culture and baculovirology distributed by Life Technologies Inc.,
Gaithersburg, page
9-10.) After appropriate incubation, blue stained plaques are picked with the
tip of a
micropipettor (e.g., Eppendorf). The agar containing the recombinant viruses
is then
resuspended in a microcentrifuge tube containing 200 u1 of Grace's medium and
the
suspension containing the recombinant baculovirus is used to infect S~ cells
seeded
in 35 mm dishes. Four days later the supernatants of these culture dishes are
harvested and then they are stored at 4 degree C.
To verify the expression of the polypeptide, Sf~ cells are grown in Grace's
medium supplemented with 10% heat-inactivated FBS. The cells are infected with
the recombinant baculovirus containing the polynucleotide at a multiplicity of
infection ("MOI") of about 2. If radiolabeled proteins are desired, 6 hours
later the
medium is removed and is replaced with SF900 II medium minus methionine and
cysteine (available from Life Technologies Inc., Rockville, MD). A$er 42
hours, 5
uCi of 35S-methionine and 5 uCi 35S-cysteine (available from Amersham) are
added.
The cells are further incubated for 16 hours and then are harvested by
centrifugation.
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The proteins in the supernatant as well as the intracellular proteins are
analyzed by
SDS-PAGE followed by autoradiogi-aphy (if radiolabeled).
Microsequencing of the amino acid sequence of the amino terminus of
purified protein may be used to determine the amino terminal sequence of the
produced protein.
Example 8: Expression of a Polyueutide in Mammalian Cells
The polypeptide of the present invention can be expressed in a mammalian
cell. A typical mammalian expression vector contains a promoter element, which
.
mediates the initiation of transcription of mRNA, a protein coding sequence,
and
signals required for the termination of transcription and polyadenylation of
the
transcript. Additional elements include enhancers, Kozak sequences and
intervening
sequences flanked by donor and acceptor sites for RNA splicing. Highly
efficient
transcription is achieved with the early and late promoters from SV40, the
long
terminal repeats (LTRs) from Retroviruses, e.g., RSV, HTLVI, HIVI and the
early
promoter of the cytomegalovirus (CMV). However, cellular elements can also be
used (e.g., the human actin promoter).
Suitable expression vectors for use, in practicing the present invention
include,
for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala, Sweden),
pRSVcat (ATCC 37152), pSV2dhfr,(ATCC 37146), pBCI2MI (ATCC 67109),
pCMVSport 2.0, and pCMVSport 3Ø Mammalian host cells that could be used
include, human Hela, 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells,
Cos 1,
Cos 7 and CV1, quail QC1-3 cells, mouse L cells and Chinese hamster ovary
(CHO)
cells.
Alternatively, the polypeptide can be expressed in stable cell lines
containing
the polynucleotide integrated into a chromosome. The co-transfection with a
selectable marker such as dhfr, gpt, neomycin, hygromycin allows the
identification
and isolation of the transfected cells.
The transfected gene can also be amplified to express large amounts-of the
encoded protein. The DHFR (dihydrofolate reductase) marker is useful in
developing
cell lines that carry several hundred or even several thousand copies of the
gene of
interest. (See, e.g., Alt, F. W., et al., J. Biol. Chem. 253:1357-1370 (1978);
Hamlin, J. _,
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L. and Ma, C., Biochem. et Biophys. Acta, 1097:107-143 (1990); Page, M. J. and
Sydenham, M. A., Biotechnology 9:64-68 (1991).) Another useful selection
marker
/is the enzyme glutamine synthase (GS) (Murphy et al., Biochem J. 227:277-279
(1991); Bebbington et al., Bio/Technology 10:169=175 (1992). Using these
markers,
the mammalian cells are grown in selective medium and the cells with the
highest
resistance are selected. These cell lines contain the amplified genes)
integrated into a
chromosome. Chinese hamster ovary (CHO) and NSO cells are often used for the
production of proteins.
Derivatives of the plasmid pSV2-dhfr (ATCC Accession No. 37146), the
expression vectors pC4 (ATCC Accession No. 209646) and pC6 (ATCC Accession
No.209647) contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen
et
al., Molecular and Cellular Biology; 438-447 (March, 1985)) plus a fragment of
the
CMV-enhancer (Boshart et al., Cell 41:521-530 (1985).) Multiple cloning sites,
e.g.,
with the restriction enzyme cleavage sites BamHI, XbaI and Asp718, facilitate
the
cloning of the gene of interest. The vectors also contain the 3' intron, the
polyadenylation and termination signal of the rat preproinsulin gene, and the
mouse
DHFR gene under control of the SV40 early promoter.
. Specifically, the plasmid pC6, for example, is digested with appropriate
restriction enzymes and then dephosphorylated using calf intestinal phosphates
by
procedures known in the art. The vector is then isolated from a 1 % agarose
gel.
A polynucleotide of the present invention is amplified according to the
protocol outlined in Example 1. If the naturally occurring signal sequence is
used to
produce the secreted protein, the vector does not need a second signal
peptide.
Alternatively, if the naturally occurring signal sequence is not used, the
vector can be
modified to include a heterologous signal sequence. (See, e.g., WO 96/34891.)
The amplified fragment is isolated from a 1 % agarose gel using a
commercially available kit ("Geneclean," BIO 101 Inc., La Jolla, Ca.). The
fragment
then is digested with appropriate restriction enzymes and again purified on a
1
agarose gel.
The amplified fragment is then digested with the same restriction enzyme and
purified on a 1 % agarose gel. The isolated fragment and the dephosphorylated
vector
are then ligated with T4 DNA ligase. E. coli HB101 or XL-1 Blue cells are then
_.
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transformed and bacteria are identified that contain the fragment inserted
into plasmid
pC6 using, for instance, restriction enzyme analysis.
Chinese hamster ovary cells lacking an active DHFR gene is used for
transfection. Five pg of the expression plasmid pC6 a pC4 is cotransfected
with 0.5
ug of the plasmid pSVneo using lipofectin (Felgner et al., supra). The plasmid
pSV2-
neo contains a dominant selectable marker, the neo gene from Tn5 encoding an
enzyme that confers resistance to a group of antibiotics including 6418. The
cells are
seeded in alpha minus MEM supplemented with 1 mg/ml 6418. After 2 days, the
cells are trypsinized and seeded in hybridoma cloning plates (Greiner,
Germany) in
alpha minus MEM supplemented with 10, 25, or 50 nglml of metothrexate plus 1
mg/ml 6418. After about 10-14 days single clones are trypsinized and then
seeded in
6-well petri dishes or 10 ml flasks using different concentrations of
methotrexate (50
nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones growing at the highest
concentrations of methotrexate are then transferred to new 6-well plates
containing
even higher concentrations of methotrexate (1 uM, 2 uM, 5 uM, 10 mM, 20 mM).
The same procedure is repeated until clones are obtained which grow at a
concentration of 100 - 200 uM. Expression of the desired gene product is
analyzed,
for instance, by SDS-PAGE and Western blot or by reversed phase HPLC analysis.
Example 9: Protein Fusions
The polypeptides of the present invention are preferably fused to other
proteins. These fusion proteins can be used for a variety of applications. For
example, fusion of the present polypeptides to His-tag, HA-tag, protein A, IgG
domains, and maltose binding protein facilitates purification. (See Example 5;
see
also EP A 394,827; Traunecker, et al., Nature 331:84-86 (1988).) Similarly,
fusion to
IgG-1, IgG-3, and albumin increases the halflife time in vivo. Nuclear
localization
signals fused to the polypeptides of the present invention can target the
protein to a
specific subcellular localization, while covalent heterodimer or homodimers
can
increase or decrease the activity of a fusion protein. Fusion proteins can
also create
chimeric molecules having more than one function. Finally, fusion proteins can
increase solubility and/or stability of the fused protein compared to the non-
fused
protein. All of the types of fusion proteins described above can be made by
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modifying the following protocol, which outlines the fusion of a polypeptide
to an
IgG molecule, or the protocol described in Example 5
Briefly, the human Fc portion of the IgG molecule can be PCR amplified,
using primers that span the 5' and 3' ends of the sequence described below.
These
primers also should have convenient restriction enzyme sites that will
facilitate
cloning into an expression vector, preferably a mammalian expression vector.
For example, if pC4 (Accession No. 209646) is used, the human Fc portion
can be ligated into the BamHI cloning site. Note that the 3' BamHI site should
be
destroyed. Next, the vector containing the human Fc portion is re-restricted
with
BamHI, linearizing the vector, and a polynucleotide of the present invention,
isolated
by the PCR protocol described in Example 1, is ligated into this BamHI site.
Note
that the polynucleotide is cloned without a stop codon, otherwise a fusion
protein will
not be produced.
If the naturally occurnng signal sequence is used to produce the secreted
protein, pC4 does not need a second signal peptide. Alternatively, if the
naturally
occurring signal sequence is not used, the vector can be modified to include a
heterologous signal sequence. (See, e.g., WO 96/34891
Human IgG Fc region:
GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACC
GTGCCCAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCC
AAA.ACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCG
TGGTGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTAC
GTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGC
AGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAG
GACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCT
CCCAACCCCCATCGAGAA.AACCATCTCCAA.AGCCAAAGGGCAGCCCCGA
GAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAA
CCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCG
CCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCAC
GCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCAC
CGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGA
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TGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCT
CCGGGTAAATGAGTGCGACGGCCGCGACTCTAGAGGAT (SEQ )17 NO:1)
Example 10: Production of an Antibody from a Polypeptide
The antibodies of the present invention can be prepared by a variety of
methods. (See, Current Protocols, Chapter 2.) As one example of such methods,
cells
expressing a polypeptide of the present invention is administered to an animal
to
induce the production of sera containing polyclonal antibodies. In a preferred
method, a preparation of the secreted protein is prepared and purified to
render it
substantially free of natural contaminants. Such a preparation is then
introduced into
an animal in order to produce polyclonal antisera of greater specific
activity.
In the most preferred method, the antibodies of the present invention are
monoclonal antibodies (or protein binding fragments thereof). Such monoclonal
antibodies can be prepared using hybridoma technology. (Kohler et al.,
Nature 256:495 (1975); Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler et
al.,
Eur. J. Immunol. 6:292 (1976); Hammerling et al., in: Monoclonal Antibodies
and T-
Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981).) In general, such
procedures
involve immunizing an animal (preferably a mouse) with polypeptide or, more
preferably, with a secreted polypeptide-expressing cell. Such cells may be
cultured in
any suitable tissue culture medium; however, it is preferable to culture cells
in Earle's
modified Eagle's medium supplemented with 10% fetal bovine serum (inactivated
at
about 56 degrees C), and supplemented with about 10 g/1 of nonessential amino
acids,
about 1,000 U/ml of penicillin, and about 100 ug/ml of streptomycin.
The splenocytes of such mice are extracted and fused with a suitable myeloma
cell line. Any suitable myeloma cell line may be employed in accordance with
the
present invention; however, it is preferable to employ the parent myeloma cell
line
(SP20), available from the ATCC. After fusion, the resulting hybridoma cells
are
selectively maintained in HAT medium, and then cloned by limiting dilution as
described by Wands et al. (Gastroenterology 80:225-232 (1981).) The hybridoma
.
cells obtained through such a selection are then assayed to identify clones
which
secrete antibodies capable of binding the polypeptide.
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Alternatively, additional antibodies capable of binding to the polypeptide can
be produced in a two-step procedure using anti-idiotypic antibodies. Such a
method
makes use of the fact that antibodies are themselves antigens, and therefore,
it is
possible to obtain an antibody which binds to a second antibody. In accordance
with
this method, protein specific antibodies are used to immunize an animal,
preferably a
mouse. The splenocytes of such an animal are then used to produce hybridoma
cells,
and the hybridoma cells are screened to identify clones which produce an
antibody
whose ability to bind to the protein-specific antibody can be blocked by the
polypeptide. Such antibodies comprise anti-idiotypic antibodies to the
protein=
specific antibody and can be used to immunize an animal to induce formation of
further protein-specific antibodies.
It will be appreciated that Fab and F(ab')2 and other fragments of the
antibodies of the present invention may be used according to the methods
disclosed
herein. Such fragments are typically produced by proteolytic cleavage, using
enzymes such as papain (to produce Fab fragments) or pepsin (to produce
F(ab')2
fragments). Alternatively, secreted protein-binding fragments can be produced
through the application of recombinant DNA technology or through synthetic
chemistry.
For in vivo use of antibodies in humans, it may be preferable to use
"humanized" chimeric monoclonal antibodies. Such antibodies can be produced
using genetic constructs derived from hybridoma cells producing the monoclonal
antibodies described above. Methods for producing chimeric antibodies are
known in
the art. (See, for review, Morrison, Science 229:1202 (1985); Oi et al.,
BioTechniques 4:214 (1986); Cabilly et al., U.S. Patent No. 4,816,567;
Taniguchi et
al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533;
Robinson
et al., WO 8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al.,
Nature
314:268 (1985).)
Example 11: Production Of Secreted Protein For High-Throughput Screening
As_ says
The following protocol produces a supernatant containing a polypeptide to be
tested. This supernatant can then be used in the Screening Assays described
herein. _.
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First, dilute Poly-D-Lysine (644 587 Boehringer-Mannheim) stock solution
( lmg/ml in PBS) 1:20 in PBS (w/o calcium or magnesium 17-516F Biowhittaker)
for .
a working solution of SOug/ml. Add 200 u1 of this solution to each well (24
well
plates) and incubate at RT for 20 minutes. Be sure to distribute the solution
over each
. well (note: a 12-channel pipetter may be used with tips on every other
channel).
Aspirate off the Poly-D-Lysine solution and rinse with lml PBS (Phosphate
Buffered
Saline). The PBS should remain in the well until just prior to plating the
cells and
plates may be poly-lysine coated in advance for up to two weeks.
Plate 293T cells (do not carry cells past P+20) at 2 x 105 cells/well in :5m1
DMEM(Dulbecco's Modified Eagle Medium)(with 4.5 G/L glucose and L-glutamine
(12-604F Biowhittaker))/10% heat inactivated FBS(14-503F Biowhittaker)/lx
Penstrep(17-602E Biowhittaker). Let the cells grow overnight.
The next day, mix together in a sterile solution basin: 300 u1 Lipofectamine
(18324-012' Gibco/BRL) and Sml Optimem I (31985070 Gibco/BRL)/96-well plate.
With a small volume mufti-channel pipetter, aliquot approximately tug of an
expression vector containing a polynucleotide insert, produced by the methods
described in Examples 8 or 9, into an appropriately labeled 96-well round
bottom
plate. With a mufti-channel pipetter, add SOuI of the Lipofectamine/Optimem I
mixture to each well. Pipette up and down gently to mix. Incubate at RT 15-45
minutes. After about 20 minutes, use a mufti-channel pipetter to add 1 SOuI
Optimem
I to each well. As a control, one plate of vector DNA lacking an insert should
be
transfected with each set of transfections.
Preferably, the transfection should be performed by tag-teaming the following
tasks. By tag-teaming, hands on time is cut in half, and the cells do not
spend too
much time on PBS. First, person A aspirates off the media.from four 24-well-
plates
of cells, and then person B rinses each well with .5-lml PBS. Person A then
aspirates
off PBS rinse, and person B, using alt-channel pip.etter with tips on every
other
channel, adds the 200u1 of DNA/Lipofectamine/Optimem I complex to the odd
wells
first, then to the even wells, to each row on the 24-well plates. Incubate at
37 degrees
C for 6 hours.
While cells are incubating, prepare appropriate media, either 1 %BSA in
DMEM with lx penstrep, or CHO-5 media (116.6 mg/L of CaCl2 (anhyd); 0.00130
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mg/L CuS04-5H20; 0.050 mg/L of Fe(N03)3-9Hz0; 0.417 mg/L of FeS04-7H20;
311.80 mg/L of Kcl; 28.64 mg/L of MgClz; 48.84 mg/L of MgS04; 6995.50 mg/L of
NaCI; 2400.0 mg/L of NaHC03; 62.50 mg/L of NaHZPOa-HzO; 71.02 mg/L of
NazHP04; .4320 mg/L of ZnS04-7H20; .002 mg/L of Arachidonic Acid ; 1.022 mg/L
of Cholesterol; .070 mg/L of DL-alpha-Tocopherol-Acetate; 0.0520 mg/L of
Linoleic
Acid; 0.010 mg/L of Linolenic Acid; 0.010 mg/L of Myristic Acid; 0.010 mg/L of
Oleic Acid; 0.010 mg/L of Palmitric Acid; 0.010 mg/L of Palinitic Acid; 100
mg/L of
Pluronic F-68; 0.010 mg/L of Stearic Acid; 2.20 mg/L of Tween 80; 4551 mg/L of
D-
Glucose; 130.85 mg/ml of L- Alanine; 147.50 mg/ml of L-Arginine-HCL; 7.50
mglml
of L-Asparagine-HzO; 6.65 mg/ml of L-Aspartic Acid; 29.56 mg/ml of L-Cystine-
2HCL-HzO; 31.29 mg/ml of L-Cystine-2HCL; 7.35 mg/ml of L-Glutamic Acid; 365.0
mg/ml of L-Glutamine; 18.75 mg/ml of Glycine; 52.48 mg/ml.of L-Histidine-HCL-
HzO; 106.97 mg/ml of L-Isoleucine; 111.45 mg/ml of L-Leucine; 163.75 mg/ml of
L-
Lysine HCL; 32.34 mg/ml of L-Methionine; 68.48 mg/ml of L-Phenylalainine; 40.0
mg/ml of L-Proline; 26.25 mg/ml of L-Serine; 101.05 mg/ml of L-Threonine;
19.22
mg/ml of L-Tryptophan; 91.79 mg/ml of L-Tryrosine-2Na-2Hz0; 99.65 mg/ml of L-
Valine; 0.0035 mg/L of Biotin; 3.24 mg/L of D-Ca Pantothenate; 11.78 mg/L of
Choline Chloride; 4.65 mg/L of Folic Acid; 15.60 mg/L of i-Inositol; 3.02 mg/L
of
Niacinamide; 3.00 mg/L of Pyridoxal HCL; 0.031 mg/L of Pyridoxine HCL; 0.319
mg/L of Riboflavin; 3.17 mg/L of Thiamine HCL; 0.365 mg/L of Thymidine; and
0.680 mg/L of Vitamin Biz; 25 mM of HEPES Buffer; 2.39 mg/L of Na
Hypoxanthine; 0.105 mg/L of Lipoic Acid; 0.081 mg/L of Sodium Putrescine-2HCL;
55.0 mg/L of Sodium Pyruvate; 0.0067 mg/L of Sodium Selenite; 20uM of
Ethanolamine; 0.122 mg/L-of Ferric Citrate; 41.70 mglL of Methyl-B-
Cyclodextrin
complexed with Linoleic Acid; 33.33 mg/L of Methyl-B-Cyclodextrin complexed
with Oleic Acid; and 10 mg/L of Methyl-B-Cyclodextrin complexed with Retinal)
with 2mm glutamine and 1 x penstrep. (BSA (81-068-3 Bayer) 100gm dissolved in
1 L
DMEM for a 10% BSA stock solution). Filter the media and collect 50 u1 for
endotoxin assay in 15m1 polystyrene conical.
The transfection reaction is terminated, preferably by tag-teaming, at the end
of the incubation period. Person A aspirates off the transfection media, while
person
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B adds 1.5m1 appropriate media to each well. Incubate at 37 degrees C for 45
or 72
hours depending on the media used: 1 %BSA for 45 hours or CHO-5 for 72 hours.
On day four, using a 300u1 multichannel pipetter, aliquot 600u1 in one lml
deep well plate and the remaining supernatant into a 2m1 deep well. The
supernatants
from each well can then be used in the assays described in Examples 13-20.
It is specifically understood that when activity is obtained in any of the
assays
described below using a supernatant, the activity originates from either the
polypeptide directly (e.g., as a secreted protein) or by the polypeptide
inducing
expression of other proteins, which are then secreted into the supernatant.
Thus, the
invention further provides a method of identifying the protein in the
supernatant
characterized by an activity in a particular assay.
Example 12: Construction of GAS Reporter Construct
One signal transduction pathway involved in the differentiation and
proliferation of cells is called the Jaks-STATs pathway. Activated proteins in
the
Jaks-STATs pathway bind to gamma activation site "GAS" elements or interferon-
sensitive responsive element ("ISRE"), located in the promoter of many genes.
The
binding of a protein to these elements alter the expression of the associated
gene.
GAS and ISRE elements are recognized by a class of transcription factors
called Signal Transducers and Activators of Transcription, or "STATs." There
are six
members of the STATs family. Statl and Stat3 are present in many cell types,
as is
Stat2 (as response to IFN-alpha is widespread). Stat4 is more restricted and
is not in
many cell types though it has. been found in T helper class I, cells after
treatment with
IL-12. StatS was originally called mammary growth factor, but has been found
at
higher concentrations in other cells including myeloid cells. It can be
activated in
tissue culture cells by many cytokines.
The STATs are activated to translocate from the cytoplasm to the nucleus
upon tyrosine phosphorylation by a set of kinases known as the Janus Kinase
("Jaks")
family. Jaks represent a distinct family of soluble tyrosine kinases and
include Tyk2,
Jakl, Jak2, and Jak3. These kinases display significant sequence similarity
and are
generally catalytically inactive in resting cells.
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The Jaks are activated by a wide range of receptors summarized in the Table
below. (Adapted from review by Schidler and Darnell, Ann. Rev. Biochem. 64:621-
51 (1995).) A cytokine receptor family, capable of activating Jaks, is divided
into two
groups: (a) Class 1 includes receptors for IL-2, IL-3, IL-4, IL-6, IL-7, IL-9,
IL-11, IL-
12, IL-15, Epo, PRL, GH, G-CSF, GM-CSF, LIF, CNTF, and thrombopoietin; and (b)
' Class 2 includes IFN-a, IFN-g, and IL-10. The Class 1 receptors share a
conserved
cysteine motif (a set of four conserved cysteines and one tryptophan) and a
WSXWS
motif (a membrane proximal region encoding Trp-Ser-Xxx-Trp-Ser (SEQ ID N0:2)).
Thus, on binding of a ligand to a receptor, Jaks are activated, which in turn
activate STATs, which then translocate and bind to GAS elements. This entire
process is encompassed in the Jaks-STATs signal transduction pathway.
Therefore, activation of the Jaks-STATs pathway, reflected by the binding of
the GAS or the ISRE element, can be used to indicate proteins involved in the
proliferation and differentiation of cells. For example, growth factors and
cytokines
are known to activate the Jaks-STATs pathway. (See Table below.) Thus, by
using
GAS elements linked to reporter molecules, activators of the Jaks-STATs
pathway
can be identified.
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JAKs STATS GAS(elements)
or ISRE
LiQand t1~1C2Jakl JaIQ Jak3
IFN family
IFN-a/B + + - - 1,2,3 ISRE
IFN-g + + - 1 GAS (IRF 1 >Lys6>IFP)
Il-10 + ? ? - 1,3
gp130 family -
IL-6 (Pleiotrophic)+ + + ? 1,3 GAS (IRF1>Lys6>IFP).
Il-11(Pleiotrophic)? + ? ? 1,3
OnM(Pleiotrophic) ? + + ? 1,3
LIF(Pleiotrophic)? + + ? 1,3
CNTF(Pleiotrophic) -/+ + + ? 1,3
G-CSF(Pleiotrophic)? + ? ? 1,3
IL-12(Pleiotrophic)+ _ + + 1,3
Q-C family
IL-2 (lymphocytes) - + - + 1,3,5GAS
IL-4 (lymph/myeloid)- + - + 6 GAS (IRF1 = IFP Ly6)(IgH)-
IL-7 (lymphocytes) - + - + 5 GAS
IL-9 (lymphocytes) - + - + 5 GAS
IL-13 (lymphocyte) - + ? ? 6 GAS
IL-15 ? + ? + 5 GAS
gp 140 family
IL-3 (myeloid) - - + - 5 GAS (IRF1>IFPLy6)
IL-5 (myeloid) - - + - S GAS
GM-CSF (myeloid) - - + - 5 GAS
Growth hormone family
GH ? - + - 5
PRL ? +/- + - 1,3,5
EPO ? - + - 5 GAS(B-CAS>IRF1=IFPLy6)
Receptor Tyrosine
Kinases
EGF ? + + - 1,3 GAS (IRF1)
PDGF ? + + . 1,3
-
CSF-1 ? + + - 1,3 GAS (not IRF1)
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To construct a synthetic GAS containing promoter element, which is used in
the Biological Assays described in Examples 13-14, a PCR based strategy is
employed to generate a GAS-SV40 promoter sequence. The 5' primer contains four
tandem copies of the GAS binding site found in the IRF1 promoter and
previously
demonstrated to bind STATs upon induction with a range of cytokines (Rothman
et
al., Immunity l :457-468 (1994).), although other GAS or ISRE elements can be
used
instead. The 5' primer also contains l8bp of sequence complementary to the
SV40
early promoter sequence and is flanked with an XhoI site. The sequence of the
5'
primer is:
5':GCGCCTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTT
CCCCGAAATGATTTCCCCGAAATATCTGCCATCTCAATTAG:3' (SEQ ID
N0:3)
The downstream primer is complementary to the SV40 promoter and is
flanked with a Hind III site: 5':GCGGCAAGCTTTTTGCAAAGCCTAGGC:3'
(SEQ ID N0:4)
PCR amplification is performed using the SV40 promoter template present in
the B-gal:promoter plasmid obtained from Clontech. The resulting PCR fragment
is
digested with XhoI/Hind III and subcloned into BLSK2-. (Stratagene.)
Sequencing
with forward and reverse primers confirms that the insert contains the
following
sequence:
5':CTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCC
GAAATGATTTCCCCGAAATATCTGCCATCTCAATTAGTCAGCAACCATAGT
CCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCA
TTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGC
CGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAG
GCCTAGGCTTTTGCAAAAAGCTT:3' (SEQ ID N0:5)
With this GAS promoter element linked to the SV40 promoter, a GAS:SEAP2
reporter construct is next engineered. Here, the reporter molecule is a
secreted
alkaline phosphatase, or "SEAP." Clearly, however, any reporter molecule can
be
instead of SEAP, in this or in any of the other Examples. Well known reporter
molecules that can be used instead of SEAP include chloramphenicol
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acetyltransferase (CAT), luciferase, alkaline phosphatase, B-galactosidase,
green
fluorescent protein (GFP), or any protein detectable by an antibody.
The above sequence confirmed synthetic GAS-SV40 promoter element is
subcloned into the pSEAP-Promoter vector obtained from Clontech using HindIII
and
XhoI, effectively replacing the SV40 promoter with the amplified GAS:SV40
promoter element, to create the GAS-SEAP vector. However, this vector does not
contain a neomycin resistance gene, and therefore, is not preferred for
mammalian
expression systems.
Thus, in order to generate mammalian stable cell lines expressing the GAS-
SEAP reporter, the GAS-SEAP cassette is removed from the GAS-SEAP vector using
SaII and NotI, and inserted into a backbone vector containing the neomycin
resistance
gene, such as pGFP-1 (Clontech), using these restriction sites in the multiple
cloning
site, to create the GAS-SEAP/Neo vector. Once this vector is transfected into
.
mammalian cells, this vector can then be used as a reporter molecule for GAS
binding
as described in Examples 13-14.
Other constructs can be made using the above description and replacing GAS
with a different promoter sequence. For example, construction of reporter
molecules
containing NFK-B and EGR promoter sequences are described in Examples 15 and
16. However, many other promoters can be substituted using the protocols
described
in these Examples. For instance, SRE, IL-2, NEAT, or Osteocalcin promoters can
be
substituted, alone or in combination (e.g., GAS/NF-KB/EGR, GAS/NF-KB, Il-
2/NFAT, or NF-KB/GAS). Similarly, other cell lines can be used to test
reporter
construct activity, such as HELA (epithelial), HUVEC (endothelial), Reh (B-
cell),
Saos-2 (osteoblast), HUVAC (aortic), or Cardiomyocyte.
Example 13: High-Throu~hnut Screening Assav for T-cell Activi
The following protocol is used to assess T-cell activity by identifying
factors,
and determining whether supernate containing a polypeptide of the invention
proliferates and/or differentiates T-cells. T-cell activity is assessed using
the
GAS/SEAP/Neo construct produced in Example 12. Thus, factors that increase
SEAP
activity indicate the ability to activate the Jaks-STATS signal transduction
pathway.
The T-cell used in this assay is Jurkat T-cells (ATCC Accession No. TIB-152),
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although Molt-3 cells (ATCC Accession No. CRL-1552) and Molt-4 cells (ATCC
Accession No. CRL-1582) cells can also be used.
Jurkat T-cells are lymphoblastic CD4+ Thl helper cells. Iri order to generate
stable cell lines, approximately 2 million Jurkat cells are transfected with
the GAS-
SEAP/neo vector using DMRIE-C (Life Technologies)(transfection procedure
described below). The transfected cells are seeded to a density of
approximately
20,000 cells per well and transfectants resistant to 1 mg/ml genticin
selected.
Resistant colonies are expanded and then tested for their response to
increasing
concentrations of interferon gamma. The dose response of a selected clone is
demonstrated.
Specifically, the following protocol will yield sufficient cells for 75 wells
containing 200 u1 of cells. Thus, it is either scaled up, or performed in
multiple to
generate sufficient cells for multiple 96 well plates. Jurkat cells are
maintained in
RPMI + 10% serum with.1 %Pen-Strep. Combine 2.5 mls of OPTI-MEM (Life
Technologies) with 10 ug of plasmid DNA in a T25 flask. Add 2.5 ml OPTI-MEM
containing 50 u1 of DMRIE-C and incubate at room temperature for 15-45 mins.
During the incubation period, count cell concentration, spin down the required
number of cells (10' per transfection), and resuspend in OPTI-MEM to a final
concentration of 10' cells/ml. Then add lml of 1 x 10' cells in OPTI-MEM to
T25
flask and incubate at 37 degrees C for 6 hrs. After the incubation, add 10 ml
of RPMI
+ 15% serum.
The Jurkat:GAS-SEAP stable reporter lines are maintained in RPMI + 10%
serum, 1 mg/ml Genticin, and 1 % Pen-Strep. These cells are treated with
supernatants containing polypeptides of the invention and/or induced
polypeptides of
the invention as produced by the protocol described in Example 11.
On the day of treatment with the supernatant, the cells should be washed and
resuspended in fresh RPMI + 10% serum fo a density of 500,000 cells per ml.
The
exact number of cells required will depend on the number of supernatants being
screened. For one 96 well plate, approximately 10 million cells (for 10
plates, 100
million cells) are required.
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Transfer the cells to a triangular reservoir boat, in order to dispense the
cells
into a 96 well dish, using a 12 channel pipette. Using a 12 channel pipette;
transfer
200 u1 of cells into each well (therefore adding 100, 000 cells per well).
After all the plates have been seeded, 50 u1 of the supernatants are
transferred
S directly from the 96 well plate containing the supernatants into each well
using a 12
channel pipette. In addition, a dose of exogenous interferon gamma (0.1, 1.0,
10 ng)
is added to wells H9, H10, and H11 to serve as additional positive controls
for the
assay.
The 96 well dishes containing Jurkat cells treated with supernatants are
placed
in an incubator for 48 hrs (note: this time is variable between 48-72 hrs). 35
u1
samples from each well are then transferred to an opaque 96 well plate using a
12
channel pipette. The opaque plates should be covered (using sellophene covers)
and
stored at -20 degrees C until SEAP assays are performed according to Example
17.
The plates containing the remaining treated cells are placed at 4 degrees C
and serve
as a source of material for repeating the assay on a specific well if desired.
As a positive control, 100 Unit/ml interferon gamma can be used which is
known to activate Jurkat T cells. Over 30 fold induction is typically observed
in the
positive control wells.
The above protocol may be used in the generation of both transient, as well
as,
stable transfected cells, which would be apparent to those of skill in the
art.
Example 14: High-Throughput ScreeninyAssay Identifying Myeloid Activity
The following protocol is used to assess myeloid activity by determining
whether polypeptides of the invention proliferates and/or differentiates
myeloid cells.
Myeloid cell activity is assessed using the GAS/SEAP/Neo construct produced in
Example 12. Thus, factors that increase SEAP activity indicate the ability to
activate
the Jaks-STATS signal transduction pathway. The myeloid cell used in this
assay is
U937, a pre-monocyte cell line, although TF-1, HL60, or KG1 can be used.
To transiently transfect U937 cells with the GAS/SEAP/Neo construct
produced in Example 12, a DEAE-Dextran method (Kharbanda et. al., 1994, Cell
Growth & Differentiation, 5:259-265) is used. First, harvest 2x 10e7 U937
cells and-
wash with PBS. The U937 cells are usually grown in RPMI 1640 medium containing
-
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10% heat-inactivated fetal bovine serum (FBS) supplemented with 100 units/ml
penicillin and 100 mg/ml streptomycin.
Next, suspend the cells in 1 ml of 20 mM Tris-HCl (pH 7.4) buffer containing
0.5 mg/ml DEAF-Dextran, 8 ug GAS-SEAP2 plasmid DNA, 140 mM NaCI, 5 mM
KCI, 375 uM Na2HP04.7H20, 1 mM MgCl2, and 675 uM CaCl2. Incubate at 37
degrees C for 45 min.
Wash the cells with RPMI 1640 medium containing 10% FBS and then
resuspend in 10 ml complete medium and incubate at 37 degrees C for 36 hr.
The GAS-SEAP/LT937 stable cells are obtained by growing the cells in 400
ug/ml 6418. The 6418-free medium is used for routine growth but every one to
two
months, the cells should be re-grown in 400 ug/ml 6418 for couple of passages.
These cells are tested byharvesting 1x10$ cells (this is enough for ten 96-
well
plates assay) and wash with PBS. Suspend the cells in 200 ml above described
growth medium, with a final density of 5x105 cells/ml. Plate 200 u1 cells per
well in
the 96-well plate (or 1x105 cells/well):
Add 50 u1 of the supernatant prepared by the protocol described in Example
11. Incubate at 37 degrees C for 48 to 72 hr., As a positive control, 100
UnitJml
interferon gamma can be used which is known to activate U937 cells. Over 30
fold
induction is typically observed in the positive control wells. SEAP assay the
supernatant according to the protocol described in Example 17.
Example 15: High-Throughput Screening Assay Identifying Neuronal Activity.
When cells undergo differentiation and proliferation, a group of genes are
activated through many, different signal transduction pathways. One of these
genes,
EGR1 (early growth response gene 1), is induced in various tissues and cell
types
upon activation. The promoter of EGR1 is responsible for such induction. Using
the
EGR1 promoter linked to reporter molecules, activation of cells can be
assessed.
Particularly, the following protocol is used to assess neuronal activity in
PC12
cell lines. PC 12 cells (rat phenochromocytoma cells) are known to proliferate
and/or
differentiate by activation with a number of mitogens, such as TPA
(tetradecanoyl
phorbol acetate), NGF (nerve growth factor), and EGF (epidermal growth
factor).
The EGR1 gene expression is activated during this treatment. Thus, by stably
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transfecting PC 12 cells with a construct containing an EGR promoter linked to
SEAP
reporter, activation of PC12 cells can be assessed.
The EGR/SEAP reporter construct can be assembled by the following
protocol. The EGR-1 promoter sequence (-633 to +1)(Sakamoto K et al., Oncogene
S 6:867-871 (1991)) can be PCR amplified from human genomic DNA using the
following primers:
5' GCGCTCGAGGGATGACAGCGATAGAACCCCGG -3' (SEQ >D N0:6)
5' GCGAAGCTTCGCGACTCCCCGGATCCGCCTC-3' (SEQ )D N0:7)
Using the GAS:SEAP/Neo vector produced in Example 12, EGRl amplified
product can then be inserted into this vector. Linearize the GAS:SEAP/Neo
vector
using restriction enzymes XhoI/HindIII, removing the GAS/SV40 stuffer.
Restrict the
EGR1 amplified product with these same enzymes. Ligate the vector and the EGR1
promoter.
To prepare 96 well-plates for cell culture, two mls of a coating solution
(1:30
dilution of collagen type I (Upstate Biotech Inc. Cat#08-115) in 30% ethanol
(filter
sterilized)) is added per one 10 cm plate or 50 ml per,well of the 96-well
plate, and
allowed to air dry for 2 hr.
PC12 cells are routinely grown in RPMI-1640 medium (Bio Whittaker)
containing 10% horse serum (JRH BIOSCIENCES, Cat. # 12449-78P), 5% heat-
inactivated fetal bovine serum (FBS) supplemented with 100 units/ml penicillin
and
100 ug/ml streptomycin on a precoated 10 cm tissue culture dish. One to four
split is
done every three to four days. Cells are removed from the plates by scraping
and
resuspended with pipetting up and down for more than 15 times.
Transfect the EGR/SEAP/Neo construct into PC 12 using the Lipofectamine
protocol described in Example 11. EGR-SEAP/PC12 stable cells are obtained by
growing the cells in 300 ug/ml 6418. The 6418-free medium is used for routine
growth but every one~to two months, the cells should be re-grown in 300 ug/ml
6418
for couple of passages.
To assay for neuronal activity, a 10 cm plate with cells around 70 to 80%
confluent is screened by removing the old medium. Wash the cells once with PBS
(Phosphate buffered saline). Then starve the cells in low serum medium (RPMI-
1640
containing 1 % horse serum and 0.5% FBS with antibiotics) overnight. _
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The next morning, remove the medium and wash the cells with PBS. Scrape
off the cells from the plate, suspend the cells well in 2 ml low serum medium.
Count
the cell number and add more low serum medium to reach final cell density as
Sx 1 OS
cells/ml.
Add 200 u1 of the cell suspension to each well of 96-well plate (equivalent to
1x105 cells/well). Add SO u1 supernatant produced by Example 11, 37oC for 48
to 72
hr. As a positive control, a growth factor known to activate PC12 cells
through EGR
can be used, such as 50 ng/ul of Neuronal Growth Factor (NGF). Over fifty-fold
induction of SEAP is typically seen in the positive control wells. SEAP assay
the
supernatant according to Example 17.
Example 16: High-Throughput Screening Assay for T-cell Activity
NF-KB (Nuclear Factor KB) is a transcription factor activated by a wide
variety of agents including the inflammatory cytokines IL-1 and TNF, CD30 and
CD40, lymphotoxin-alpha and lymphotoxin-beta, by exposure to LPS or thrombin,
and by expression of certain viral gene products. As a transcription factor,
NF-KB
regulates the expression of genes involved in immune cell activation, control
of
apoptosis (NF- KB appears to shield cells from apoptosis), B and T-cell
development,
anti-viral and antimicrobial responses, and multiple stress responses.
In non-stimulated conditions, NF- KB is retained in the cytoplasm with I-KB
(Inhibitor KB). However, upon stimulation, I- KB is phosphorylated and
degraded,
causing NF- KB to shuttle to the nucleus, thereby activating transcription of
target
genes. Target genes activated by NF- KB include IL-2, IL-6, GM-CSF, ICAM=1 and
class 1 MHC.
Due to its central role and ability to respond to a range of stimuli, reporter
constructs utilizing the NF-KB promoter element are used to screen the
supernatants
produced in Example 11. Activators or inhibitors of NF-KB would be useful in
treating diseases. For example, inhibitors of NF-KB could be used to treat
those
diseases related to the acute or chronic activation of NF-KB, such as
rheumatoid
30. arthritis.
To construct a vector containing the NF-KB promoter element, a PCR based
strategy is employed. The upstream primer contains four tandem copies of the
NF-
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KB binding site (GGGGACTTTCCC) (SEQ ID N0:8), 18 by of sequence
complementary to the 5' end of the SV40 early promoter sequence, and is
flanked
with an XhoI site:
5':GCGGCCTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCC
GGGACTTTCCATCCTGCCATCTCAATTAG:3' (SEQ ID N0:9)
The downstream primer is complementary to the 3' end of.the SV40 promoter
and is flanked with a Hind III site:
S':GCGGCAAGCTTTTTGCAAAGCCTAGGC:3' (SEQ ID N0:4)
PCR amplification is performed using the SV40 promoter template present in
the pB-gal:promoter plasmid obtained from Clontech. The resulting PCR fragment
is
digested with XhoI and Hind III and subcloned into BLSK2-. (Stratagene)
Sequencing with the T7 and T3 primers confirms the insert contains the
following
sequence:.
~ 5':CTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGAC
TTTCCATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTC
CGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATG
GCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTG
AGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGC
AAAA.AGCTT:3' (SEQ ID NO:10)
Next, replace the SV40 minimal promoter element present in the pSEAP2-
promoter plasmid (Clontech) with this NF-KB/SV40 fragment using XhoI and
HindIII. However, this vector does not contain a neomycin resistance gene, and
therefore, is not preferred for mammalian expression systems.
In order to generate stable mammalian cell lines, the NF-KB/SV40/SEAP
cassette is removed from the above NF-KB/SEAP vector using restriction enzymes
SaII and NotI, and inserted into a vector containing neomycin resistance.
Particularly,
the NF-KB/SV40/SEAP cassette was inserted into pGFP-1 (Clontech), replacing
the
GFP gene, after restricting pGFP-1 with SaII and NotI.
Once NF-KB/SV40/SEAP/Neo vector is created, stable Jurkat T-cells are
created and maintained according to the protocol described in Example 13.
Similarly, _
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the method for assaying supernatants with these stable Jurkat T-cells is also
described
in Example ,13. As a positive control, exogenous TNF alpha (0.1, l, 10 ng) is
added to
wells H9, H10, and H11, with a 5-10 fold activation typically observed.
Example 17: Assay for SEAP Activity
As a reporter molecule for the assays described in Examples 13-16, SEAP
activity is assayed using the Tropix Phospho-light Kit (Cat. BP-400) according
to the
following general procedure. The Tropix Phospho-light Kit supplies the
Dilution,
Assay, and Reaction Buffers used below.
Prime a dispenser with the 2.5x Dilution Buffer and dispense 15 u1 of 2.5x
dilution buffer into Optiplates containing 35 u1 of a supernatant. Seal the
plates with
a plastic sealer and incubate at 65 degree C for 30 min. Separate the
Optiplates to
avoid uneven heating.
Cool the samples to room temperature for 15 minutes. Empty the dispenser
and prime with the Assay Buffer. Add 50 ml Assay Buffer and incubate at room
temperature 5 min. Empty the dispenser and prime with the Reaction Buffer (see
the
table below). Add 50 u1 Reaction Buffer and incubate at room temperature for
20
minutes. Since the intensity of the chemiluminescent signal is time dependent,
and it
takes about 10 minutes to read 5 plates on luminometer, one should treat 5
plates at
each time and start the second set 10 minutes later.
Read the relative light unit in the luminometer. Set H12 as blank, and print
the results. An increase in chemiluminescence indicates reporter activity.
Reaction Buffer Formulation:
# of Rxn buffer diluent CSPD
fates (ml) (ml)
10 60 3
11 65 3.25
12 . 70 3.5
13 . ' 75 3.75
~
14 80 4
15 - 85 4.25
16 90 4.5
17 95 4.75
18 100 S
19 105 5.25
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20 110 5.5
. 21 115 5.75
22 120 6
23 125 6.25
24 130 6.5
25 135 6.75
26 140 7
27 145 7.25
28 150 7.5
29 155 7.75
30 160 8
31 165 8.25
32 170 8.5
33 175 8.75
34 180 9
35 . 185 9.25
36 190 9.5
37 195 9.75
38 200 10
39 205 ~ 10.25
40 210 10.5
41 215 10.75
42 220 11
43 225 11.25
44 230 11.5
45 235 11.75
46 240 12
47 245 12.25
48 250 12.5
49 255 12.75
50 260 13
Example 18: High-Throughput Screening Assay Identifyin~ Changes in Small
-Molecule Concentration and Membrane Permeability
Binding of a ligand to a receptor is known to alter intracellular levels of
small
molecules, such as calcium, potassium, sodium, and pH, as well as alter
membrane
potential. These alterations can be measured in an assay to identify
supernatants
which bind to receptors of a particular cell. Although the following protocol
describes an assay for calcium, this protocol can easily be modified to detect
changes
in potassium, sodium, pH, membrane potential, or any other small molecule
which is
detectable by a fluorescent probe.
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The following assay uses Fluorometric Imaging Plate Reader ("FLIPR") to
measure changes in fluorescent molecules (Molecular Probes) that bind small
molecules. Clearly, any fluorescent molecule detecting a small molecule can be
used
instead of the calcium fluorescent molecule, fluo-4 (Molecular Probes, Inc.;
catalog
no. F-14202), used here.
For adherent cells, seed the cells at 10,000 -20,000 cells/well in a Co-star
black 96-well plate with clear bottom. The plate is incubated in a COZ
incubator for
20 hours. The adherent cells are washed two times in Biotek washer with 200 u1
of
HBSS (Hank's Balanced Salt Solution) leaving 100 u1 of buffer after the final
wash.
A stock solution of 1 mg/ml fluo-4 is made in 10% pluronic acid DMSO. To
load the cells with fluo-4 , 50 u1 of 12 ug/ml fluo-4 is added to each well.
The plate
is incubated at 37 degrees C in a COZ incubator for 60 min. The plate is
washed four
times in the Biotek washer with HBSS leaving 100 u1 of buffer.
For non-adherent cells, the cells are spun down from culture media. Cells are
re-suspended to 2-Sx 106 cells/ml with HBSS ~in a 50-ml conical tube. 4 u1 of
f mg/ml
fluo-4 solution in 10% pluronic acid DMSO is added to each ml of cell
suspension.
The tube is then placed in a 37 degrees C water bath for 30-60 min. The cells
are
washed twice with HBSS, resuspended to 1 x 1 O6 cells/ml, and dispensed into a
microplate, 100 ul/well. The plate is centrifuged at 1000 rpm for 5 min. The
plate is
then washed once in Denley CellWash with 200 u1, followed by an aspiration
step to
100 u1 final volume.
For a non-cell based assay, each yell contains a fluorescent molecule, such as
fluo-4 . .The supernatant is added to the well, and a change in fluorescence
is
detected.
To measure the fluorescence of intracellular calcium; the FLIPR is set for the
following parameters: (1) System gain is 300-800 mW; (2) Exposure time is 0.4
second; (3) Camera F/stop is F/2; (4) Excitation is 488 nm; (5) Emission is
530 nm;
and (6) Sample addition is 50 u1. Increased emission at 530 nm indicates an
extracellular signaling event which has resulted in an increase in the
intracellular
Ca++ concentration.
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Example 19: High-Throughput Screening Assay Identifying Tyrosine Kinase
Activi
The Protein Tyrosine Kinases (PTK) represent a diverse group of
transmembrane and cytoplasmic kinases. Within the Receptor Protein Tyrosine
Kinase RPTK) group are receptors for a range of mitogenic and metabolic growth
factors including the PDGF, FGF, EGF, NGF, HGF and Insulin receptor
subfamilies.
In addition there are a large family of RPTKs for which the corresponding
ligand is
unknown. Ligands for RPTKs include mainly secreted small proteins, but also
membrane-bound and extracellular matrix proteins.
Activation of RPTK by ligands involves ligand-mediated receptor
dimerization, resulting in transphosphorylation of the receptor subunits and
activation
of the cytoplasmic tyrosine kinases. The cytoplasmic tyrosine kinases include
receptor associated tyrosine kinases of the src-family (e.g., src, yes, lck,
lyn, fyn) and
non-receptor linked and cytosolic protein tyrosine kinases, such as the Jak
family,
1 S members of which mediate signal transduction triggered by the cytokine
superfamily
of receptors (e.g., the Interleukins, Interferons, GM-CSF, and Leptin).
Because of the wide range of known factors capable of stimulating tyrosine
kinase activity, the identification of novel human secreted proteins capable
of
activating tyrosine kinase signal transduction pathways are of interest.
Therefore, the
following protocol is designed to identify those novel human secreted proteins
capable of activating the tyrosine kinase signal transduction pathways.
Seed target cells (e.g., primary keratinocytes) at a density of approximately.
25,000 cells per well in a 96 well Loprodyne Silent Screen Plates purchased
from
Nalge Nunc (Naperville, IL). The plates are sterilized with two 30 minute
rinses with
100% ethanol, rinsed with water and dried overnight. Some plates are coated
for 2 hr
with 100 ml of cell culture grade type I collagen (50 mg/ml), gelatin (2%) or
polylysine (50 mg/ml), all of which can be purchased from Sigma Chemicals (St.
Louis, MO) or 10% Matrigel purchased from Becton Dickinson (Bedford,MA), or
calf serum, rinsed with PBS and stored at 4 degree C. Cell growth on these
plates is
assayed by seeding 5,000 cells/well in growth medium and indirect quantitation
of
cell number through use of alamarBlue as described by the manufacturer Alamar
Biosciences, Inc. (Sacramento, CA) after 48 hr. 'Falcon plate covers #3071
from
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Becton Dickinson (Bedford,MA) are used to cover the Loprodyne Silent Screen
Plates. Falcon Microtest III cell culture plates can also be used in some
proliferation
experiments.
To prepare extracts, A431 cells are seeded onto the nylon membranes of
Loprodyne plates (20,000/200m1/well) and cultured overnight in complete
medium.
Cells are quiesced by incubation in serum-free basal medium for 24 hr. After 5-
20
minutes treatment with EGF (60ng/ml) or 50 u1 of the supernatant produced in
Example 11, the medium was removed and 100 ml of extraction buffer ((20 mM
HEPES pH 7.5, 0.15 M NaCI, 1% Triton X-100, 0.1% SDS, 2 mM Na3V04, 2 mM
Na4P2O7 and a cocktail of protease inhibitors (# 1836170) obtained from
Boeheringer Mannheim (Indianapolis, IN) is added to each well and the plate is
shaken on a rotating shaker for 5 minutes at 4 degrees C. The plate is then
placed in a
vacuum transfer manifold and the extract filtered through the 0.45 mm membrane
bottoms of each well using house vacuum. Extracts are collected in a 96-well
catch/assay plate in the bottom of the vacuum manifold and immediately placed
on
ice. To obtain extracts clarified by centrifugation, the content of each well,
after
detergent solubilization for 5 minutes, is removed and centrifuged for 15
minutes at 4
degrees C at 16,000 x g.
Test the filtered extracts for levels of tyrosine kinase activity. Although
many
methods of detecting tyrosine kinase activity are known, one method is
described
here.
Generally, the tyrosine kinase activity of a supernatant is evaluated by
determining its ability to phosphorylate a tyrosine residue on a specific
substrate (a
biotinylated peptide). Biotinylated peptides that can be used for this purpose
include
PSK1 (corresponding to amino acids 6-20 of the cell division kinase cdc2-p34)
and
PSK2 (corresponding to amino acids 1-17 of gastrin). Both peptides are
substrates for
a range of tyrosine kinases and are available from Boehringer Mannheim
The tyrosine kinase reaction is set up by adding the following components in
order. First, add 10u1 of 5uM Biotinylated Peptide, then 10u1 ATP/Mg2+ (SmM
ATP/SOmM MgCl2), then 10u1 of Sx Assay Buffer (40mM imidazole hydrochloride,
pH7.3, 40 mM beta-glycerophosphate, 1mM EGTA, 100mM MgCl2, 5 mM MnCl2~
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0.5 mg/ml BSA), then Sul of Sodium Vanadate(1mM), and then Sul of water. Mix
the
components gently and preincubate the reaction mix at 30 degrees C for 2 min.
Initial
the reaction by adding 10u1 of the control enzyme or the filtered supernatant.
The tyrosine kinase assay reaction is then terminated by adding 10 u1 of
120mm EDTA and place the reactions on ice.
Tyrosine kinase activity is determined by transferring SO u1 aliquot of
reaction
mixture to a microtiter plate (MTP) module and incubating at 37 degrees C for
20
min. This allows the streptavadin coated 96 well plate to associate with the
biotinylated peptide. Wash the MTP module with 300u1/well of PBS four times.
Next add 75 u1 of anti-phospotyrosine antibody conjugated to horse radish
peroxidase(anti-P-Tyr-POD(O.Su/ml)) to each well and incubate at 37 degrees C
for
one hour. Wash the well as above.
Next add 100u1 of peroxidase substrate solution (Boehringer Mannheim) and
incubate at room temperature for at least 5 mins (up to 30 min). Measure the
absorbance of the sample at 405 nm by using ELISA reader. The level of bound
peroxidase activity is quantitated using an ELISA reader and reflects the
level of
tyrosine kinase activity.
Example 20: High-Throughput Screening Assay Identifyin~ Phosphorylation
Activi
As a potential alternative and/or compliment to the assay of protein tyrosine
kinase activity described in Example 19, an assay which detects activation
(phosphorylation) of major intracellular signal transduction intermediates can
also be .
used. For example, as described below one particular assay can detect tyrosine
phosphorylation of the Erk-1 and Erk-2 kinases. However, phosphorylation of
other
molecules, such as Raf; JNK, p38 MAP, Map kinase kinase (MEK), MEK kinase,
Src, Muscle specific kinase (MuSK), IRAK, Tec, and Janus, as well as any other
phosphoserine, phosphotyrosine, or phosphothreonine molecule, can be detected
by
substituting these molecules for Erk-1 or Erk-2 in the following assay.
Specifically, assay plates are made by coating the wells of a 96-well ELISA
plate with O.lml of protein G (lug/ml) for 2 hr at room temp, (RT). The plates
are
then rinsed with PBS and blocked with 3% BSA/PBS for 1 hr at RT. The protein G
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plates are then treated with 2 commercial monoclonal antibodies ( 1 OOng/well)
against
Erk-land Erk-2 (1 hr at RT) (Santa Cruz Biotechnology). (To detect other
molecules,
this step can easily be modified by substituting a monoclonal antibody
detecting any
of the above described molecules.) After 3-5 rinses with PBS, the plates are
stored at
4 degrees C until use.
A431 cells are seeded at 20,000/well in a 96-well Loprodyne filterplate and
cultured overnight in growth medium. The cells are then starved for 48 hr in
basal medium (DMEM) and then treated with EGF (6ng/well) or 50 u1 of the
supernatants obtained in Example 11 for 5-20 minutes. The cells are then
solubilized
and extracts filtered directly into the assay plate.
After incubation with the extract for 1 hr at RT; the wells are again rinsed.
As
a positive control, a commercial preparation of MAP kinase ( l Ong/well) is
used in
place of A431 extract. Plates are then treated with a commercial polyclonal
(rabbit)
antibody (lug/ml) which specifically recognizes the phosphorylated epitope of
the
Erk-1 and Erk-2 kinases (1 hr at RT). This antibody is biotinylated by
standard
procedures. The bound polyclonal antibody is then quantitated by successive
incubations with Europium-streptavidin and Europium fluorescence enhancing
reagent in the Wallac DELFIA instrument (time-resolved fluorescence). An
increased
fluorescent signal over background indicates a phosphorylation.
Example 21: Method of Determining Alterations in a Gene Corresponding to a
Polynucleotide ,
RNA isolated from entire families or individual patients presenting with a
phenotype of interest (such as a disease) is be isolated. cDNA is then
generated from
these RNA samples using protocols known in the art. (See, Sambrook.) The cDNA
.
is then used as a template for PCR, employing primers surrounding regions of
interest
in SEQ >D NO:X. Suggested PCR conditions consist of 35 cycles at 95 degrees C
for
seconds; 60-120 seconds at 52-58 degrees C; and 60-120 seconds at 70 degrees
C,
using buffer solutions described in Sidransky et al., Science 252:706 ( 1991
).
30 ~ PCR products are then sequenced using primers labeled at their 5' end
with T4
polynucleotide kinase, employing SequiTherm Polymerise. (Epicentre
Technologies). The intron-exon borders of selected exons is also determined
and _
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genomic PCR products analyzed to confirm the results. PCR products harboring'
suspected mutations is then cloned and sequenced to validate the results of
the direct
sequencing.
PCR products is cloned into T-tailed vectors as described in Holton et al.,
Nucleic Acids Research, 19:1156 (1991) and sequenced with T7 polymerase
(United
States Biochemical). Affected individuals are identified by mutations not
present in
unaffected individuals.
Genomic rearrangements are also observed as a method of determining
alterations in a gene corresponding to a polynucleotide. Genomic clones
isolated
according to Example 2 are nick-translated with digoxigenindeoxy-uridine 5'-
triphosphate (Boehringer Manheim), and FISH performed as described in Johnson
et
al., Methods Cell Biol. 35:73-99 (1991). Hybridization with the labeled probe
is
carried out using a vast excess of human cot-1 DNA for specific hybridization
to the
corresponding genoriiic locus.
Chromosomes are counterstained with 4,6-diamino-2-phenylidole and
propidium iodide, producing a combination of C- and R-bands. Aligned images
for
precise mapping are obtained using a triple-band filter set (Chroma
Technology,
Brattleboro, VT) in combination with a cooled charge-coupled device camera
(Photometrics, Tucson, AZ) and variable excitation wavelength filters.
(Johnson et
al., Genet. Anal. Tech. Appl., 8:75 (1991).) Image collection, analysis and
chromosomal fractional length measurements are performed using the ISee
Graphical
Program System. (Inovision Corporation, Durham, NC.) Chromosome alterations of
the genomic region hybridized by the probe are identified as insertions,
deletions, and
translocations. These alterations are used as a diagnostic marker for an
associated
disease.
Example 22: Method of Detecting Abnormal Levels of a Polvnentide in a
Biological Sample
A polypeptide of the present invention can be detected in a biological sample,
and if an increased or decreased level of the polypeptide is detected, this
polypeptide
is a marker for a particular phenotype. Methods of detection are numerous, and
thus,
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it is understood that one skilled in the art can modify the following assay to
fit their
particular needs.
For example, antibody-sandwich ELISAs are used to detect polypeptides in a
sample, preferably a biological sample. Wells of a microtiter plate are coated
with
specific antibodies, at a final concentration of 0.2 to l0~ug/ml. The
antibodies are
either monoclonal or polyclonal and are produced by the method described in
Example 10. The wells are blocked so that non-specific binding of the
polypeptide to
the well is reduced.
The coated wells are then incubated for > 2 hours at RT with a sample
containing the polypeptide. Preferably, serial dilutions of the sample should
be used
to validate results. The plates are then washed three times with deionized or
distilled
water to remove unbounded polypeptide.
Next, 50 u1 of specific antibody-alkaline phosphatase conjugate, at a
concentration of 25-400 ng, is added and incubated for 2 hours at room
temperature.
The plates are again washed three times with deionized or distilled water to
remove
unbounded conjugate.
Add 75 u1 of 4-methylumbelliferyl phosphate (MUP) or p-nitrophenyl
phosphate (NPP) substrate solution to each well and incubate 1 hour at room
temperature. Measure the reaction by a microtiter plate reader. Prepare a
standard
curve, using serial dilutions of a control sample, and plot polypeptide
concentration
on the X-axis (log scale) and fluorescence or absorbance of the Y-axis (linear
scale).
Interpolate the concentration of the polypeptide in the sample using the
standard
curve.
Example 23: Formulation
The invention also provides methods of treatment and/or prevention of
diseases or disorders (such as, for example, any one or more of the diseases
or
disorders disclosed herein) by administration to a subject of an effective
amount of a
Therapeutic. By therapeutic is meant polynucleotides or polypeptides of the
invention (including fragments and variants), agonists or antagonists thereof,
and/or
antibodies thereto, in combination with a pharmaceutically acceptable Garner
type
(e.g., a sterile Garner). _
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The Therapeutic will be formulated and dosed in a fashion consistent with
good medical practice, taking into account the clinical condition of the
individual
patient (especially the side effects of treatment with the Therapeutic alone),
the site of
delivery, the method of administration, the scheduling of administration, and
other
factors known to practitioners. The "effective amount" for purposes herein is
thus
determined by such considerations.
As a general proposition, the total pharmaceutically effective amount of the
Therapeutic administered parenterally per dose will be in the range of about
lug/kg/day to 10 mg/kg/day of patient body weight, although, as noted above,
this
will be subject to therapeutic discretion. More preferably, this dose is at
least 0.01
mg/kg/day, and most preferably for humans between about 0.01 and 1 mg/kg/day
for
the hormone. If given continuously, the Therapeutic is typically administered
at a
dose rate of about 1 ug/kg/hour to about 50 uglkg/hour, either by 1-4
injections per
day or by continuous subcutaneous infusions, for example, using a mini-pump.
An
intravenous bag solution may also be employed. The length of treatment needed
to
observe changes and the interval following treatment for responses to occur
appears
to vary depending on the desired effect.
Therapeutics can be are administered orally, rectally; parenterally,
intracistemally, intravaginally, intraperitoneally, topically (as by powders,
ointments,
gels, drops or transdermal patch), bucally, or as an oral or nasal spray.
"Pharmaceutically acceptable carrier" refers to a non-toxic solid, semisolid
or liquid
filler, diluent, encapsulating material or formulation auxiliary of any. The
term .
"parenteral" as used herein refers to modes of administration which include
intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and
intraarticular injection and infusion.
Therapeutics of the invention are also suitably administered by sustained-
release systems. Suitable examples of sustained-release Therapeutics are
administered orally, rectally, parenterally, intracistemally, intravaginally,
intraperitoneally, topically (as by powders, ointments, gels, drops or
transdermal
patch), bucally, or as an oral or nasal spray. "Pharmaceutically acceptable
carrier"
refers to a non-toxic solid, semisolid or liquid filler, diluent,
encapsulating material or
formulation auxiliary of any type. The term "parenteral" as used herein refers
to
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modes of administration which include intravenous, intramuscular,
intraperitoneal,
intrasternal, subcutaneous and intraarticular injection and infusion.
Therapeutics of the invention are also suitably administered by sustained-
release systems. Suitable examples of sustained-release Therapeutics include
suitable
polymeric materials (such as, for example, semi-permeable polymer matrices in
the
form of shaped articles, e.g., films, or mirocapsules), suitable hydrophobic
materials
(for example as an emulsion in an acceptable oil) or ion exchange resins, and
sparingly soluble derivatives (such as, for example, a sparingly soluble
salt).
Sustained-release matrices include polylactides (U.S. Pat. No. 3,773,919, EP
58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et
al.,
Biopolymers 22:547-556 (1983)), poly (2- hydroxyethyl methacrylate) (Larger et
al.,
J. Biomed. Mater. Res. 15:167-277 (1981), and Larger, Chem. Tech. 12:98-105
(1982)), ethylene vinyl acetate (Larger et al., Id.) or poly-D- (-)-3-
hydroxybutyric
acid (EP 133,988).
Sustained-release Therapeutics also include liposomally entrapped
Therapeutics of the invention (see generally, Larger, Science 249:1527-1533
(1990);
Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer,
Lopez-
Berestein and Fidler (eds.), Liss, New York, pp. 317 -327 and 353-365 (1989)).
Liposomes containing the Therapeutic are prepared by methods known per se: DE
3,218,121; Epstein et al., Proc. Natl. Acad. Sci. (LISA) 82:3688-3692 (1985);
Hwang
et al., Proc. Natl. Acad. Sci.(USA) 77:4030-4034 (1980); EP 52,322; EP 36,676;
EP
88,046; EP 143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos.
4,485,045 and 4,544,545; and EP 102,324. Ordinarily, the liposomes are of the
small
(about 200-800 Angstroms) unilamellar type in which the lipid content is
greater than
about 30 mol. percent cholesterol, the selected proportion being adjusted for
the
optimal Therapeutic.
In yet an additional embodiment, the Therapeutics of the invention are
delivered by way of a pump (see Larger, supra; Sefton, CRC Crit. Ref. Biomed.
Erg.
14:201 (1987); Buchwald et al., Surgery 88:507 (.1980); Saudek et al., N.
Engl. J.
Med. 321:574 (1989)).
Other controlled release systems are discussed in the review by Larger
(Science 249:1527-1533 (1990)). _
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For parenteral administration, in one embodiment, the Therapeutic is
formulated generally by mixing. it at the desired degree of purity, in a unit
dosage
injectable form (solution,'suspension, or emulsion), with a.pharmaceutically
acceptable Garner, i.e., one that is non-toxic to recipients at the dosages
and
S concentrations employed and is compatible with other ingredients of the
formulation.
For example, the formulation preferably does not include oxidizing agents and
other
compounds that are known to be deleterious to the Therapeutic.
Generally, the formulations are prepared by contacting the Therapeutic
uniformly and intimately with liquid carriers or finely divided solid carriers
or both.
Then, if necessary, the product is shaped into the desired formulation.
Preferably the
carrier is a parenteral carrier, more preferably a solution that is isotonic
with the blood
of the recipient. Examples of such Garner vehicles include water, saline,
Ringer's
solution, and dextrose solution. Non-aqueous vehicles such as fixed oils and
ethyl
oleate are also useful herein, as well as liposomes.
The carrier suitably contains minor amounts of additives such as substances
that enhance isotonicity and chemical stability. Such materials are non-toxic
to
recipients at the dosages and concentrations employed, and include buffers
such as
phosphate, citrate, succinate, acetic acid, and other organic acids or their
salts;
antioxidants such as ascorbic acid; low molecular weight (less than about ten
residues) polypeptides, e.g., polyarginine or tripeptides; proteins, such as
serum
albumin, gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic
acid, or
arginine; monosaccharides, disaccharides, and other carbohydrates including
cellulose
or its derivatives, glucose, manose, or dextrins; chelating agents such as
EDTA; sugar
alcohols such as mannitol or sorbitol; counterions such as sodium;, and/or
nonionic
surfactants such as polysorbates, poloxamers, or PEG.
The Therapeutic is typically formulated in such vehicles at a concentration of
about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, at a pH of about 3 to 8.
It will
be understood that the use of certain of the foregoing excipients, carriers,
or
stabilizers will result in the formation of polypeptide salts.
Any pharmaceutical used for therapeutic administration can be sterile.
Sterility is readily accomplished by filtration through sterile filtration
membranes.
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(e.g., 0.2 micron membranes). Therapeutics generally are placed into a
container
having a sterile access port, for example, an intravenous solution bag or vial
having a
stopper pierceable by a hypodermic injection needle.
Therapeutics ordinarily will be stored in unit or mufti-dose containers, for
example, sealed ampoules or vials, as an aqueous solution or as a lyophilized
formulation for reconstitution. As an example of a lyophilized formulation, 10-
ml
vials are filled with S ml of sterile-filtered 1 % (w/v) aqueous Therapeutic
solution,
and the resulting mixture is lyophilized. The infusion solution is prepared by
reconstituting the lyophilized Therapeutic using bacteriostatic Water-for-
Injection.
The invention also provides a pharmaceutical pack or kit comprising one or
more containers-filled with one or more of the ingredients of the Therapeutics
of the
invention. Associated with such containers) can be a notice in the form
prescribed by
a governmental agency regulating the manufacture, use or sale of
pharmaceuticals or
biological products, which notice reflects approval by the agency of
manufacture, use
or sale for human administration. In addition, the Therapeutics may be
employed in
conjunction with other therapeutic compounds.
The Therapeutics of the inventiommay be administered alone or in
combination with adjuvants. Adjuvants that may be administered with the
Therapeutics of the invention include, but are not limited to, alum, alum plus
deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21 (Generitech, Inc.), BCG
(e.g., THERACYS~), MPL and nonviable prepartions of Corynebacterium parvum.
In a specific embodiment, Therapeutics of the invention are administered in
combination with alum. In another specific embodiment, Therapeutics of the
invention are administered in combination with QS-21. Further adjuvants-that
may be
administered with the Therapeutics of the invention include, but are not
limited to,
Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18, CRL1005,
Aluminum salts, MF-59, and Virosomal adjuvant technology. Vaccines that may be
administered with the Therapeutics of the invention include, but are not
limited to,
vaccines directed toward protection against MMR (measles, mumps, rubella),
polio,
varicella, tetanus/diptheria, hepatitis A, hepatitis B, haemophilus influenzae
B,
whooping cough, pneumonia, influenza, Lyme's Disease, rotavirus, cholera,
yellow
fever, Japanese encephalitis, poliomyelitis, rabies, typhoid fever, and
pertussis.
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Combinations may be administered either concomitantly, e.g., as an admixture,
separately but simultaneously or concurrently; or sequentially. This includes
presentations in which the combined agents are administered together as a
therapeutic
mixture, and also procedures in which the combined agents are administered
separately but simultaneously, e.g., as through separate intravenous lines
into the
same individual. Administration "in combination" further includes the separate
administration of one of the compounds or agents given first, followed by the
second.
The Therapeutics of the invention may be administered alone or in
combination with other therapeutic agents. Therapeutic agents that may be
administered in combination with the Therapeutics of the invention, include
but not
limited to, chemotherapeutic agents, antibiotics, steroidal and non-steroidal
anti=
inflammatories, conventional immunotherapeutic agents, and/or therapeutic
treatments described below. Combinations may be administered either
concomitantly, e.g., as an admixture, separately but simultaneously or
concurrently;
of sequentially. This includes presentations in which the combined agents are
administered together as a therapeutic mixture, and'also procedures in which
the
combined agents are administered separately but simultaneously, e.g., as
through
separate intravenous lines into the same individual. Administration "in
combination"
further includes the separate administration of one of the compounds or agents
given
first, followed by the second.
In certain embodiments, Therapeutics of the invention are administered in
combination with antiretroviral agents, nucleoside/nucleotide reverse
transcriptase
inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs),
and/or
protease inhibitors (PIs). NRTIs that may be administered in combination with
the
Therapeutics of the invention, include, but are not limited to, RETROVIRT""
(zidovudine/AZT), VIDEXT"" (didanosine/ddI), HIVIDT"~ (zalcitabine/ddC),
ZERITT""
(stavudine/d4T), EPNIRT"" (lamivudine/3TC), and COMBNIRT""
(zidovudine/lamivudine). NNRTIs that may be administered in combination with
the
Therapeutics of the invention, include, but are not limited to, VIRAMUNET"~
(nevirapine), RESCRIPTORT"" (delavirdine), and SUSTNAT"" (efavirenz). Protease
inhibitors that may be administered in combination with the Therapeutics of
the
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invention, include, but are not limited to, CRIXIVANT"" (indinavir), NORVIRT""
(ritonavir), INVIRASET"" (saquinavir), and VIRACEPTT"" (nelfinavir). In a
specific
embodiment, antiretroviral agents, nucleoside reverse transcriptase
inhibitors, non-
nucleoside reverse transcriptase inhibitors, and/or protease inhibitors may be
used in
any combination with Therapeutics of the invention to treat AIDS and/or to
prevent
or treat HIV infection. ,
Additional NRTIs include LODENOSINET"" (F-ddA; an acid-stable adenosine
NRTI; Triangle/Abbott; COVIRACILT"" (emtricitabine/FTC; structurally related
to
lamivudine (3TC) but with 3- to 10-fold greater activity in vitro;
Triangle/Abbott);
dOTC (BCH-10652, also structurally related to lamivudine but retains activity
against a substantial proportion of lamivudine-resistant isolates; Biochem
Pharma);
Adefovir (refused approval for anti-HIV therapy by FDA; Gilead Sciences);
PREVEON~ (Adefovir Dipivoxil, the active prodrug of adefovir; its active form
is
PMEA-pp); TENOFOVIRT"" (bis-POC PMPA, a PMPA prodrug; Gilead);
DAPD/DXG (active metabolite of DAPD; Triangle/Abbott); D-D4FC (related to
3TC, with activity against AZT/3TC-resistant virus); GW420867X (Glaxo
Wellcome); ZIAGENT"' (abacavir/159U89; Glaxo Wellcome Inc.); CS-87 (3'azido-
2',3'-dideoxyuridine; WO 99/66936); and S-acyl-2-thioethyl (SATE)-bearing
prodrug forms of (3-L-FD4C and ~3-L-FddC (WO 98/17281).
Additional NNRTIs include COACTINONT"" (Emivirine/MKC-442, potent
NIVRTI of the HEPT class; Triangle/Abbott); CAPRAVIRINET"" (AG-1549/S-1153,
a next generation NNRTI with activity against viruses,containing the K103N
mutation; Agouron); PNU-142721 (has 20- to 50-fold greater activity than its
predecessor delavirdine and is active against K103N mutants; Pharmacia &
Upjohn);
DPC-961 and DPC-963 (second-generation derivatives of efavirenz, designed to
be
active against viruses with the K103N mutation; DuPont); GW-420867X' (has 25-
fold
greater activity than HBY097 and is active against K103N mutants; Glaxo
Wellcome); CALANOLIDE A (naturally occurring agent from the latex tree; active
against viruses containing either or both the Y181C and K103N mutations); and
Propolis (WO 99/49830).
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Additional protease inhibitors include LOPINAVIRT"" (ABT378/r; Abbott
Laboratories); BMS-232632 (an azapeptide; Bristol-Myres Squibb); TIPRANAVIRT""
(PNU-140690, a non-peptic dihydropyrone; Pharmacia & Upjohn); PD-178390 (a
nonpeptidic dihydropyrone; Parke-Davis); BMS 232632 (an azapeptide; Bristol-
Myers Squibb); L-756,423 (an indinavir analog; Merck); DMP-450 (a cyclic urea
compound; Avid & DuPont); AG-1776 (a peptidomimetic with in vitro activity
against protease inhibitor-resistant viruses; Agouron); VX-175/GW-433908
(phosphate prodrug of amprenavir; Vertex & Glaxo Welcome); CGP61755 (Ciba);
and AGENERASET"" (amprenavir; Glaxo Wellcome Inc.).
Additional antiretroviral agents include fusion inhibitors/gp41 binders.
Fusion inhibitors/gp41 binders include T-20 (a peptide from residues 643-678
of the
HIV gp41 transmembrane protein ectodomain which binds to gp41 in its resting
state
and prevents transformation to the fusogenic state; Trimeris) and T-1249 (a
second-
generation fusion inhibitor; Trimeris).
Additional antiretroviral agents include fusion inhibitors/chemokine receptor
antagonists. Fusion inhibitors/chemokine receptor antagonists include CXCR4
antagonists such as AMD 3100 (a bicyclam), SDF-1 and its analogs, and ALX40-4C
(a cationic peptide), T22 (an 18 amino acid peptide; Trimeris) and the T22
analogs
T134 and T140; CCRS antagonists such as RANTES (9-68), AOP-RANTES, NNY-
RANTES, and TAK-779; and CCRS/CXCR4 antagonists such as NSC 651016 (a
distamycin analog). Also included are CCR2B, CCR3; and CCR6 antagonists.
Chemokine recpetor agonists such as RANTES, SDF-1, MIP-la, MIP-1(3, etc., may
also inhibit fusion.
Additional antiretroviral agents include integrase inhibitors. Integrase
, inhibitors include dicaffeoylquinic (DFQA) acids; L-chicoric acid (a
dicaffeoyltartaric (DCTA) acid); quinalizarin (QLC) and related
anthraquinones;
ZINTEVIRT"" (AR 177, an oligonucleotide that probably acts at cell surface
rather
than being a true integrase inhibitor; Arondex); and naphthols such as those
disclosed
in WO 98/50347.
Additional antiretroviral agents include hydroxyurea-like compunds such as
BCX-34 (a purine nucleoside phosphorylase inhibitor; Biocryst); ribonucleotide
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reductase inhibitors such as DIDOXT"" (Molecules for Health); inosine
monophosphate dehydrogenase (IMPDH) inhibitors sucha as VX-497 (Vertex); and
mycopholic acids such as CellCept (mycophenolate mofetil; Roche).
Additional antiretroviral agents include inhibitors of viral integrase,
inhibitors
of viral genome nuclear translocation such as arylene bis(methylketone)
compounds;
inhibitors of HN entry such as AOP-RANTES, NNY-RANTES, RANTES-IgG
fusion protein, soluble complexes of RANTES and glycosaminoglycans (GAG), and
AMD-3100; nucleocapsid zinc finger inhibitors such as dithiane compounds;
targets
of HIV Tat and Rev; and pharmacoenhancers such as ABT-378.
Other antiretroviral therapies and adjunct therapies include cytokines and
lymphokines such as MIP-la, MIP-1(3; SDF-la, IL-2,°PROLEUKINT""
(aldesleukin/L2-7001; Chiron), IL-4, IL-10, IL-12, and IL-13; interferons such
as
IFN-a2a; antagonists of TNFs, NFxB, GM-CSF, M-CSF, and IL-10; agents that
modulate immune activation such as cyclosporin and prednisone; vaccines such
as
RemuneT"" (HIV Immunogen), APL 400-003 (Apollon), recombinant gp120 and
fragments, bivalent (B/E) recombinant envelope glycoprotein; rgp120CM235, MN
rgp120, SF-2 rgp120, gp120/soluble CD4 complex, Delta JR-FL protein, branched
synthetic peptide derived from discontinuous gp120 C3/C4 domain, fusion-
competent immunogens, and Gag, Pol, Nef, and Tat vaccines; gene-based
therapies
such as genetic suppressor elements (GSEs;, WO 98/54366), and intrakines
(genetically modified CC chemokines targetted to the ER to block surface
expression
of newly synthesized CCRS (Yang et al., PNAS 94:11567-72 (1997); Chen et al.,
Nat. Med. 3:1110-16 (1997)); antibodies such as the anti-CXCR4 antibody 1265,
the
anti-CCRS antibodies 2D7, 5C7, PAB, PA9, PA10, PA11, PA12, and PA14, the anti-
CD4 antibodies Q4120 and RPA-T4, the anti-CCR3 antibody 7B 11, the anti-gp 120
antibodies 17b, 48d, 447-52D, 257-D, 268-D and 50.1, anti-Tat antibodies, anti-
TNF-
a antibodies, and monoclonal antibody 33A; aryl hydrocarbon (AH) receptor
agonists and antagonists such as TCDD, 3,3',4,4',5-pentachlorobiphenyl,
3,3',4,4'-
tetrachlorobiphenyl, and a-naphthoflavone (WO 98/30213); and antioxidants such
as
y-L-glutamyl-L-cysteine ethyl ester (y-GCE; WO 99/56764).
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In a further embodiment, the Therapeutics of the invention are administered
in combination with an antiviral agent. Antiviral agents that may be
administered
with the Therapeutics of the invention include, but are not limited to,
acyclovir,
ribavirin, amantadine, and remantidine.
In other embodiments, Therapeutics of the invention may be administered in
combination with anti-opportunistic infection agents. Anti-opportunistic
agents that
may be administered in combination with the Therapeutics of the invention,
include,
but are not limited to, TRIMETHOPRIM-SULFAMETHOXAZOLET"",
DAPSONET"", PENTAMIDINET"', ATOVAQUONET"", ISONIAZIDT"',
RIFAMPINT"", PYRAZINAMIDET"~, ETHAMBUTOLT"", RIFABUTINT"", .
CLARITHROMYCINT"", AZITHROMYCINT"", GANCICLOVIRT"",
FOSCARNETT"~, CIDOFOVIRT"", FLUCONAZOLET"", ITRACONAZOLET"",
KETOCONAZOLET"", ACYCLOVIRT"", FAMCICOLVIRT"", PYRIMETHAMINET"~,
LEUCOVORINT"", NEUPOGENT"" (filgrastim/G-CSF), and LEUKINET""
(sargramostim/GM-CSF). In a specific embodiment, Therapeutics of the invention
are used in any combination with TRIMETHOPRIM-SULFAMETHOXAZOLET"",
DAPSbNET"", PENTAMIDINET"", and/or ATOVAQUONET"" to prophylactically
treat or prevent an opportunistic Pneumocystis carinii pneumonia infection. In
another specific embodiment, Therapeutics of the invention are used in any
combination with ISONIAZIDT"", R1FAMPINT"", PYRAZINAMIDET"", and/or
ETHAMBUTOLT"" to prophylactically treat or prevent an opportunistic
Mycobacterium avium complex infection. In another specific embodiment,
Therapeutics of the invention are used in any combination with RIFABUTINT"",
CLARITHROMYCINT"", and/or AZITHROMYC1NT"" to prophylactically treat or
prevent an opportunistic Mycobacterium tuberculosis infection. In another
specific
embodiment, Therapeutics of the invention are used in any combination with
GANCICLOVIRT"", FOSCARNETT"", and/or CIDOFOVIRT"" to prophylactically treat
or prevent an opportunistic cytomegalovirus infection. In another specific
embodiment, Therapeutics of the invention are used in any combination with
FLUCONAZOLET"", ITRACONAZOLET"", and/or KETOCONAZOLET"' to
prophylactically treat or prevent an opportunistic fungal infection. In
another
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specific embodiment, Therapeutics of the invention are used in any combination
with
ACYCLOVIRT"~ and/or FAMCICOLVIRTM to prophylactically treat or prevent an
opportunistic herpes simplex virus type, I and/or type II infection. In
another specific
embodiment, Therapeutics of the invention are used in any combination with
PYRIMETHAMINET"" and/or LEUCOVORINT"" to prophylactically treat or prevent
an opportunistic Toxoplasma gondii infection. In another specific embodiment,
Therapeutics of the invention are used in any combination with LEUCOVORINT""
and/or NEUPOGENT"" to prophylactically treat or prevent an opportunistic
bacterial
infection.
In a further embodiment, the Therapeutics of the invention are administered
in combination with an antibiotic agent. Antibiotic agents that may be
administered
with the Therapeutics of the invention include, but are not limited to,
amoxicillin,
beta-lactamases, aminoglycosides, beta-lactam (glycopeptide), beta-lactamases,
Clindamycin, chloramphenicol, cephalosporins, ciprofloxacin, erythromycin,
fluoroquinolones, macrolides, metronidazole, penicillins, quinolones,
rapamycin,
rifampin, streptomycin, sulfonamide, tetracyclines, trimethoprim, trimethoprim-
sulfamethoxazole, and vancomycin. .
In other embodiments, Therapeutics of the invention are administered in
combination with immunosuppressive agents. Immunosuppressive agents that may
be administered'in combination with the Therapeutics of the invention
include,. but are
not limited to, steroids, cyclosporine, cyclosporine analogs, cyclophosphamide
methylprednisone, prednisone, azathioprine, FK-506, 15-deoxyspergualin, and
other
immunosuppressive, agents that act by suppressing the function of responding T
cells.
Other immunosuppressive agents that may be administered in combination with
the
Therapeutics of the invention include, but are not limited to, prednisolone,
methotrexate, thalidomide, methoxsalen, rapamycin, leflunomide, mizoribine
(BREDININTM), brequinar, deoxyspergualin, and azaspirane (SKF 105685),
ORTHOCLONE OKT~ 3 (muromonab-CD3), SANDIMMUNET"", NEORALT"",
SANGDYAT"" (cyclosporine), PROGRAF~ (FK506, tacrolimus), CELLCEPT~
(mycophenolate motefil, of which the active metabolite is mycophenolic acid),
IMURANTM (azathioprine), glucocorticosteroids, adrenocortical steroids such as
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DELTASONETM (prednisone) and HYDELTRASOLTM (prednisolone), FOLEXTM
and MEXATETM (methotrxate), OXSORALEN-ULTR.ATM (methoxsalen) and
~TM (sirolimus). ~ In a specific embodiment, immunosuppressants may be
used to prevent rejection of organ or bone marrow transplantation.
~In an additional embodiment, Therapeutics of the invention are administered
alone or in combination with one or more intravenous immune globulin
preparations.
Intravenous immune globulin preparations that may be administered with the
Therapeutics of the invention include, but not limited to, GAMMARTM,
IVEEGAMT"", SANDOGLOBULINT"", GAMMAGARD S/DT"~, ATGAMTM
(antithymocyte glubulin), and GAMIMUNET"". In a specific embodiment,
Therapeutics of the invention are administered in combination with intravenous
immune globulin preparations in transplantation therapy (e.g., bone marrow
transplant). ,
In certain embodiments, the Therapeutics of the invention are administered
alone or in combination with an anti-inflammatory agent. Anti-inflammatory
agents
that may be administered with the Therapeutics of the invention include, but
are not
limited to, corticosteroids (e.g. betamethasone, budesonide, cortisone,
dexamethasone, hydrocortisone, methylprednisolone, prednisolone, prednisone,
and
triamcinolone), nonsteroidal anti-inflammatory drugs (e.g., diclofenac,
diflunisal,
etodolac, fenoprofen, floctafenine, flurbiprofen, ibuprofen, indomethacin,
ketoprofen,
meclofenamate, mefenamic acid, meloxicam, nabumetone, naproxen, oxaprozin,
phenylbutazone, piroxicam, sulindac, tenoxicam, tiaprofenic acid, and
tolmetin.), as
well as antihistamines, aminoarylcarboxylic acid derivatives, arylacetic acid
derivatives, arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic
acid
derivatives, pyrazoles, pyrazolones, salicylic acid derivatives,
thiazinecarboxamides,
e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyric acid,
amixetrine, bendazac, benzydamine, bucolome, difenpiramide, ditazol,
emorfazone,
guaiazulene, nabumetone, nimesulide, orgotein, oxaceprol, paranyline,
perisoxal,
pifoxime, proquazone, proxazole, and tenidap.
, In an additional embodiment, the compositions of the invention are
administered alone or in combination with an anti-angiogenic agent. Anti-
angiogenic
agents that may be administered with the compositions of the invention
include, but
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are not limited to, Angiostatin (Entremed, Rockville, MD), Troponin-1 (Boston
Life
Sciences, Boston, MA), anti-Invasive Factor, retinoic acid and derivatives
thereof,
paclitaxel (Taxol), Suramin, Tissue Inhibitor of Metalloproteinase-1, Tissue
Inhibitor
of Metalloproteinase-2, VEGI, Plasminogen Activator Inhibitor-1, Plasminogen
Activator Inhibitor-2, and various forms of the lighter "d group" transition
metals.
Lighter "d group" transition metals include, for example, vanadium,
molybdenum, tungsten, titanium, niobium, and tantalum species. Such transition
metal species may form transition metal complexes. Suitable complexes of the
above-mentioned transition metal species include oxo transition metal
complexes.
Representative examples of vanadium complexes include oxo vanadium
complexes such as vanadate and vanadyl complexes. Suitable vanadate complexes
include metavanadate and orthovanadate complexes such as, for example,
ammonium
metavanadate, sodium metavanadate, and sodium orthovanadate. Suitable vanadyl
complexes include, for example, vanadyl acetylacetonate and vanadyl sulfate
including vanadyl sulfate hydrates such as vanadyl sulfate mono- and
trihydrates.
Representative examples of tungsten and molybdenum complexes also include
oxo complexes. Suitable oxo tungsten complexes include tungstate and tungsten
oxide complexes. Suitable tungstate complexes include ammonium tungstate,
calcium tungstate, sodium tungstate dihydrate, and tungstic acid. Suitable
tungsten
oxides include tungsten (IV) oxide and tungsten (VI) oxide. Suitable oxo
molybdenum complexes include molybdate, molybdenum oxide, and molybdenyl
complexes. Suitable molybdate complexes include ammonium molybdate and its
_ hydrates, sodium molybdate and its hydrates, and potassium molybdate and its
hydrates. Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum
(VI) oxide, and molybdic acid. Suitable molybdenyl complexes include, for
example,
molybdenyl acetylacetonate. Other suitable tungsten and molybdenum complexes
include hydroxo derivatives derived from, for example, glycerol, tartaric
acid, and
sugars.
A wide variety.of other anti-angiogenic factors may also be utilized within
the
' context of the present invention. Representative examples include, but are
not limited
to, platelet factor 4; protamine sulphate; sulphated chitin derivatives
(prepared from
queen crab shells), (Murata et al., Cancer Res. 51:22-26, ( 1991 )); Sulphated
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Polysaccharide Peptidoglycan Complex (SP- PG) (the function of this compound
may
be enhanced by the presence of steroids such as estrogen, and tamoxifen
citrate);
Staurosporine; modulators of matrix metabolism, including for example, proline
analogs, cishydroxyproline, d,L-3,4-dehydroproline, Thiaproline, alpha,alpha-
dipyridyl, aminopropionitrile fumarate; 4-propyl-5-(4-pyridinyl)-2(3H)-
oxazolone;
Methotrexate; Mitoxantrone; Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-
3
(Pavloff et al., J. Bio. Chem. 267:17321-17326, (1992)); Chymostatin
(Tomkinson et
al., Biochem J. 286:475-480, (1992)); Cyclodextrin Tetradecasulfate;
Eponemycin;
Camptothecin; Fumagillin (Ingber et al., Nature 348:555-557, (1990)); Gold
Sodium
Thiomalate ("GST"; Matsubara and Ziff, J. Clin. Invest. 79:1440-1f46, (1987));
anticollagenase-serum; alpha2-antiplasmin (Holmes et al., J. Biol. Chem.
262(4):1659-1664, (1987)); Bisantrene (National Cancer Institute); Lobenzarit
disodium (N-(2)-carboxyphenyl-4- chloroanthronilic acid disodium or "CCA";
(Takeuchi et al., Agents Actions 36:312-316, (1992)); and metalloproteinase
inhibitors such as BB94.
Additional anti-angiogenic factors that may also be utilized within the
context
of the present invention include Thalidomide, (Celgene, Warren, NJ);
Angiostatic
steroid; AGM-1470 (H. Brem and J. Folkman JPediatr. Surg. 28:445-51 (1993));
an
integrin alpha v beta 3 antagonist (C. Storgard et al., J Clin. Invest. 103:47-
54
(1999)); carboxynaminolmidazole; Carboxyamidotriazole (CAI) (National Cancer
Institute, Bethesda, MD); Conbretastatin A-4 (CA4P) (OXiGENE, Boston, MA); '
Squalamine (Magainin Pharmaceuticals, Plymouth Meeting, PA); TNP-470, (Tap
Pharmaceuticals, Deerfield, IL); ZD-0101 AstraZeneca (London, LJK); APRA
(CT2584); Benefin, Byrostatin-1 (5.C339555); CGP-41251 (PKC 412); CM101;
Dexrazoxane (ICRF187); DMXAA; Endostatin; Flavopridiol; Genestein; GTE;
ImmTher; Iressa (ZD1839); Octreotide (Somatostatin); Panretin; Penacillamine;
Photopoint; PI-88; Prinomastat (AG-3340) Purlytin; Suradista (FCE26644);
Tamoxifen (Nolvadex); Tazarotene; Tetrathiomolybdate; Xeloda (Capecitabine);
and
5-Fluorouracil.
Anti-angiogenic agents that may be administed in combination with the
compounds of the invention may work through a variety of mechanisms including,
but not limited to, inhibiting proteolysis of the extracellular matrix,
blocking the
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function of endothelial cell-extracellular matrix adhesion molecules, by
antagonizing
the function of angiogenesis inducers such as growth factors, and inhibiting
integrin
receptors expressed on proliferating endothelial cells. Examples of anti-
angiogenic
inhibitors that interfere with extracellular matrix proteolysis and which may
be
administered in combination with the compositons of the invention include, but
are
not united to, AG-3340 (Agouron, La Jolla, CA), BAY-12-9566 (Bayer, West
Haven,
CT), BMS-275291 (Bristol Myers Squibb, Princeton, NJ), CGS-27032A (Novartis,
East Hanover, NJ), Marimastat (British Biotech, Oxford, UK), and Metastat
(Aeterna,
St-Foy, Quebec). Examples of anti-angiogenic inhibitors that act by blocking
the
function of endothelial cell-extracellular matrix adhesion molecules and which
may
be administered in combination with the compositons of the invention include,
but are
not lmited to, EMD-121974, (Merck KcgaA Darmstadt, Germany) and Vitaxin
(Ixsys,
La Jolla, CA/Medimmune, Gaithersburg, MD). Examples of anti-angiogenic agents
that act by directly antagonizing or inhibiting angiogenesis inducers and
which may
be administered in combination with the compositons of the invention include,
but are
not lmited to, Angiozyme (Ribozyme, Boulder, CO), Anti-VEGF antibody
(Genentech, S. San Francisco, CA), PTK-787/ZK-225846 (Novartis, Basel,
Switzerland), SU-101 (Sugeil, S. San Francisco, CA), SU-5416 (Sugen/ Pharmacia
Upjohn, Bridgewater, NJ), and SU-6668 (Sugen). Other anti-angiogenic agents
act to
indirectly inhibit angiogenesis. Examples of indirect inhibitors of
angiogenesis which
may be administered in combination with the compositons of the invention
include,
but are not limited to, IM-862 (Cytran, Kirkland, WA), Interferon-alpha, IL-12
(Roche, Nutley, NJ), and Pentosan polysulfate (Georgetown University,
Washington;
DC). '
In particular embodiments, the use of compositions of the invention in
combination with anti-angiogenic agents is contemplated for the treatment,
prevention, and/or amelioration of an autoimmune disease, such as for example,
an
autoimmune disease described herein.
In a particular embodiment, the use of compositions of the invention in
combination with anti-angiogenic agents is, contemplated for the treatment,
prevention, and/or amelioration of arthritis. In a more particular embodiment,
the use
of compositions of the invention in combination with anti-angiogenic agents is
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contemplated for the treatment, prevention, and/or amelioration of rheumatoid
arthritis.
In another embodiment, the polynucleotides encoding a polypeptide of the
present invention are administered in combination with an angiogenic protein,
or
S polynucleotides encoding an angiogenic protein. Examples of angiogenic
proteins that
may be administered with the compositions of the invention include, but are
not
limited to, acidic and basic fibroblast growth factors, VEGF-1, VEGF-2, VEGF-
3,
epidermal growth factor alpha and beta, platelet-derived endothelial cell
growth
factor, platelet-derived growth factor, tumor necrosis factor alpha,
hepatocyte growth
factor, insulin-like growth factor, colony stimulating factor, macrophage
colony
stimulating factor, granulocyte/macrophage colony stimulating factor, and
nitric oxide
synthase.
In additional embodiments, compositions of the invention are administered in
combination with a chemotherapeutic agent. Chemotherapeutic agents that may be
administered with the Therapeutics of the invention include, but are not
limited to
alkylating agents such as nitrogen mustards (for example, Mechlorethamine,
cyclophosphamide, Cyclophosphamide Ifosfamide, Melphalan (L-sarcolysin), and
Chlorambucil), ethylenimines and methylmelamines (for example,
Hexamethylmelamirie and Thiotepa), alkyl sulfonates (for example, Busulfan),
nitrosoureas (for example, Carmustine (BCNU), Lomustine (CCNL~, Semustine
(methyl-CCNU), and Streptozocin (streptozotocin)), triazenes (for example,
Dacarbazine (DTIC; dimethyltriazenoimidazolecarboxamide)), folic acid analogs
(for
example, Methotrexate (amethopterin)), pyrimidine analogs (for example,
Fluorouacil (5-fluorouracil; 5-FU), Floxuridine (fluorodeoxyuridine; FudR),
and
Cytarabine (cytosine arabinoside)), purine analogs and related inhibitors (for
example, Mercaptopurine (6-mercaptopurine-; 6-MP), Thioguanine (6-thioguanine;
TG), and Pentostatin (2'-deoxycoformycin)), vinca alkaloids (for example,
Vinblastine (VLB, vinblastine sulfate)) and Vincristine (vincristine
sulfate)),
epipodophyllotoxins (for example, Etoposide and Teniposide), antibiotics (for
example, Dactinomycin (actinomycin D), Daunorubicin (daunomycin; rubidomycin),
Doxorubicin, Bleomycin, Plicamycin (mithramycin), and Mitomycin (mitomycin C),
enzymes (for example, L-Asparaginase), biological response modifiers (for
example,
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Interferon-alpha and interferon-alpha-2b), platinum coordination compounds
(for
example, Cisplatin (cis-DDP) and Carboplatin), anthracenedione (Mitoxantrone),
substituted ureas (for example, Hydroxyurea), methylhydrazine derivatives (for
example, Procarbazine (N-methylhydrazine; MIH), adrenocorticosteroids (for
example, Prednisone), progestins (for example, Hydroxyprogesterone caproate,
Medroxyprogesterone, Medroxyprogesterone acetate, and Megestrol acetate),
estrogens (for example, Diethylstilbestrol (DES), Diethylstilbestrol
diphosphate,
Estradiol, and Ethinyl estradiol), antiestrogens (for example, Tamoxifen),
androgens
(Testosterone proprionate, and Fluoxymesterone), antiandrogens (for example,
Flutamide), gonadotropin-releasing horomone analogs (for example, Leuprolide),
other hormones and hormone analogs (for example, methyltestosterone,
estramustine,
estramustine phosphate sodium, chlorotrianisene, and testolactone), and others
(for
example, dicarbazine, glutamic acid, and mitotane).
In one embodiment, the compositions of the invention are administered in
combination with one or more of the'following drugs: infliximab (also known as
RemicadeTM Centocor, Inc.), Trocade (Roche, RO-32-3555), Leflunomide (also
known as AravaTM from Hoechst Marion Roussel), KineretTM (an IL-1 Receptor
antagonist also known as Anakinra from Amgen, Inc.)
In a specific embodiment, compositions of the invention are administered in
combination with CHOP (cyclophosphamide, doxorubicin, vincristine, and
prednisone) or combination of one or more of the components of CHOP. In one
embodiment, the compositions of the invention are administered in combination
with
anti-CD20 antibodies, human monoclonal anti-CD20 antibodies. In another
embodiment, the compositions of the invention are administered in combination
with
anti-CD20 antibodies and CHOP, or anti-CD20 antibodies and any combination of
one or more of the components of CHOP, particularly cyclophosphamide and/or
prednisone. In a specific embodiment, compositions of the invention are
administered
in combination with Rituximab. In a further embodiment, compositions of the
invention are administered with Rituximab and CHOP, or Rituximab and any
combination of one or more of the components of CHOP, particularly
cyclophosphamide and/or prednisone. In a specific embodiment, compositions of
the
invention are administered in combination with tositumomab. In a further
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embodiment, compositions of the invention are administered with tositumomab
and
CHOP, or tositumomab and any combination of one or more of the components of
CHOP, particularly cyclophosphamide and/or prednisone. The anti-CD20
antibodies
may optionally be associated with radioisotopes, toxins or cytotoxic prodrugs.
In another specific embodiment, the compositions of the invention are
administered in combination ZevalinT"". In a further embodiment, compositions
of the
invention are administered with ZevalinT"" and CHOP, or ZevalinT"" and any
combination of one or more of the components of CHOP, particularly
cyclophosphamide and/or prednisone. ZevalinT"" may be associated with one or
more
radisotopes. Particularly preferred isotopes are 9°Y and 1' ~In.
In an additional embodiment, the Therapeutics of the invention are
administered in combination with cytokines. Cytokines that may be administered
with the Therapeutics of the invention include, but are not limited to, IL2,
IL3, IL4,
ILS, IL6, IL7, IL 10, IL 12, IL 13, IL 1 S, anti-CD40, CD40L, IFN-gamma and
TNF-
alpha. In another embodiment, Therapeutics of the invention may be
administered
with any interleukin, including, but not limited to, IL-lalpha, IL-lbeta, IL-
2, IL-3,
IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15,
IL-16,
IL-17, IL-18, IL-19, IL-20, and IL-21.
In one embodiment, the Therapeutics of the invention are administered in
combination with members of the TNF family. TNF, TNF-related or TNF-like
molecules that may be administered with the Therapeutics of the invention
include,
but are not limited to, soluble forms of TNF-alpha, lymphotoxin-alpha (LT-
alpha,
also known as TNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-
beta),
OPGL, Fast, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma
(International Publication No. WO 96/14328), AIM-I (International Publication
No.
WO 97/33899), endokine-alpha (International Publication No. WO 98/07880), OPG,
and neutrokine-alpha (International Publication No. WO 98/18921, OX40, and
nerve
growth factor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2
(International Publication No. WO 96/34095), DR3 (International Publication
No.
WO 97/33904), DR4 (International Publication No. WO 98/32856), TRS
(International Publication No. WO 98/30693), TRANK, TR9 (International
Publication No. WO 98/56892),TR10 (International Publication No. WO 98/54202),
-
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312C2 (International Publication No. WO 98/06842), and TR12, and soluble forms
CD154, CD70, and CD153.
In an additional embodiment, the Therapeutics of the invention are
administered in combination with angiogenic proteins. Angiogenic proteins that
may
S be administered with the Therapeutics of the invention include, but are not
limited to,
Glioma Derived Growth Factor (GDGF), as disclosed in European Patent Number
EP-399816; Platelet Derived Growth Factor-A (PDGF-A), as disclosed in European
Patent Number EP-682110; Platelet Derived Growth Factor-B (PDGF-B), as
disclosed in European Patent Number EP-282317; Placental Growth Factor (P1GF),
as
disclosed in International Publication Number WO 92/06194; Placental Growth
Factor-2 (P1GF-2), as disclosed in Hauser et al., Growth Factors, 4:259-268
(1993);
Vascular Endothelial Growth Factor (VEGF), as disclosed in International
Publication
Number WO 90/13649; Vascular Endothelial Growth Factor-A (VEGF-A), as
disclosed in European Patent Number EP-506477; Vascular Endothelial Growth
Factor-2 (VEGF-2), as disclosed in International Publication Number WO
96/39515;
Vascular Endothelial Growth Factor B (VEGF-3); Vascular Endothelial Growth
Factor B-186 (VEGF-B186), as disclosed in International Publication Number WO
96/26736; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed in
International Publication Number WO 98/02543; Vascular Endothelial Growth
Factor-D (VEGF-D), as disclosed in International Publication Number WO
98/07832;
and Vascular Endothelial Growth Factor-E (VEGF-E), as disclosed in German
Patent
Number DE19639601. The above mentioned references are herein incorporated by
reference in their entireties.
In an additional embodiment, the Therapeutics of the invention are
administered in combination with Fibroblast Growth Factors. Fibroblast Growth
Factors that may be administered with the Therapeutics of the invention
include, but
are not limited to, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8,
FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, and FGF-15.
In an additional embodiment, the Therapeutics of the invention are
administered in combination with hematopoietic growth factors. Hematopoietic
growth factors that may be administered with the Therapeutics of the invention
include, but are not limited to, granulocyte macrophage colony stimulating
factor
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(GM-CSF) (sargramostim, LEZJKINETM, PROKINETM), granulocyte colony
stimulating factor (G-CSF) (filgrastim, NEUPOGENT"~), macrophage colony
stimulating factor (M-CSF, CSF-1) erythropoietin (epoetin alfa, EPOGENTM,
PROCRITTM), stem cell factor (SCF, c-kit ligand, steel factor), megakaryocyte
colony stimulating factor, PIXY321 (a GMCSF/IL-3 fusion protein),
interleukins,
especially any one or more of IL-1 through IL-12, interferon-gamma, or
thrombopoietin.
In certain embodiments, Therapeutics of the present invention are
administered in combination with adrenergic Mockers, such as, for example,
acebutolol, atenolol, betaxolol, bisoprolol, carteolol, labetalol, metoprolol,
nadolol,
oxprenolol, penbutolol, pindolol, propranolol, sotalol, and timolol.
In another embodiment, the Therapeutics of the invention are administered in
combination with an antiarrhythmic drug (e.g., adenosine, amidoarone,
bretylium,
digitalis, digoxin, digitoxin, diliazem, disopyramide, esmolol, flecainide,
lidocaine,
mexiletine, moricizine, phenytoin, procainamide, N-acetyl procainamide,
propafenone, propranolol, quinidine, sotalol, tocainide, and verapamil).
In another embodiment, the Therapeutics of the invention are administered in
combination with diuretic agents, such as carbonic anhydrase-inhibiting agents
(e.g.,
acetazolamide, dichlorphenamide, and methazolamide), osmotic diuretics (e.g.,
glycerin, isosorbide, mannitol, and urea), diuretics that inhibit Na+-K+-2Ch
symport
(e.g., furosemide, bumetanide, azosemide, piretanide, tripamide, ethacrynic
acid,
muzolimine, and torsemide), thiazide and thiazide-like diuretics (e.g.,
bendroflumethiazide; benzthiazide, chlorothiazide, hydrochlorothiazide,
hydroflumethiazide, methyclothiazide, polythiazide, trichormethiazide,
chlorthalidone, indapamide; metolazone, and quinethazone), potassium sparing
diuretics (e.g., amiloride and triamterene), and mineralcorticoid receptor
antagonists
(e.g., spironolactone, canrenone, and potassium canrenoate).
In one embodiment, the Therapeutics of the invention are administered in
combination with treatments for endocrine and/or hormone imbalance disorders.
Treatments for endocrine and/or hormone imbalance disoi-ders include, but are
not
limited to, 1271, radioactive isotopes of iodine such as i3~I and ~23I;
recombinant
growth hormone, such as HLTMATROPET"" (recombinant somatropin); growth
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hormone analogs such as PROTROPINT"" (somatrem); dopamine agonists such as
PARLODELT"" (bromocriptine); somatostatin analogs such as SANDOSTATINT""
(octreotide); gonadotropin preparations such as PREGNYLT"", A.P.L.T~~ and
PROFASIT"" (chorionic gonadotropin (CG)), PERGONALT"" (menotropins), and
METROD1NT"" (urofollitropin (uFSH)); synthetic human gonadotropin releasing
hormone preparations such as FACTRELT"" and LUTREPULSET"" (gonadorelin
hydrochloride); synthetic gonadotropin agonists such as LUPRONT"" (leuprolide
acetate), SUPPRELINT"" (histrelin acetate), SYNARELT"" (nafarelin acetate),
and
ZOLADEXT~" (goserelin acetate); synthetic preparations of thyrotropin-
releasing
hormone such as RELEFACT TRHT"" and THYPINONET"" (protirelin); recombinant
human TSH such as THYROGENT""; synthetic preparations of the sodium salts of
the
natural isomers of thyroid hormones such as L-T4T"", SYNTHROIDT"" and
LEVOTHROIDT"" (levothyroxine sodium), L-T3T"', CYTOMELT"" and TRIOSTATT""
(liothyroine sodium), and THYROLART"~ (liotrix); antithyroid compounds such as
6-
n-propylthiouracil (propylthiouracil), 1-methyl-2-mercaptoimidazole and
TAPAZOLET"" (methimazole), NEO-MERCAZOLET"" (carbimazole); beta-adrenergic
receptor antagonists such as propranolol and esmolol; Ca2+ channel blockers;
dexamethasone and iodinated radiological contrast agents such as TELEPAQUET""
(iopanoic acid) and ORAGRAFINT"" (sodium ipodate).
Additional treatments for endocrine and/or hormone imbalance disorders
include, but are not limited to, estrogens or congugated estrogens such as
ESTRACET"" (estradiol), ESTINYLT"" (ethinyl estradiol), PREMARINT"",
ESTRATABT"", ORTHO-ESTT"", OGENT"" and estropipate (estrone), ESTROVIST""
(quinestrol), ESTRADERMT"" (estradiol), DELESTROGENT"" and VALERGENT""
(estradiol valerate), DEPO-ESTRADIOL CYPIONATET"" and ESTROJECT LAT""
(estradiol cypionate); antiestrogens such as NOLVADEXT"" (tamoxifen),
SEROPHENET"" and CLOMIDT"" (clomiphene); progestins such as DURALUTINT""
(hydroxyprogesterone caproate), MPAT"" and DEPO-PROVERAT""
(medroxyprogesterone acetate), PROVERAT"" and CYCRINT"" (MPA), MEGACET""
(megestrol acetate), NORLUTINT"" (norethindrone), and NORLUTATET"" and
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AYGEST1NT"" (norethindrone acetate); progesterone implants such as NORPLANT
SYSTEMT"" (subdermal implants of norgestrel); antiprogestins such as RU 486T""
(mifepristone); hormonal contraceptives such as ENOVIDT"" (norethynodrel plus
mestranol), PROGESTASERTT"" (intrauterine device that releases progesterone),
LOESTRINT"', BREVICONT"", MODICONT"', GENORAT"", NELONAT"",
NORINYLT"~, OVACON-35T"" and OVACON-SOT"" (ethinyl estradiol/norethindrone),
LEVLENT~", NORDETTET"", TRI-LEVLENT"" and TRIPHASIL-21T"" (ethinyl
estradiol/levonorgestrel) LO/OVRALT"" and OVRALT"" (ethinyl
estradiol/norgestrel),
DEMULENT"" (ethinyl estradiol/ethynodiol diacetate), NORINYLT"", ORTHO-
NOVUMT"", NORETHINT"", GENORAT"", and NELOVAT"" (norethindrone/mestranol),
DESOGENT"" and ORTHO-CEPTT"" (ethinyl estradiol/desogestrel), ORTHO-
CYCLENT"" and ORTHO-TRICYCLENT"" (ethinyl estradiol/norgestimate),
MICRONORT"" and NOR-QDT"" (norethindrone), and OVRETTET"" (norgestrel).
Additional treatments for endocrine and/or hormone imbalance disorders
include, but are not limited to, testosterone esters such as methenolone
acetate and
testosterone undecanoate; parenteral and oral androgens such as TESTOJECT-
SOT""
(testosterone), TESTEXT~" (testosterone propionate), DELATESTRYLT""
(testosterone
enanthate), DEPO-TESTOSTERONET"" (testosterone cypionate), DANOCRINET""
(danazol), HALOTESTINT"" (fluoxymesterone), ORETON METHYLT"", TESTREDT""
and VIRILONT"" (methyltestosterone), and OXANDRINT"" (oxandrolone);
testosterone transdermal systems such as TESTODERMT""; androgen receptor
antagonist and 5-alpha-reductase inhibitors such as ANDROCURT"" (cyproterone
acetate), EULEXINT"" (flutamide), and PROSCART"" (finasteride);
adrenocorticotropic
hormone preparations such as CORTROSYNT"" (cosyntropin); adrenocortical
steroids
and their synthetic analogs such as ACLOVATET"" (alclometasone dipropionate),
CYCLOCORTT"" (amcinonide), BECLOVENTT"" and VANCERILT"' (beclomethasone
dipropionate), CELESTONET"" (betamethasone), BENISONET"" and UTICORTT""
(betamethasone benzoate), DIPROSONET"" (betamethasone dipropionate),
CELESTONE PHOSPHATET"' (betamethasone sodium phosphate), CELESTONE
SOLUSPANT"~ (betamethasone sodium phosphate and acetate), BETA-VALT"" and
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VALISONET"" (betamethasone valerate), TEMOVATET"" (clobetasol propionate),
CLODERMT"" (clocortolone pivalate), CORTEFT"" and HYDROCORTONET""
(cortisol (hydrocortisone)), HYDROCORTONE ACETATET"" (cortisol
(hydrocortisone) acetate), LOCOIDT"" (cortisol (hydrocortisone) butyrate),
HYDROCORTONE PHOSPHATET"" (cortisol (hydrocortisone) sodium phosphate),
A-HYDROCORTT"" and SOLU CORTEFT"" (cortisol (hydrocortisone) sodium
succinate), WESTCORTT"" (cortisol (hydrocortisone) valerate), CORTISONE
ACETATET"" (cortisone acetate), DESOWENT"" and TRIDESILONT"" (desonide),
TOPICORTT"" (desoximetasone), DECADRONT"" (dexamethasone), DECADRON
LAT"" (dexamethasone acetate), DECADRON PHOSPHATET"" and HEXADROL
PHOSPHATET"" (dexamethasone sodium phosphate), FLORONET"" and
MAXIFLORT~" (diflorasone diacetate), FLORINEF ACETATET"" (fludrocortisone
acetate), AEROB>DT"" and NASALIDET"" (flunisolide), FLUONIDT"" and
SYNALART"" (fluocinolone acetonide), LIDEXT"" (fluocinonide), FLUOR-OPT"" and
FMLT"" (fluorometholone), CORDRANT"" (flurandrenolide), HALOGT""
(halcinonide),
HMS LIZUIFILMT"" (medrysone), MEDROLT"" (methylprednisolone), DEPO-
MEDROLT"" and MEDROL ACETATET"' (methylprednisone acetate), A-
METHAPREDT"' and SOLUMEDROLT"" (methylprednisolbne sodium succinate),
ELOCONT"" (mometasone furoate), HALDRONET"" (paramethasone acetate),
DELTA-CORTEFT"" (prednisolone), ECONOPREDT"" (prednisolone acetate),
HYDELTRASOLT"" (prednisolone sodium phosphate), HYDELTRA-T.B.AT""
(prednisolone tebutate), DELTASONET"" (prednisone), ARISTOCORTT"" and
KENACORTT"" (triamcinolone), KENALOGT"" (triamcinolone acetonide),
ARISTOCORTT"" and KENACORT DIACETATET"" (triamcinolone diacetate), and
ARISTOSPANT~" (triamcinolone hexacetonide); inhibitors of biosynthesis and
action
of adrenocortical steroids such as CYTADRENT"" (aminoglutethimide), NIZORALT""
(ketoconazole), MODRASTANET"" (trilostane), and METOPIRONET"" (metyrapone);
bovine, porcine or human insulin or mixtures thereof; insulin analogs;
recombinant
human insulin such as HUMULINT"" and NOVOLINT""; oral hypoglycemic agents
such as ORAMIDET"" and ORINASET"" (tolbutamide), DIABINESET""
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(chlorpropamide), TOLAMIDET"" and TOLINASET"" (tolazamide), DYMELORT""
(acetohexamide), glibenclamide, MICRONASET"", DIBETAT"" and GLYNASET""
(glyburide), GLUCOTROLT"" (glipizide), and DIAMICRONT"" (gliclazide),
GLUCOPHAGET"" (metformin), ciglitazone, pioglitazone, and alpha-glucosidase
inhibitors; bovine or porcine glucagon; somatostatins such as SANDOSTATINT""
(octreotide); and diazoxides such as PROGLYCEMT"" (diazoxide).
In one embodiment, the Therapeutics of the invention are administered in
combination with treatments for uterine motility disorders. Treatments for
uterine
motility disorders include, but are not limited to, estrogen drugs such as
conjugated
estrogens (e.g., PREMARIN° and. ESTRATAB°), estradiols (e.g.,
CLIMARA° and
ALORA°), estropipate; and chlorotrianisene; progestin drugs (e.g.,
AMEN°
(medroxyprogesterone), MICRONOR° (norethidrone acetate),
PROMETRIUM°
progesterone, and megestrol acetate); and estrogen/progesterone combination
therapies such as, for example, conjugated estrogens/medroxyprogesterone
(e.g.,
PREMPROT"" and PREMPHASE°) and norethindrone acetate/ethinyl estsradiol
(e.g.,
FEMHRTT"").
In an additional embodiment, the Therapeutics of the invention are
administered in combination with drugs effective in treating iron deficiency
and
hypochromic anemias, including but not limited to, ferrous sulfate (iron
sulfate,
FEOSOLTM), ferrous fumarate (e.g., FEOSTATTM), ferrous gluconate (e.g.,
FERGONTM), polysaccharide-iron complex (e.g., NIFEREXTM), iron dextran
injection (e.g., INFEDTM), cupric sulfate, pyroxidine, riboflavin, Vitamin
B,Z,
cyancobalamin injection (e.g., REDISOLTM, RUBRAMIN PCTM), hydroxocobalamin,
folic acid (e.g., FOLVITETM), leucovorin (folinic acid, 5-CHOH4PteGlu,
citrovorum
factor) or WELLCOVORIN (Calcium salt of leucovorin), transferrin or ferritin.
In certain embodiments, the Therapeutics of the invention are administered in
combination with agents used to treat psychiatric disorders. Psychiatric drugs
that
may be administered with the Therapeutics of the invention include, but are
not
limited to, antipsychotic agents (e.g., chlorpromazine, chlorprothixene,
clozapine,
fluphenazine, haloperidol, loxapine, mesoridazine, molindone, olanzapine,
perphenazine, pimozide, quetiapine, risperidone, thioridazine, thiothixene,
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trifluoperazine, and triflupromazine), antimanic agents (e.g., carbamazepine,
divalproex sodium, lithium carbonate, and lithium citrate), antidepressants
(e.g.,
amitriptyline, amoxapine, bupropion, citalopram, clomipramine, desipramine,
doxepin, fluvoxamine, fluoxetine, imipramine, isocarboxazid, maprotiline,
5' mirtazapine, nefazodone, nortriptyline, paroxetine, phenelzine,
protriptyline,
sertraline, tranylcypromine, trazodone, trimipramine, and venlafaxine),
antianxiety
agents (e.g., alprazolam, buspirone, chlordiazepoxide, clorazepate, diazepam,
halazepam, lorazepam, oxazepam, and prazepam), and stimulants (e.g., d-
amphetamine, methylphenidate, and pemoline).
In other embodiments, the Therapeutics of the invention are administered in
combination with agents used to treat neurological disorders. Neurological
agents
that may be administered with the Therapeutics of the invention include, but
are not
limited to, antiepileptic agents (e.g., carbamazepine, clonazepam,
ethosuximide,
phenobarbital, phenytoin, primidone, valproic acid, divalproex sodium,
felbamate,
gabapentin, lamotrigine, levetiracetam, oxcarbazepine, tiagabine, topiramate,
zonisamide, diazepam, lorazepam, and clonazepam), antiparkinsonian agents
(e.g:,
levodopa/carbidopa, selegiline, amantidine, bromocriptine, pergolide,
ropinirole,
pramipexole, benztropine; biperiden; ethopropazine; procyclidine;
trihexyphenidyl,
tolcapone), and ALS therapeutics (e.g. riluzole).
In another embodiment, Therapeutics of the invention are administered in
combination with vasodilating agents and/or calcium channel blocking agents.
Vasodilating agents that may be administered with the Therapeutics of the
invention
include, but are not limited to, Angiotensin Converting Enzyme (ACE)
inhibitors
(e.g., papaverine, isoxsuprine, benazepril, captopril, cilazapril, enalapril,
enalaprilat,
fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril,
spirapril, trandolapril,
and nylidrin), and nitrates (e.g., isosorbide dinitrate, isosorbide
mononitrate, and
nitroglycerin). Examples of calcium channel blocking agents that may be
administered in combination with the Therapeutics of the invention include,
but are
not limited to amlodipine, bepridil, diltiazem, felodipine, flunarizine,
isradipine,
nicardipine, nifedipine, nimodipine, and verapamil.
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In additional embodiments, the Therapeutics of the invention are administered
in combination with other therapeutic or prophylactic regimens, such as, for
example,
radiation therapy.
Example 24: Method of Treating Decreased Levels of the Polypeptide
The present invention relates to a method for treating an individual in need
of
an increased level of a polypeptide of the invention in the body comprising
administering to such an individual a composition comprising a therapeutically
effective amount of an agonist of the invention (including polypeptides of the
invention). Moreover, it will be appreciated that conditions caused by a
decrease in
the standard or normal expression level of a secreted protein in an individual
can be
treated by administering the polypeptide of the present invention, preferably
in the
secreted form. Thus, the invention also provides a method of treatment of an
individual in need of an increased level of the polypeptide comprising
administering
to such an individual a Therapeutic comprising an amount of the polypeptide to
increase the activity level of the polypeptide in such an individual.
For example, a patient with decreased levels of a polypeptide receives a daily
dose 0.1-100 ug/kg of the polypeptide for six consecutive days. Preferably,
the
polypeptide is in the secreted form. The exact details of the dosing scheme,
based on .
administration and formulation, are provided in Example 23.
Example 25: Method of Treating Increased Levels of the Polypeptide
The present invention also relates to a method of treating an individual in
need
of a decreased level of a polypeptide of the invention in the body comprising
administering to such an individual a composition comprising a therapeutically
effective amount of an antagonist of the invention (including polypeptides and
antibodies of the invention).
In one example, antisense technology is used to inhibit production of a
polypeptide of the present invention. This technology is one example of a
method of
decreasing levels of a polypeptide, preferably a secreted form, due to a
variety of
etiologies, such as cancer. For example, a patient diagnosed with abnormally
increased levels of a polypeptide is administered intravenously antisense
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polynucleotides at 0.5, 1.0, 1.5, 2.0 and 3.0 mg/kg day for 21 days. This
treatment is
repeated after a 7-day rest period if the treatment was well tolerated. The
formulation
of the antisense polynucleotide is provided in Example 23.
Example 26:' Method of Treatment Using Gene Therapy-Ex Vivo
One method of gene therapy transplants fibroblasts, which are capable of
expressing a polypeptide, onto a patient. Generally, fibroblasts are obtained
from a
subject by skin biopsy. The resulting tissue is placed in tissue-culture
medium and
separated into small pieces. Small chunks of the tissue are placed on a wet
surface of
a tissue culture flask, approximately ten pieces are placed in each flask. The
flask is
turned upside down, closed tight and left at room temperature over night.
After 24
hours at room temperature, the flask is inverted and the chunks of tissue
remain fixed
to the bottom of the flask and fresh media (e.g., Ham's F12 media, with 10%
FBS,
penicillin and streptomycin) is added. The flasks are then incubated at 37
degree C
for approximately one week.
At this time, fresh media is added and subsequently changed every several
days: After an additional two weeks in culture, a monolayer of fibroblasts
emerge.
The monolayer is trypsinized and scaled into larger flasks.
pMV-7 (Kirschmeier, P.T. et al., DNA, 7:219-25 (1988)), flanked by the long
terminal repeats of the Moloney murine sarcoma virus, is digested with EcoRI
and
HindIII and subsequently treated with calf intestinal phosphatase. The linear
vector is
fractionated on agarose gel and purified, using glass beads.
The cDNA encoding a polypeptide of the present invention can be amplified
using PCR primers which correspond to the 5' and 3' end sequences respectively
as set
forth in Example 1 using primers and having appropriate restriction sites and
initiation/stop codons, if necessary. Preferably, the 5' primer contains an
EcoRI site
and the 3' primer includes a HindIII'site. Equal quantities of the Moloney
murine
sarcoma virus linear backbone and the amplified EcoRI and HindIII fragment are
added together, in the presence of T4 DNA ligase. The resulting mixture is
maintained under conditions appropriate for ligation of the two fragments. The
ligation mixture is then used to transform bacteria HB101; which are then
plated onto
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agar containing kanamycin for the purpose of confirming that the vector has
the gene
of interest properly inserted. .
The amphotropic pA317 or GP+arnl2 packaging cells are grown in tissue
culture to confluent density in Dulbecco's Modified Eagles Medium (DMEM) with
10% calf serum (CS), penicillin and streptomycin. The MSV vector containing
the
gene is then added to the media and the packaging cells transduced with the
vector.
The packaging cells now produce infectious viral particles containing the gene
(the
packaging cells are now referred to as producer cells).
Fresh media is added to the transduced producer cells, and subsequently, the
media is harvested from a 10 cm plate of confluent producer cells. The spent
media,
containing the infectious viral particles, is filtered through a millipore
filter to remove
detached producer cells and this media is then used to infect fibroblast
cells. Media is
removed from a sub-confluent plate of fibroblasts and quickly replaced with
the
media from the producer cells. This media is removed and replaced with fresh
media.
If the titer of virus is high, then virtually all fibroblasts will be infected
and no
selection is required. If the titer is very low, then it is necessary to use a
retroviral
vector that has a selectable marker, such as neo or his. Once the fibroblasts
have been
efficiently infected, the fibroblasts are analyzed to determine whether
protein is
produced.
The engineered fibroblasts are then transplanted onto the host, either alone
or
after having been grown to confluence on cytodex 3 microcarrier beads.
Example 27: Gene Therapy Using Endogenous Genes Corresponding To
Polynucleotides of the Invention
Another method of gene therapy according to the present invention involves
operably associating the endogenous polynucleotide sequence of the invention
with a
promoter via homologous recombination as described, for example, in U.S.
Patent
NO: 5,641,670, issued June 24, 1997; International Publication NO: WO
96/29411,
published September 26, 1996; International Publication NO: WO 94/12650,
published August 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA, 86:8932-
8935,
(1989); and Zijlstra et al., Nature, 342:435-438 (1989). This method involves
the
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activation of.a gene which is present in the target cells, but which is not
expressed in
the cells, or is expressed at a lower level than desired.
Polynucleotide constructs are made which contain a promoter and targeting
sequences, which are homologous to the 5' non-coding sequence of endogenous
polynucleotide sequence, flanking the promoter. The targeting sequence will be
sufficiently near the 5' end of the polynucleotide sequence so the promoter
will be
operably linked to the endogenous sequence upon homologous recombination. The
promoter and the targeting sequences can be amplified using PCR. Preferably,
the
amplified promoter contains distinct restriction enzyme sites on the 5' and 3'
ends.
Preferably, the 3' end of the first targeting sequence contains the same
restriction
enzyme site as the 5' end of the amplified promoter and the 5' end of the
second
targeting sequence contains the same restriction site as the 3' end of the
amplified
promoter.
The amplified promoter and the amplified targeting sequences are digested
with the appropriate restriction enzymes and subsequently treated with calf
intestinal
phosphatase. The digested promoter and digested targeting sequences are added
together in the presence of T4 DNA lipase. The resulting mixture is maintained
under
conditions appropriate for ligation of the two fragments. The construct is
size
fractionated on an agarose gel then purified by phenol extraction and ethanol
precipitation.
In this Example, the polynucleotide constructs are administered as naked
polynucleotides via electroporation. However, the polynucleotide constructs
may also
be administered with transfection-facilitating agents, such as liposomes,
viral
sequences, viral particles, precipitating agents, etc. Such methods of
delivery are
known in the art.
Once the cells are transfected, homologous recombination will take place
which results in the promoter being operably linked to the endogenous
polynucleotide
sequence. This results in the expression of polynucleotide corresponding to
the
polynucleotide in the cell. Expression may be detected by immunological
staining, or
any other method known in the art.
Fibroblasts are obtained from a subject by skin biopsy. The resulting tissue
is
placed in DMEM + 10% fetal calf serum. Exponentially growing or early
stationary
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phase fibroblasts are trypsinized and rinsed from the plastic surface with
nutrient
medium. An aliquot of the cell suspension is removed for counting, and the
remaining
cells are subjected to centrifugation. The supernatant is aspirated and the
pellet is
resuspended in 5 ml of electroporation buffer (20 mM HEPES pH 7.3, 137 mM
NaCI,
5 mM KC1, 0.7 mM Na2 HP04, 6 mM dextrose). The cells are recentrifuged, the
supernatant aspirated, and the cells resuspended in electroporation buffer
containing 1
mg/ml acetylated bovine serum albumin. The final cell suspension contains,
approximately 3X106 cells/ml: Electroporation should be performed immediately
following resuspension.
Plasmid DNA is prepared according to standard techniques. For example, to
construct a plasmid for targeting to the locus corresponding to the
polynucleotide of
the invention, plasmid pUCl8 (MBI Fermentas, Amherst, NY) is digested with
HindIII. The CMV promoter is amplified by PCR with an XbaI site on the 5' end
and
a BamHI site on the 3'end. Two non-coding sequences are amplified via PCR: one
non-coding sequence (fragment 1 ) is amplified with a HindIII site at the 5'
end and an
Xba site at the 3'end; the other non-coding sequence (fragment 2) is amplified
with a
BamHI site at the 5'end and a HindIII site at the 3'end. The CMV promotei and
the
fragments (1 and 2) are digested with the appropriate enzymes (CMV promoter -
XbaI
and BamHI; fragment 1 - XbaI; fragment 2 - BamHI) and ligated together. The
resulting ligation product is digested with HindIII, and ligated with the
HindIII-
digested pUCl8 plasmid.
Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrode gap
(Bio-Rad). The final DNA concentration is generally at least 120 ~g/ml. 0.5 ml
of the
cell suspension (containing approximately 1.5.X106 cells) is then added to the
cuvette,
and the cell suspension and DNA solutions are gently mixed. Electroporation is
performed with a Gene-Pulser apparatus (Bio-Rad). Capacitance and voltage are
set at
960 pF and 250-300 V, respectively. As voltage increases, cell survival
decreases, but
the percentage of surviving cells that stably incorporate the introduced DNA
into their
genome increases dramatically. Given these parameters, a pulse time of
approximately 14-20 mSec should be observed.
Electroporated cells are maintained at room temperature for approximately 5
min, and the contents of the cuvette are then gently removed with a sterile
transfer
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pipette. The cells are added directly to 10 ml of prewarmed nutrient media
(DMEM
with 15% calf serum) in a 10 cm dish and incubated at 37 degree C. The
following
day, the media is aspirated and replaced with 10 ml of fresh media and
incubated for a
further 16-24 hours.
The engineered fibroblasts are then injected into the host, either alone or
after
having been grown to confluence on cytodex 3 microcarrier beads. The
fibroblasts
now produce the protein product. The fibroblasts can then be introduced into a
patient as described above.
Example 28: Method of Treatment Using Gene Therapy - In Vivo
Another aspect of the present invention is using in vivo gene therapy methods
to treat disorders, diseases and conditions. The gene therapy method relates
to the
introduction of naked nucleic acid (DNA, RNA, and antisense DNA or RNA)
sequences into an animal to increase or decrease the expression of the
polypeptide.
The polynucleotide of the present invention may be operatively linked to a
promoter
or any other genetic elements necessary for the expression of the polypeptide
by the
target tissue. Such gene therapy and delivery techniques and methods are known
in
the art, see, for example, W090/11092, W098/11779; U.S. Patent NO. 5693622,
5705151, 5580859; Tabata et al., Cardiovasc. Res. 35(3):470-479 (1997); Chao
et al.,
Pharmacol. Res. 35(6):517-522 (1997); Wolff, Neuromuscul. Disord. 7(5):314-318
(1997); Schwartz et al., Gene Ther. 3(5):405-411 (1996); Tsurumi et al.,
Circulation
94(12):3281-3290 (1996) (incorporated herein by reference).
The polynucleotide constructs may be delivered by any method that delivers
injectable materials to the cells of an animal, such as, injection into the
interstitial
space of tissues (heart, muscle, skin, lung, liver, intestine and the like).
The
polynucleotide constructs can be delivered in a pharmaceutically acceptable
liquid or
aqueous carrier.
The term "naked" polynucleotide, DNA or RNA, refers to sequences that are
free from any delivery vehicle that acts to assist, promote, or facilitate
entry into the
cell, including viral sequences, viral particles, liposome formulations,
lipofectin or
precipitating agents and the like. However, the polynucleotides of the present
.
invention may also be delivered in liposome formulations (such as those taught
in _.
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Felgner P.L. et al. (1995) Ann. NY Acad. Sci. 772:126-139 and Abdallah B. et
al.
(1995) Biol. Cell 85(1):1-7) which can be prepared by methods well known to
those
skilled in the art.
The polynucleotide vector constructs used in the gene therapy method are
preferably constructs that will not integrate into the host genome nor will
they contain
sequences that allow for replication. Any strong promoter known to those
skilled in
the art can be used for driving the expression of DNA. Unlike other gene
therapies
techniques, one major advantage of introducing naked nucleic acid sequences
into
target cells is the transitory nature of the polynucleotide synthesis in the
cells. Studies
have shown that non-replicating DNA sequences can be introduced into cells to
provide production of the desired polypeptide for periods of up to six months.
The polynucleotide construct can be delivered to the interstitial space of
tissues within the an animal, including of muscle, skin, brain, lung, liver,
spleen, bone
marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall
bladder,
stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland,
and
connective tissue. Interstitial space of the tissues comprises the
intercellular fluid,
mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic
fibers
in the walls of vessels or chambers, collagen fibers of fibrous tissues, or
that same
matrix within connective tissue ensheathing muscle cells or in the lacunae of
bone. It
is similarly the space occupied by the plasma of the circulation and the lymph
fluid of
the lymphatic channels. Delivery to the interstitial space of muscle tissue is
preferred
for the reasons discussed below. They may be conveniently delivered by
injection
into the tissues comprising these cells. They are preferably delivered to and
expressed in persistent, non-dividing cells which are differentiated, although
delivery
and expression maybe achieved in non-differentiated or less completely
differentiated cells, such as, for example, stem cells of blood or skin
fibroblasts. In
vivo muscle cells are particularly competent in their ability to take up and
express
polynucleotides.
For the naked polynucleotide injection, an effective dosage amount of DNA or
RNA will be in the range of from about 0.05 g/kg body weight to about 50 mg/kg
body weight. Preferably the dosage will be from about 0.005 mg/kg to about 20
mg/kg and more preferably from about 0.05 mg/kg to about 5 mg/kg. Of course,
as
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the artisan of ordinary skill will appreciate, this dosage will vary according
to the
tissue site of injection. The appropriate and effective dosage of nucleic acid
sequence
can readily be determined by those of ordinary skill in the art and may depend
on the
condition being treated and the route of administration. The preferred route
of
administration is by the parenteral route of injection into the interstitial
space of
tissues. However, other parenteral routes may also be usef, such as,
inhalation of an
aerosol formulation particularly for delivery to lungs or bronchial tissues,
throat or
mucous membranes of the nose. In addition, naked polynucleotide constructs can
be
delivered to arteries during angioplasty by the catheter used ~in the
procedure.
The dose response effects of injected polynucleotide in muscle in vivo is
determined as follows. Suitable template DNA for production of mRNA coding for
polypeptide of the present invention is prepared in accordance with a standard
recombinant DNA methodology. The template DNA, which may be either circular or
linear, is either used as naked DNA or complexed with liposomes. The
quadriceps
muscles of mice are then injected with various amounts of the template DNA.
Five to six week old female and male Balb/C mice are anesthetized by
intraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cm incision is
made on
the anterior thigh, and the quadriceps muscle is directly visualized. The
template
DNA is injected in 0.1 ml of carrier in a 1 cc syringe through a 27 gauge
needle over
one minute, approximately 0.5 cm from the distal insertion site of the muscle
into the
. knee and about 0.2 cm deep. A suture is placed over the injection site for
future
localization, and the skin is closed with, stainless steel clips.
After an appropriate incubation time (e.g., 7 days) muscle extracts are
prepared by excising the entire quadriceps. Every fifth 15 um cross-section of
the
individual quadriceps muscles is histochemically stained for protein
expression. A
time course for protein expression may be done in a similar fashion except
that
quadriceps from different mice are harvested at different times. Persistence
of DNA
in muscle following injection may be determined by Southern blot analysis
after
preparing total cellular DNA and HIRT supernatants from injected and control
mice.
The results of the above experimentation in mice can be use to extrapolate
proper
dosages and other treatment parameters in humans and other animals using naked
DNA.
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Example 29: Trans~enic Animals.
The polypeptides of the invention can also be expressed in transgenic animals.
Animals of any species, including, but not limited to, mice, rats, rabbits,
hamsters,
guinea pigs, pigs, micro-pigs, goats, sheep, cows and non-human primates,
e.g.,
baboons, monkeys, and chimpanzees may be used to generate transgenic animals.
In a
specific embodiment, techniques described herein or otherwise known in the
art, are
used to express polypeptides of the invention in humans, as part of a gene
therapy
protocol.
Any technique known in the art may be used to introduce the transgene (i.e.,
polynucleotides of the invention) into animals to produce the founder lines of
transgenic animals. Such techniques include, but are not limited to,
pronuclear
microinjection (Paterson et al., Appl. Microbiol. Biotechnol. 40:691-698
(1994);
Carver et al., Biotechnology (NY) 11:1263-1270 (1993); Wright et al.,
Biotechnology
(NY) 9:830-834 (1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989));
retrovirus
mediated gene transfer into germ lines (Van der Putten et al., Proc. Natl.
Acad. Sci.,
USA 82:6148-6152 (1985)), blastocysts or embryos; gene targeting in embryonic
stem cells (Thompson et al., Cell 56:313-321 ( 1989)); electroporation of
cells or
embryos (Lo, 1983, Mol Cell. Biol. 3:1803-1814 (1983)); introduction of the
polynucleotides of the invention using a gene gun (see, e.g., Ulmer et al.,
Science
259:1745 (1993); introducing nucleic acid constructs into embryonic
pleuripotent
stem cells and transferring the stem cells back into the blastocyst; and sperm-
mediated gene transfer (Lavitrano et al., Cell 57:717-723 (1989); etc. For a
review of
such techniques, see Gordon, "Transgenic Animals," Intl. Rev. Cytol. 115:171-
229
(1989), which is incorporated by reference herein in its entirety.
Any technique known in the art may be used to produce transgenic clones
containing polynucleotides of the invention, for example, nuclear transfer
into
enucleated oocytes of nuclei from cultured embryonic, fetal, or adult cells
induced to
quiescence (Campell et al., Nature 380:64-66 (1996); Wilmut et al., Nature
385:810-
813 (1997)).
The present invention provides for transgenic animals that carry the transgene
in all their cells, as well as animals which carry the transgene in some, but
not all their
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cells, i.e., mosaic animals or chimeric. The transgene may be integrated as a
single
transgene or as multiple copies such as in concatamers, e.g., head-to-head
tandems or
head-to-tail tandems. The transgene may also be selectively introduced into
and
activated in a particular cell type by following, for example, the teaching of
Lasko et
al. (Lasko et al., Proc. Natl. Acad. Sci. USA 89:6232-6236 (1992)). The
regulatory
sequences required for such a cell-type specific activation will depend upon
the
particular cell type of interest, and will be apparent to those of skill in
the art. When
it is desired that the polynucleotide transgene be integrated into the
chromosomal site
of the endogenous gene, gene targeting is preferred. Briefly, when such a
technique is
to be utilized, vectors containing some nucleotide sequences homologous to the
endogenous gene are designed for the purpose of integrating, via homologous
recombination with chromosomal sequences, into and disrupting the function of
the
nucleotide sequence of the endogenous gene. The transgene may also be
selectively
introduced into a particular cell type, thus inactivating the endogenous gene
in only
that cell type, by following, for example, the teaching of Gu et al. (Gu et
al., Science
265:103-106 (1994)). The regulatory sequences required for such a cell-type
specific
inactivation will depend upon the particular cell type of interest, and will
be apparent
to those of skill in the art.
Once transgenic animals have been generated, the expression of the
recombinant gene may be assayed utilizing standard techniques. Initial
screening
may be accomplished by Southern blot analysis or PCR techniques to analyze
animal
tissues to verify that integration of the transgene has taken place. The level
of mRNA
expression of the transgene iri the tissues of the transgenic animals may also
be
assessed using techniques which include, but are not limited to, Northern blot
analysis
of tissue samples obtained from the animal, in situ hybridization analysis,
.and reverse
transcriptase-PCR (rt-PCR). Samples of transgenic gene-expressing tissue may
also
be evaluated immunocytochemically or immunohistochemically using antibodies
specific for the transgene product.
Once the founder animals are produced, they may be bred, inbred, outbred, or
30- crossbred to produce colonies of the particular animal. Examples of such
breeding
strategies include, but are not limited to: outbreeding of founder animals
with more
than one integration site in order to establish separate lines; inbreeding of
separate
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lines in order to produce compound transgenics that express the transgene at
higher
levels because of the effects of additive expression of each transgene;
crossing of
heterozygous transgenic animals to produce animals homozygous for a given
integration site in order to both augment expression and eliminate the need
for
screening of animals by DNA analysis; crossing of separate homozygous lines to
produce compound heterozygous or homozygous lines; and breeding to place the
transgene on a distinct background that is appropriate for an experimental
model of
interest.
Transgenic animals of the invention have uses which include, but are not
limited to, animal model systems useful in elaborating the biological function
of
polypeptides of the present invention, studying diseases, disorders, and/or
conditions
associated with aberrant expression, and in screening for compounds effective
in
ameliorating such diseases, disorders, and/or conditions.
Examule 30: Knock-Out Animals.
Endogenous gene expression can also be reduced by inactivating or "knocking
out" the gene and/or its promoter using targeted homologous recombination.
(E.g.,
see Smithies et al., Nature 317:230-234 (1985); Thomas & Capecchi, Cell 51:503-
512 (1987); Thompson et al., Cell 5:313-321 (1989); each of which is
incorporated by
reference herein in its entirety). For example, a mutant, non-functional
polynucleotide of the invention (or a completely unrelated DNA sequence)
flanked by
DNA homologous to the endogenous polynucleotide sequence (either the coding
regions or regulatory regions of the gene) can be used, with or without a
selectable
marker and/or a negative selectable marker, to transfect cells that express
polypeptides of the invention in vivo. In another embodiment, techniques known
in
the art are used to generate knockouts in cells that contain, but do not
express the gene
of interest. Insertion of the DNA construct, via targeted homologous
recombination,
results in inactivation of the targeted gene. Such approaches are particularly
suited in
research and agricultural fields where modifications to embryonic stem cells
can be
used to generate animal offspring with an inactive targeted gene (e.g., see
Thomas &
Capecchi 1987 and Thompson 1989, supra). However this approach can be
routinely
adapted for use in humans provided the recombinant DNA constructs are directly
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administered or targeted to the required site in vivo using appropriate viral
vectors that
will be apparent to those of skill in the art.
In further embodiments of the invention, cells that are genetically engineered
to express the polypeptides of the invention, or alternatively, that are
genetically
engineered not fo express the polypeptides of the invention (e.g., knockouts)
are
administered to a patient in vivo. Such cells may be obtained from the patient
(i.e.,
animal, including human) or an MHC compatible donor and can include, but are
not
limited to fibroblasts, bone marrow cells, blood cells (e.~, lymphocytes),
adipocytes,
muscle cells, endothelial cells etc. The cells are genetically engineered in
vitro using
recombinant DNA techniques to introduce the coding sequence of polypeptides of
the
invention into the cells, or alternatively, to disrupt the coding sequence
and/or
endogenous regulatory sequence associated with the polypeptides of the
invention,
e.~, by transduction (using viral vectors, and preferably vectors that
integrate the
transgene into the cell genome) or transfection procedures, including, but not
limited
to, the use of plasmids, cosmids, YACs, naked DNA, electroporation, liposomes,
etc.
The coding sequence of the polypeptides of the invention can.be placed under
the .
control of a strong constitutive or inducible promoter or prorrioter/enhancer
to achieve
expression, and preferably secretion, of the polypeptides~of the invention.
The
engineered cells which express and preferably secrete the polypeptides of the
invention can be introduced into the patient systemically, e.g., in the
circulation, or
intraperitoneally..
Alternatively, the cells can be incorporated into a matrix and implanted in
the
body, ~, genetically engineered fibroblasts can be implanted as part of a skin
graft;
genetically engineered endothelial cells can be implanted as part of a
lymphatic or
vascular graft. (See, for example, Anderson et al. U.S. Patent No. 5,399,349;
and
Mulligan & Wilson, U.S. Patent No. 5,460,959 each of which is incorporated by
reference herein in its entirety).
When the cells to be administered are non-autologous or non-MHC
compatible cells, they can be administeredwsing well known techniques which
prevent the development of a host immune response against the introduced
cells. For
example, the cells may be introduced in an encapsulated form which, while
allowing
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for an exchange of components with the immediate extracellular environment,
does
not allow the introduced cells to be recognized by the host immune system.
Transgenic and "knock-out" animals of the invention have uses which include,
but are not limited to, animal model systems useful in elaborating the
biological
function of polypeptides of the present invention, studying diseases,
disorders, and/or
conditions associated with aberrant expression, and in screening for compounds
effective in ameliorating such diseases, disorders, and/or conditions.
Example 31: Production of an Antibody
Hybridoma Technology
The antibodies of the present invention can be prepared by a variety of
methods. (See, Current Protocols, Chapter 2.) As one example of such methods,
cells expressing polypeptide(s) of the invention are administered to an animal
to
induce the production of sera containing polyclonal antibodies. In a preferred
method,
a preparation of polypeptide(s) of the invention is prepared and purified to
render it
substantially free of natural contaminants. Such a preparation is then
introduced into
an animal in order to produce polyclonal antisera of greater specific
activity.
Monoclonal antibodies specific for polypeptide(s) of the invention are
prepared using hybridoma technology. (Kohler et al., Nature 256:495 (1975);
Kohler
et al., Eur. J. Immunol. 6:511 (1976); Kohler et al., Eur. J. Immunol. 6:292
(1976);
Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier,
N.Y., pp. 563-681 (1981)). In general, an animal (preferably a mouse) is
immunized
with polypeptide(s) of the invention, or, more preferably, with a secreted
polypeptide-
expressing cell. Such polypeptide-expressing cells are cultured in any
suitable tissue
culture medium, preferably in Earle's modified Eagle's medium supplemented
with
10% fetal bovine serum (inactivated at about 56°C), and supplemented
with about 10
g/I of nonessential amino acids, about 1,000 U/ml of penicillin, and about 100
~g/ml
of streptomycin.
The splenocytes of such mice are extracted and fused with a suitable myeloma
cell line. Any suitable myeloma cell line may be employed in accordance with
the
present invention; however, it is preferable to employ the parent myeloma cell
line
(SP20), available from the ATCC. After fusion, the resulting hybridoma cells
are ' _,
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selectively maintained in HAT medium, and then cloned by limiting dilution as
described by Wands et al. (Gastroenterology 80:225-232 (1981)). The hybridoma
cells obtained through such a selection are then assayed to identify clones
which
secrete antibodies capable of binding the polypeptide(s) of the invention.
Alternatively, additional antibodies capable of binding polypeptide(s) of the
invention can be produced in a two-step procedure using anti-idiotypic
antibodies.
Such a method makes use of the fact that antibodies are themselves antigens,
and
therefore, it is possible to obtain an antibody which binds to a second
antibody. In
accordance with this method, protein specific antibodies are used to immunize
an
animal, preferably a mouse. The splenocytes of such an animal are then used to
produce hybridoma cells, and the hybridoma cells are screened to identify
clones
which produce an antibody whose ability to bind to the polypeptide(s) of the
invention protein-specific antibody can be blocked by polypeptide(s) of the
invention.
Such antibodies comprise anti-idiotypic antibodies to the polypeptide(s) of
the
invention protein-specific antibody and are used to immunize an animal to
induce
formation of further polypeptide(s),of the invention protein-specific
antibodies.
For in vivo use of antibodies in humans, an antibody is "humanized". Such
antibodies can be produced using genetic constructs derived from hybridoma
cells
producing the monoclonal antibodies described above. Methods for producing
chimeric and humanized antibodies are known in the art and are discussed
herein.
(See, for review, Mornson, Science 229:1202 ( 1985); Oi et al., BioTechniques
4:214
(1986); Cabilly et al., U.S. Patent No. 4,816,567; Taniguchi et al., EP
171496;
Mornson et al., EP 173494; Neuberger et al:, WO 8601533; Robinson et al., WO
8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et,al., Nature
314:268
(1985).)
Isolation Of Antibody Fragments Directed polypeptide(s) of the invention From
A
Library Of scFvs
Naturally occurring V-genes isolated from human PBLs are constructed into a
library of antibody fragments which contain reactivities against
polypeptide(s) of the
invention to which the donor may or may not have been exposed (see e.g., U.S.
Patent
5,885,793 incorporated herein by reference in its entirety).
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Rescue of the Library. A library of scFvs is constructed from the RNA of
human PBLs as described in PCT publication WO 92/01047. To rescue phage
displaying antibody fragments, approximately 109 E. coli harboring the
phagemid are
used to inoculate 50 ml of 2xTY containing 1% glucose and 100 p.g/ml of
ampicillin
(2xTY-AMP-GLU) and grown to an O.D. of 0.8 with shaking. Five ml of this
culture
is used to irmoculate 50 ml of 2xTY-AMP-GLU, 2 x 108 TU of delta gene 3 helper
(M13 delta gene III, see PCT publication WO 92/01047) are added and the
culture
incubated at 37°C for 45 minutes without shaking and then at
37°C for 45 minutes
with shaking. The culture is centrifuged at 4000 r.p.m. for 10 min. and the
pellet
resuspended in 2 liters of 2xTY containing 100 ~ug/ml ampicillin and 50 ug/ml
kanamycin and grown overnight. Phage are prepared as described in PCT
publication
WO 92/01047.
M 13 delta gene III is prepared as follows: M 13 delta gene III helper phage
does not encode gene III protein, hence the phage(mid) displaying antibody
fragments have a greater avidity of binding to antigen. Infectious M13 delta
gene III
particles are made by growing the helper phage in cells harboring a pUCl9
derivative
supplying the wild type gene III protein during phage motphogenesis. The
culture is
incubated for 1 hour at 37° C without shaking and then for a further
hour at 37°C with
shaking. Cells are spun down (IEC-Centra 8,400 r.p.m. for 10 min), resuspended
in
300 ml 2xTY broth containing 100 pg ampicillin/ml and 25 pg kanamycin/ml (2xTY-
AMP-KAN) and grown overnight, shaking at 37°C. Phage particles are
purified and
concentrated from the culture medium by two PEG-precipitations (Sambrook et
al.,
1990), resuspended in 2 ml PBS and passed through a 0.45 pm filter (Minisart
NML;
Sartorius) to give a final concentration of approximately 1013 transducing
units/ml
(ampicillin-resistant clones).
Panning of the Library. Immunotubes (Nunc) are coated overnight in PBS
with 4 ml of either 100 p.g/ml or 10 pg/ml of a polypeptide of the present
invention.
Tubes are blocked with 2% Marvel-PBS for 2 hours at 37°C and then
washed 3'times
in PBS. Approximately 1013 TU of phage is applied to the tube and incubated
for 30
minutes at room temperature tumbling on an over and under turntable and then
left to
stand for another 1.5 hours. Tubes are washed 10 times with PBS 0.1% Tween-20
and 10 times with PBS. Phage are eluted by adding 1 ml of 100 mM triethylamine
and ..
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rotating 15 minutes on an under and over turntable after which the solution is
immediately neutralized with 0.5 ml of 1.0M Tris-HCI, pH 7.4. Phage are then
used
to infect 10 ml of mid-log E. coli TG1 by incubating eluted phage with
bacteria for 30
minutes at 37°C. The E. coli are then plated on TYE plates containing
1% glucose
and 100 pg/ml ampicillin. The resulting bacterial library is then rescued with
delta
gene 3 helper phage as described above to prepare phage for a subsequent round
of
selection. This process is then repeated for a total of 4 rounds of affinity
purification ,
with tube-washing increased to 20 times with PBS, 0.1 % Tween-20 and 20 times
with
PBS for rounds 3 and 4.
Characterization of Binders. Eluted phage from the 3rd and 4th rounds of
selection are used to infect E. coli HB 2151 and soluble scFv is produced
(Marks, et
al., 1991) from single colonies for assay. ELISAs are performed with
microtitre
plates coated with either 10 pg/ml of the polypeptide of the present invention
in 50
mM bicarbonate pH 9.6. Clones positive in ELISA are further characterized by
PCR
fingerprinting (see, e.g., PCT publication WO 92/01047) and then by
sequencing.
These ELISA positive clones may also be further characterized by techniques
known
in the art, such as, for example, epitope mapping, binding affinity, receptor
signal
transduction, ability to block or competitively inhibit antibody/antigen
binding, and
competitive agonistic or antagonistic activity.
Example 32: Assays Detecting Stimulation or Inhibition of B cell Proliferation
and Differentiation
Generation of functional humoral immune responses requires both soluble and
cognate signaling between B-lineage cells and their microenvironment. Signals
may
impart a positive stimulus that allows a B-lineage cell to continue its
programmed
development, or a negative stimulus that instructs the cell to arrest its
current
developmental pathway. To date, numerous stimulatory and inhibitory signals
have been
found to influence B cell responsiveness including IL-2, IL-4, IL-5, IL-6, IL-
7, IL10, IL-
13,~IL-14 and IL-15. Interestingly, these signals are by themselves weak
effectors but can,
in combination with various co-stimulatory proteins, induce activation,
proliferation,
differentiation, homing, tolerance and death among B cell populations.
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One of the best studied classes of B-cell co-stimulatory proteins is the TNF-
superfamily. Within this family CD40, CD27, and CD30 along with their
respective
ligands CD154, CD70, and CD153 have been found to regulate a variety of immune
responses. Assays which allow for the detection and/or observation of the
proliferation
and differentiation of these B-cell populations and their precursors are
valuable tools in
determining the effects various proteins may have on these B-cell populations
in terms of
proliferation and differentiation. Listed below are two assays designed to
allow for the
detection of the differentiation, proliferation, or inhibition of B-cell
populations and their
precursors.
In Vitro Assay- Purified polypeptides of the invention, or truncated forms
thereof, is assessed for its ability to induce activation, proliferation,
differentiation or
inhibition and/or death in B-cell populations and their precursors. The
activity of the
polypeptides of the invention on purified human tonsillar B cells, measured
qualitatively over the dose range from 0.1 to 10,000 ng/mL, is assessed in a
standard
B-lymphocyte co-stimulation assay in which purified tonsillar B cells are
cultured in
the presence of either formalin-fixed Staphylococcus aureus Cowan I (SAC) or
immobilized anti-human IgM antibody as the priming agent. Second signals such
as
IL-2 and IL-15 synergize with SAC and IgM crosslinking to elicit B cell
proliferation
as measured by tritiated-thymidine incorporation. Novel synergizing agents can
be
readily identified using this assay. The assay involves isolating human
tonsillar B
cells by magnetic bead (MACS) depletion of CD3-positive cells. The resulting
cell
population is greater than 95% B cells as assessed by expression of
CD45R(B220).
Various dilutions of each sample are placed into individual wells of a 96-well
plate
to which are added 105 B-cells suspended in culture medium (RPMI 1640
containing 10%
FBS, 5 X 10-5M 2ME, 100U/ml penicillin, l0ug/ml streptomycin, and 10-5
dilution of
SAC) in a total volume of 150u1. Proliferation or inhibition is quantitated by
a 20h pulse
(luCi/well) with 3H-thymidine (6.7 Ci/mM) beginning 72h post factor addition.
The
positive and negative controls are IL2 and medium respectively.
In Vivo Assay- BALB/c mice are injected (i.p.) twice per day with buffer
only, or 2 mg/Kg of a polypeptide of the invention, or truncated forms
thereof. Mice
receive this treatment for 4 consecutive days, at which time they are
sacrificed and
various tissues and serum collected for analyses. Comparison of H&E sections
from
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normal spleens and spleens treated with polypeptides of the invention identify
the
results of the activity o.f the polypeptides on spleen cells, such as the
diffusion of peri-
arterial lymphatic sheaths, and/or significant increases in the nucleated
cellularity of
the red pulp regions, which may indicate the activation of the differentiation
and
proliferation of B-cell populations. Immunohistochemical studies using a B
cell
marker, anti-CD45R(B220), are used to determine whether any physiological
changes
to splenic cells, such as splenic disorganization, are due to increased B-cell
representation within loosely defined B-cell zones that infiltrate established
T-cell
regions.
~ Flow cytometric analyses of the spleens from mice treated with polypeptide
is used
to indicate whether the polypeptide specifically increases the proportion of
ThB+,
CD45R(B220)dull B cells over that which is observed in control mice.
Likewise, a predicted consequence of increased mature B-cell representation in
vivo is a relative increase in serum Ig titers. Accordingly, serum IgM and IgA
levels are
compared between buffer and polypeptide-treated mice.
The studies described in this example tested activity of a polypeptide of the
invention. However, one skilled in the art could easily modify the exemplified
studies to
test the activity of polynucleotides of the invention (e.g., gene therapy),
agonists, and/or
antagonists of polynucleotides or polypeptides of the invention.
Example 33: T Cell Proliferation Assay
Proliferation assay for Resting PBLs.
A CD3-induced proliferation assay is performed on PBMCs and is measured by the
uptake of 3H-thymidine. The assay is performed as follows. Ninety-six well
plates are coated
with 100 microliters per well of mAb to CD3 (HIT3a, Pharmingen) or isotype-
matched control
mAb (B33.1) overnight at 4 C (1 microgram/ml in .05M bicarbonate buffer, pH
9.5), then washed
three times with PBS. PBMC are isolated by F/H gradient centrifugation from
human peripheral
blood and added to quadruplicate wells (5 x 104/well) of mAb coated plates in
RPMI containing
10% FCS and P/S in the presence of varying concentrations~of TNF Delta and/or
TNF Epsilon
protein (total volume 200 microliters). Relevant protein buffer and medium
alone are controls.
After 48 hr. culture at 37 C, plates are spun for 2 min. at 1000 rpm and 100
microliters of
supernatant is removed and stored -20 C for measurement of IL-2 (or other
cytokines) if effect on
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proliferation is observed. Wells are supplemented with 100 microliters of
medium containing 0.5
microcuries of 3H-thymidine and cultured at 37 C for 18-24 hr. Wells are
harvested and
incorporation of 3H-thymidine used as a measure of proliferation. Anti-CD3
alone is the positive
control for proliferation. IL-2 (100 U/ml) is also used as a control which
enhances proliferation.
Control antibody which does not induce proliferation of T cells is used as the
negative controls
for the effects of TNF Delta and/or TNF Epsilon proteins.
Alternatively, a proliferation assay on resting PBL (peripheral blood
lymphocytes) is measured by the up-take of 3H-thymidine. The assay is
performed as
follows. PBMC are isolated by Ficoll (LSM, ICN Biotechnologies, Aurora, Ohio)
gradient centrifugation from human peripheral blood, and are cultured
overnight in
10% (Fetal Calf Serum, Biofluids, Rockville, MD)1RPMI (Gibco BRL,
Gaithersburg,
MD). This overnight incubation period allows the adherent cells to attach to
the
plastic, which results in a lower background in the assay as there are fewer
cells that
can act as antigen presenting cells or that might be producing growth factors.
The
following day the non-adherent cells are collected, washed and used in the
proliferation assay. The assay is performed in a 96 well plate using 2 x 104
cells/well
in a final volume of 200 microliters. The supernatants (e.g., CHO or 293T
supernatants) expressing the protein of interest are tested at a 30% final
dilution,
therefore 60u1 are added to 140u1 of 10% FCS/RPMI containing the cells.
Control
supernatants are used at the same final dilution and express the following
proteins:
vector (negative control), IL-2 (*), IFN , TNF , IL-10 and TR2. In addition to
the
control supernatants, recombinant human IL-2 (R & D Systems, Minneapolois, MN)
at a final concentration of 100ng/ml is also used. After 24 hours of culture,
each well
is pulsed with luCi of 3H-thymidine (Nen, Boston, MA). Cells are then
harvested 20
hours following pulsing and incorporation of 3H-thymidine is used as a measure
of
proliferation. Results are expressed as an average of triplicate samples plus
or minus
standard error.
(*) The amount of the control cytokines IL-2, IFN , TNF and IL-10 produced in
each
transfection varies between 300pg to 5ng/ml.
.
Costimulation assay.
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A costimulation assay on resting PBL (peripheral blood lymphocytes) is
performed in the presence of immobilized antibodies to CD3 and CD28. The use
of
antibodies specific for the invariant regions of CD3 mimic the induction of T
cell
activation that would occur through stimulation of the T cell receptor by an
antigen.
Cross-linking of the TCR (first signal) in the absence of a costimulatory
signal
(second signal) causes very low induction of proliferation and will eventually
result in
a state of "anergy", which is characterized by the absence of growth and
inability to
produce cytokines. The addition of a costimulatory signal such as an antibody
to
CD28, which mimics the action of the costimulatory molecule. B7-1 expressed on
activated APCs, results in enhancement of T cell responses including cell
survival and
production of IL-2. Therefore this type of assay allows to detect both
positive and
negative effects caused by addition of supernatants expressing the proteins of
interest
on T cell proliferation.
The assay is performed as follows. Ninety-six yell plates are coated with
~ 100ng/ml anti-CD3 and Sug/ml anti-CD28 (Pharmingen, San Diego, CA) in a
final
volume of 100u1 and incubated overnight at 4C. Plates are washed twice with
PBS
before use. PBMC are isolated by Ficoll (LSM, ICN Biotechnologies, Aurora,
Ohio)
gradient centrifugation from human peripheral blood, and are cultured
overnight in
10%-FCS(Fetal Calf Serum, Biofluids, Rockville, MD)/RPMI (Gibco BRL,
Gaithersburg, MD). This overnight incubation period allows the adherent cells
to
attach to the plastic, which results in a lower background in the assay as
there are
fewer cells that can act as antigen presenting cells or that might be
producing growth
factors. The following day the non adherent cells are collected, washed and
used in
the proliferation assay. The assay is performed in a 96 well plate using 2 x
104
cells/well in a final volume of 200u1. The supernatants (e.g., CHO or 293T
supernatants) expressing the protein of interest are tested at a 30% final
dilution,
therefore 60u1 are added to 140u1 of 10% FCS/RPMI containing the cells.
Control
supernatants are used at the same final dilution and express the following
proteins:
vector only (negative control), IL-2, IF'N , TNF , IL-10 and TR2. In addition
to the
control supernatants recombinant human IL-2 (R & D Systems, Minneapolis, MN)
at
a final concentration of l Ong/ml is also used. After 24 hours of culture,
each well is
pulsed with luCi of 3H-thymidine (Nen, Boston, MA). Cells are then harvested
20 _. .
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hours following pulsing and incorporation of 3H-thymidine is used as a measure
of
proliferation. Results are expressed as an average of triplicate samples plus
or minus
standard error.
Costimulation assay: IFN y and IL-2 ELISA
The assay is performed as follows. Twenty-four well plates are coated with
either 300ng/ml or 600ng/ml anti-CD3 and Sug/ml anti-CD28 (Pharmingen, San
Diego, CA) in a final volume of SOOuI and incubated overnight at 4C. Plates
are
washed twice with PBS before use. PBMC are isolated by Ficoll (LSM, ICN
Biotechnologies, Aurora, Ohio) gradient centrifugation from human peripheral
blood,
and are cultured overnight in 10% FCS(Fetal Calf Serum, Biofluids, Rockville,
MD)/RPMI (Gibco BRL, Gaithersburg, MD). This overnight incubation period
allows the adherent cells to attach to the plastic, which results in a lower
background
in the assay as there are fewer cells that can act as antigen presenting cells
or that
might be producing growth factors. The following day the non adherent cells
are
collected, washed and used in the costimulation assay. The assay is performed
in the
pre-coated twenty-four well plate using 1 x 105 cells/well in a final volume
of 900u1.
The supernatants (293T supernatants) expressing the protein of interest are
tested at a
30% final dilution, therefore 300u1 are added to 600u1 of 10% FCS/RPMI
containing
the cells. Control supernatants are used at the same final dilution and
express the
following proteins: vector only(negative control), IL-2, IFN , IL-12 and IL-
18. In
addition to the control supernatants recombinant human IL-2 (all cytokines
were
purchased from R & D Systems, Minneapolis, MN) at a final concentration of
lOng/ml, IL-12 at a final concentration of lng/ml and IL-18 at a final
concentration of
SOng/ml are also used. Controls and unknown samples are tested in duplicate.
Supernatant samples (250u1) are collected 2 days and 5 days after the
beginning of the
assay. ELISAs to test for IFN and IL-2 secretion are performed using kits
purchased from R & D Systems, (Minneapolis, MN). Results are expressed as an
average of duplicate samples plus or minus standard error.
Proliferation assay for preactivated-resting T cells.
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A proliferation assay on preactivated-resting T cells is performed on cells
that
are previously activated with the lectin phytohemagglutinin (PHA). Lectins are
polymeric plant proteins that can bind to residues on T cell surface
glycoproteins
including the TCR and act as polyclonal activators. PBLs treated with PHA and
then
cultured in the presence of low doses of IL-2 resemble effector T cells. These
cells
are generally more sensitive to further activation induced by growth factors
such as
IL-2. This is due to the expression of high affinity IL-2 receptors that
allows this
population to respond to amounts of IL-2 that are 100 fold lower than what
would
have an effect on a-naive T cell. Therefore the use of this type of cells
might enable
to detect the effect of very low doses of an unknown growth factor, that would
not be
sufficient to induce proliferation on resting (naive ) T cells.
The assay is performed as follows. PBMC are isolated by F/H gradient
centrifugation from human peripheral blood, and are cultured inl0% FCS(Fetal
Calf
Serum, Biofluids, Rockville, MD)/RPMI (Gibco BRL, Gaithersburg, MD) in the
presence of 2ug/ml PHA (Sigma, Saint Louis, MO) for three days. The cells are
then
washed in PBS and cultured inl0% FCS/RPMI in the presence of Sng/ml of human
recombinant IL-2 (R & D Systems, Minneapolis, MN) for 3 days. The cells are
washed and rested in starvation medium (1%FCS/RPMI) forl6 hours prior to the
beginning of the proliferation assay. An aliquot of the cells is analyzed by
FACS to
determine the percentage of T cells (CD3 positive cells) present; this usually
ranges
between 93-97% depending on the donor. The assay is performed in a 96 well
plate
using 2 x104 cells/well in a final volume of 200u1. The supernatants (e.g.,
CHO or
293T supernatants) expressing the protein of interest are tested at a 30%
final dilution,
therefore 60u1 are added to 140u1 of inl0% FCS/RPMI containing the cells.
Control
supernatants are used at the same final dilution and express the following
proteins:
vector (negative control), IL-2, IFN , TNF , IL-10 and TR2. In addition to the
control supernatants recombinant human IL-2 at a final concentration of
lOng/ml is
also used. After 24 hours of culture, each well is pulsed with luCi of 3H-
thymidine(Nen, Boston, MA). Cells are then harvested 20 hours following
pulsing
and incorporation of 3H-thymidine is used as a measure of proliferation.
Results are
expressed as an average of triplicate samples plus or minus standard error.
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The studies described in this example test activity of polypeptides of the
invention. However, one skilled in the art could easily modify the exemplified
studies
to test the activity of polynucleotides of the invention (e.g., gene therapy),
agonists,
and/or antagonists of polynucleotides or polypeptides of the invention.
Example 34: Effect of Polypeptides of the Invention on the Expression of MHC
Class II, Costimulatory and Adhesion Molecules and Cell Differentiation of
Monocytes and Monocyte-Derived Human Dendritic Cells
Dendritic cells are generated by the expansion of proliferating precursors
found in
the peripheral blood: adherent PBMC or elutriated monocytic fractions are
cultured for 7-
10 days with GM-CSF (50 ng/ml) and IL-4 (20 ng/ml). These dendritic cells have
the
characteristic phenotype of immature cells (expression of CD1, CD80, CD86,
CD40 and
MHC class II antigens). Treatment with activating factors, such as TNF-a,
causes a rapid
change in surface phenotype (increased expression of MHC class I and II,
costimulatory
and adhesion molecules, downregulation of FCyRII, upregulation of CD83). These
changes correlate with increased antigen-presenting capacity and with
functional
maturation of the dendritic cells.
FACS analysis of surface antigens is performed as follows. Cells are treated 1-
3
days with increasing concentrations of polypeptides of the invention or LPS
(positive
control), washed with PBS containing 1 % BSA and 0.02 mM sodium azide, and
then
incubated with 1:20 dilution of appropriate FITC- or PE-labeled monoclonal
antibodies
for 30 minutes at 4 degrees C. After an additional wash, the labeled cells are
analyzed by
flow cytometry on a FACScan (Becton Dickinson).
Effect on the production of cvtokines. Cytokines generated by dendritic cells,
in particular IL-12, are important in the initiation of T-cell dependent
immune
responses. IL-12 strongly influences the development of Thl helper T-cell
immune
response, and induces cytotoxic T and NK cell function. An ELISA is used to
measure the IL-12 release as follows. Dendritic cells (106/m1) are treated
with
increasing concentrations of polypeptides of the invention for 24 hours. LPS
(100
ng/ml) is added to the cell culture as positive control. Supernatants from the
cell
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cultures are then collected and analyzed for IL-12 content using commercial
ELISA
kit (e..g, R & D Systems (Minneapolis, MN)). The standard protocols provided
with
the kits are used.
Effect on the expression of MHC Class II, costimulatory and adhesion
molecules. Three major families of cell surface antigens can be identified on
monocytes: adhesion molecules, molecules involved in antigen presentation, and
Fc
receptor. Modulation of the expression of MHC class II antigens and other
costimulatory molecules, such as B7 and ICAM-1, may result in changes in the
antigen presenting capacity of monocytes and ability to induce T cell
activation.
Increase expression of Fc receptors may correlate with improved monocyte
cytotoxic
activity, cytokine release and phagocytosis.
FACS analysis is used to examine the surface antigens as follows. Monocytes
are treated 1-5 days with increasing concentrations of polypeptides of the
invention or
LPS (positive control), washed with PBS containing 1% BSA and 0.02 mM sodium
azide, and then incubated with 1:20 dilution of appropriate FITC- or PE-
labeled
monoclonal antibodies for 30 minutes at 4 degreesC. After an additional wash,
the
labeled cells are analyzed by flow cytometry on a FACScan (Becton Dickinson).
Monocyte activation and/or increased survival. Assays for molecules that
activate (or alternatively, inactivate) monocytes and/or increase monocyte
survival (or
alternatively, decrease monocyte survival) are known in the art and may
routinely be
applied to determine whether a molecule of the invention functions as an
inhibitor or
activator of monocytes. Polypeptides, agonists, or antagonists of the
invention can be
screened using the three assays described below: For each of these assays,
Peripheral
blood mononuclear cells (PBMC) are purified from single donor leukopacks
(American Red Cross, Baltimore, MD) by centrifugation through a Histopaque
gradient (Sigma): Monocytes are isolated from PBMC by counterflow centrifugal
elutriation.
Monocyte Survival Assay. Human peripheral blood monocytes progressively
lose viability when cultured in absence of serum or other stimuli. Their death
results
from internally regulated process (apoptosis). Addition to the culture of
activating
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factors, such as TNF-alpha dramatically improves cell survival and prevents
DNA
fragmentation. Propidium iodide (PI) staining is used to measure apoptosis as
follows. Monocytes are cultured for 48 hours in polypropylene tubes in serum-
free
medium (positive control), in the presence of 100 ng/ml TNF-alpha (negative
control), and in the presence of varying concentrations of the compound to be
tested.
Cells are suspended at a concentration of 2 x 106/m1 in PBS containing PI at a
final
concentration of 5 p.g/ml, and then incubaed at room temperature for 5 minutes
before
FACScan analysis. PI uptake has been demonstrated to correlate with DNA
fragmentation in this experimental paradigm.
Effect on cytokine release. An important function of monocytes/macrophages
is their regulatory activity on other cellular populations of the immune
system through
the release of cytokines after stimulation. An ELISA to measure cytokine
release is
performed as follows. Human monocytes are incubated at a density of Sx105
cells/ml
1 S with increasing concentrations of the a polypeptide of the invention and
under the
same conditions, but in the absence of the polypeptide. For IL-12 production,
the
cells are primed overnight with IFN (100 U/ml) in presence of a polypeptide of
the
invention. LPS (10 ng/ml) is then added. Conditioned media are collected after
24h
and kept frozen until use. Measurement of TNF-alpha, IL-10, MCP-1 and IL-8 is
then
performed using a commercially available ELISA kit (e..g, R & D Systems
(Minneapolis, MN)) and applying the standard protocols provided with the kit.
Oxidative burst. Purified monocytes are plated in 96-w plate at 2-1x105
cell/well. Increasing concentrations of polypeptides of the invention are
added to the
wells in a total volume of 0.2 ml culture medium (RPMI 1640 + 10% FCS,
glutamine
and antibiotics). After 3 days incubation, the plates are centrifuged and the
medium is
removed from the wells. To the macrophage monolayers, 0.2 ml per well of
phenol
red solution (140 mM NaCI, 10 mM potassium phosphate buffer pH 7.0, 5.5 mM
dextrose, 0.56 mM phenol red and 19 U/ml of HRPO) is added, together with the
stimulant (200 nM PMA). The plates are incubated at 37°C for 2 hours
and the
reaction is stopped by adding 20 p.1 1 N NaOH per well. The absorbance is read
at 610
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nm. To calculate the amount of H202 produced by the macrophages, a standard
curve
of a H20z solution of known molarity is performed for each experiment.
The studies described in this example tested activity of a polypeptide of the
invention. However, one skilled in the art could easily modify the exemplified
studies
to test the activity of polypeptides, polynucleotides (e.g., gene therapy),
agonists,
and/or antagonists of the invention.
Example 35: Biological Effects of Polypeptides of the Invention '
Astrocyte and Neuronal Assays
Recombinant polypeptides of the invention; expressed in Escherichia coli and
purified as described above, can be tested for activity in promoting the
survival, neurite
outgrowth, or phenotypic differentiation of cortical neuronal cells and for
inducing the
proliferation of glial fibrillary acidic protein immunopositive cells,
astrocytes. The
selection of cortical cells for the bioassay is based.on the prevalent
expression of FGF-1
~d FGF-2 in cortical structures and on the previously reported enhancement of
cortical
neuronal survival resulting from FGF-2 treatment. A thymidine incorporation
assay, for
example, can be used to elucidate a polypeptide of the invention's activity on
these cells.
Moreover, previous reports describing the biological effects of FGF-2 (basic
FGF)
on cortical or hippocampal neurons in vitro have demonstrated increases in
both neuron
survival and neurite outgrowth (Walicke et al., "Fibroblast growth factor
promotes
survival of dissociated hippocampal neurons and enhances neurite extension."
Proc. Natl.
Acad. Sci. USA 83:3012-3016. (1986), assay herein incorporated by reference in
its
entirety). However, reports from experiments done on PC-12 cells suggest that
these two
responses are not necessarily synonymous and may depend on not only which FGF
is
being tested but also on which receptors) are expressed on the target cells.
Using the
primary cortical neuronal culture paradigm, the ability of a polypeptide of
the invention to
induce neurite outgrowth can be compared to the response achieved with FGF-2
using, for
example, a thymidine incorporation assay.
Fibroblast and endothelial cell assays~
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Human lung fibroblasts are obtained from Clonetics (San Diego, CA) and
maintained in growth media from Clonetics. Dermal microvascular endothelial
cells are
obtained from Cell Applications (San Diego, CA). For proliferation assays, the
human
lung fibroblasts and dermal microvascular endothelial cells can be cultured at
5,000
cells/well in a 96-well plate for one day in growth medium. The cells are then
incubated
for one day in 0.1% BSA basal medium. After replacing the medium with fresh
0.1% BSA
medium, the cells are incubated with the test proteins for 3 days. Alamar Blue
(Alamar
Biosciences, Sacramento, CA) is added to each well to a final concentration of
10%. The
cells are incubated for 4 hr. Cell viability is measured by reading in a
CytoFluor
fluorescence reader. For the PGEz assays, the human lung fibroblasts are
cultured at
5,000 cells/well in a 96-well plate for one day. After a medium change to 0.1
% BSA
basal medium, the cells are incubated with FGF-2 or polypeptides of the
invention with or
without IL-la for 24 hours. The supernatants are collected and assayed for
PGEZ by EIA
kit (Cayman, Ann Arbor, MI). For the IL-6 assays, the human lung fibroblasts
are
cultured at 5,000 cells%vvell in a 96-well plate for one day. After a medium
change to
0.1% BSA basal medium, the cells are incubated with FGF-2 or with or without
polypeptides of the invention IL-la for 24 hours. The supernatants are
collected and
assayed for IL-6 by ELISA kit (Endogen, Cambridge, MA).
Human lung fibroblasts are cultured with FGF-2 or polypeptides of the
invention
for 3 days in basal medium before the addition of Alamar Blue to assess
effects on growth
of the fibroblasts. FGF-2 should show a stimulation at 10 - 2500 ng/ml which
can be used
to compare stimulation with polypeptides of the invention.
Parkinson Models.
The loss of motor function in Parkinson's disease is attributed to a
deficiency of
striatal dopamine resulting from the degeneration of the nigrostriatal
dopaminergic
projection neurons. An animal model for Parkinson's that has been extensively
characterized involves the systemic administration of 1-methyl-4 phenyl
1,2,3,6-
tetrahydropyridine (MPTP). In the CNS, MPTP is taken-up by astrocytes and
catabolized
by monoamine oxidase B to 1-methyl-4-..phenyl pyridine (MPP+) and released.
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Subsequently, MPP+ is actively accumulated in dopaminergic neurons by the high-
affinity
reuptake transporter for dopamine. MPP+ is then concentrated in mitochondria
by the
electrochemical gradient and selectively inhibits nicotidamide adenine
disphosphate:
ubiquinone oxidoreductionase (complex I), thereby interfering with electron
transport and
eventually generating oxygen radicals.
It has been demonstrated in tissue culture paradigms that FGF-2 (basic FGF)
has
trophic activity towards nigral dopaminergic neurons (Ferrari et al., Dev.
Biol. 1989).
Recently, Dr. Unsicker's group has demonstrated that administering FGF-2 in
gel foam
implants in the striatum results in the near complete protection of nigral
dopaminergic
neurons from the toxicity associated with MPTP exposure (Otto and Unsicker, J.
Neuroscience, 1990).
Based on the data with FGF-2, polypeptides of the invention can be evaluated
to
determine whether it has an action similar to that of FGF-2 in enhancing
dopaminergic
neuronal survival in vitro and it can also be tested in vivo for protection of
dopaminergic
neurons in the striatum from the damage associated with MPTP treatment. The
potential
effect of a polypeptide of the invention is first examined in vitro in a
dopaminergic
neuronal cell culture paradigm. The cultures are prepared by dissecting the
midbrain floor
plate from gestation day 14 Wistar rat embryos. The tissue is dissociated with
trypsin and
seeded at a density of 200,000 cells/cm2 on polyorthinine-laminin coated glass
coverslips.
The cells are maintained in Dulbecco's Modified Eagle's medium and F12 medium
containing hormonal supplements (N1). The cultures are fixed with
paraformaldehyde
after 8 days in vitro and are processed for tyrosine hydroxylase, a specific
marker for
dopminergic neurons, immunohistochemical staining. Dissociated cell cultures
are
prepared from embryonic rats. The culture medium is changed every third day
and the
factors are also added at that time.
Since the dopaminergic neurons are isolated from animals at gestation day 14,
a
developmental time which is past the stage when the dopaminergic precursor-
cells are
proliferating, an increase in the number of tyrosine hydroxylase
immunopositive neurons
would represent an increase in the number of dopaminergic neurons surviving in
vitro.
Therefore, if a polypeptide of the invention acts to prolong the survival of
dopaminergic
neurons, it would suggest that- the polypeptide may be involved in Parkinson's
Disease.
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The studies described in this example tested activity of a polypeptide of the
invention. However, one skilled in the art could easily modify the exemplified
studies to
test the activity of polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of the
invention.
Example 36: The Effect of Polypeptides of the Invention on the Growth of
Vascular Endothelial Cells
On day 1, human umbilical vein endothelial cells (HUVEC) are seeded at 2-5x104
cells/35 mm dish density in M199 medium containing 4% fetal bovine serum
(FBS), 16
units/ml heparin, and 50 units/ml endothelial cell growth supplements (ECGS,
Biotechnique, Inc.). On day 2, the medium is replaced with M199 containing 10%
FBS, 8
units/ml heparin. A polypeptide having the amino acid sequence of SEQ ff~
NO:Y, and
positive controls, such as VEGF and basic FGF (bFGF) are added, at varying
. concentrations. On days 4 and 6, the medium is replaced. On day 8, cell
number is
determined with a Coulter Counter.
An increase in the number of HUVEC cells indicates that the polypeptide of the
invention may proliferate vascular endothelial cells.
The studies described in this example tested activity of a polypeptide of the
invention. However, one skilled in the art could easily modify the exemplified
studies to
test the activity of polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of the
invention.
Example 37: Stimulatory Effect of Polypeptides of the Invention on the
Proliferation of Vascular Endothelial Cells
For evaluation of mitogenic activity of growth factors, the colorimetric MTS
(3-(4,5-dimethylthiazol-2-yl)-S-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-
tetrazolium) assay with the electron coupling reagent PMS (phenazine
methosulfate) was
performed (CellTiter 96 AQ; Promega). Cells are seeded in a 96-well plate
(5,000
cells/well) in 0.1 mL serum-supplemented medium and are allowed to attach
overnight.
After serum-starvation for 12 hours in 0.5% FBS, conditions (bFGF, VEGFibs or
a
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polypeptide of the invention in 0.5% FBS) with or without Heparin (8 U/ml) are
added to
wells for 48 hours. 20 mg of MTS/PMS mixture (1:0.05) are added per well and
allowed
to incubate for 1 hour at 37°C before measuring the absorbance at 490
nm in an ELISA
plate reader. Background.absorbance from control wells (some media, no cells)
is
subtracted, and seven wells are performed in parallel for each condition. See,
Leak et al.
In Vitro Cell. Dev. Biol. 30A:512-518 (1994).
The studies described in this example tested activity of a polypeptide of the
invention. However, one skilled in the art could easily modify the exemplified
studies to
test the activity of polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of the .
invention.
Example 38: Inhibition of PDGF-induced Vascular Smooth Muscle Cell
Proliferation Stimulatory Effect
HAoSMC proliferation can be measured, for example, by BrdUrd incorporation.
Briefly, subconfluent, quiescent cells grown_on the 4-chamber slides are
transfected with
CRP or FITC-labeled AT2-3LP. Then, the cells are pulsed with 10% calf serum
and 6
mg/ml BrdUrd. After 24 h, immunocytochemistry is performed by using BrdUrd
Staining
Kit (Zymed Laboratories). In brief, the cells are incubated with the
biotinylated mouse
anti-BrdUrd antibody at 4 degrees C for 2 h after being exposed to denaturing
solution and
then incubated with the streptavidin-peroxidase and diaminobenzidine. After
counterstaining with hematoxylin, the cells are mounted for microscopic
examination, and
the BrdUrd-positive cells are counted. The BrdUrd index is calculated as a
percent of the
BrdUrd-positive cells to the total cell number. In addition, the simultaneous
detection of
the BrdUrd staining (nucleus) and the FITC uptake (cytoplasm) is performed for
individual cells by the concomitant use of bright field illumination and dark
field-UV
fluorescent illumination. See, Hayashida et al., J. Biol. Chem.
6:271(36):21985-21992
( 1996).
The studies described in this example tested activity of a polypeptide of the
invention. However, one skilled in the art could easily modify the exemplified
studies to
test the activity of polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of the
invention.
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Example 39: Stimulation of Endothelial Migration
This example will be used to explore the possibility that a polypeptide of the
invention may stimulate lymphatic endothelial cell migration.
Endothelial cell migration assays are performed using a 48 well
microchemotaxis
chamber (Neuroprobe Inc:, Cabin John, MD; Falk, W., et al., J. Immunological
Methods
1980;33:239-247). Polyvinylpyrrolidone-free polycarbonate filters with a pore
size of 8
um (Nucleopore Corp. Cambridge, MA) are coated with 0.1% gelatin for at least
6 hours
at room temperature and dried under sterile air. Test substances are diluted
to appropriate
concentrations in M199 supplemented with 0.25% bovine serum albumin (BSA), and
25
~ul of the final dilution is placed in the lower chamber of the modified
Boyden apparatus.
Subconfluent, early passage (2-6) HWEC or BMEC cultures are washed and
trypsinized
for the minimum time required to achieve cell detachment. After placing the
filter
between lower and upper chamber, 2.5 x 105 cells suspended in 50 u1 M199
containing 1%
FBS are seeded in the upper compartment. The apparatus is then incubated for.
S hours at
37°C in a humidified chamber with 5% C02 to allow cell migration. After
the incubation
period, the filter is removed and the upper side of the filter with the non-
migrated cells is
scraped with a rubber policeman. The filters are fixed with methanol and
stained with a
Giemsa solution (Diff Quick, Baxter, McGraw Park, IL). Migration is quantified
by
counting cells of three random high-power fields (40x) in each well, and all
groups are
performed in quadruplicate.
The studies described in this example tested activity of a polypeptide of the
invention. However, one skilled in the art could easily modify the exemplified
studies to
test the activity of polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of the
invention.
Example 40: Stimulation of Nitric Oxide Production by Endothelial Cells
Nitric oxide released,by the vascular endothelium is believed to be a mediator
of
vascular endothelium relaxation. Thus, activity of a polypeptide of the
invention can be
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assayed by determining nitric oxide production by endothelial cells in
response to the
polypeptide.
Nitric oxide is measured in 96-well plates of confluent microvascular
endothelial
cells after 24 hours starvation and a subsequent 4 hr exposure to various
levels of a
positive control (such as VEGF-1) and the polypeptide of the invention. Nitric
oxide in
the medium is determined by use of the Griess reagent to measure total nitrite
after
reduction of nitric oxide-derived nitrate by nitrate reductase. The effect of
the polypeptide
of the invention on nitric oxide release is examined on HUVEC.
J
Briefly, NO release from cultured HLTVEC monolayer is measured with a NO-
specific polarographic electrode connected to a NO meter (Iso-NO, World
Precision
Instruments Inc.) (1049). Calibration of the NO elements is performed
according to the
following equation:
2KN0z+2KI+2HZS0462N0+Iz+2Hz0+2KZS04
The standard calibration curve is obtained by adding graded concentrations of
KN02 (0, 5, 10, 25, 50, 100, 250, and 500 nmol/L) into the calibration
solution containing
KI and HZS04. The specificity of the Iso-NO electrode to NO is previously
'determined by
measurement of NO from authentic NO gas (1050). The culture medium is removed
and
HUVECs are washed twice with Dulbecco's phosphate buffered saline. The cells
are then
bathed in S ml of filtered Krebs-Henseleit solution in 6-well plates, and the
cell plates are
kept on a slide warmer (Lab Line Instruments Inc.) To maintain the temperature
at 37°C.
The NO sensor probe is inserted vertically into the wells, keeping the tip of
the electrode 2
mm under the surface of the solution, before addition of the different
conditions.
S-nitroso acetyl penicillamin (SNAP) is used as a positive control. The amount
of
released NO is expressed as picomoles per 1x106 endothelial cells: All values
reported are
means of four to six measurements in each group (number of cell culture
wells). See,
Leak et al. Biochem. and Biophys. Res..Comm. 217:96-105 (1995).
The studies described in this example tested activity of polypeptides of the
invention. However, one skilled in the art could easily modify the exemplified
studies to
test the activity of polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of the
invention.
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Example 41: Effect of Polypepides of the Invention on Cord Formation in
An~io~enesis
Another step in angiogenesis is cord formation, marked by differentiation of
endothelial cells. This bioassay measures the ability of microvascular
endothelial cells to
form capillary-like structures (hollow structures) when cultured in vitro.
CADMEC (microvascular endothelial cells) are purchased from Cell Applications,
Inc. as proliferating (passage 2) cells and are cultured in Cell Applications'
CADMEC
Growth Medium and used at passage S. For the in vitro angiogenesis assay, the
wells of a
48-well cell culture plate are coated with Cell Applications' Attachment
Factor Medium
(200 ml/well) for 30 min. at 37°C. CADMEC are seeded onto the coated
wells at 7,500
cells/well and cultured overnight in Growth Medium. The Growth Medium is then
replaced with 300 mg Cell Applications' Chord Formation Medium containing
control
buffer or a polypeptide of the invention (0.1 to 100 ng/ml) and the cells are
cultured for an
additional 48 hr. The numbers and lengths of the capillary-like.chords are
quantitated
through use of the Boeckeler VIA-170 video image analyzer. All assays are done
in
triplicate.
Commercial (R&D) VEGF (50 nglml) is used as a positive control. b-esteradiol
(1
ng/ml) is used as a negative control. The appropriate buffer (without protein)
is also
utilized as a control.
The studies described in this example tested activity of a polypeptide of the
invention. However, one skilled in the art could easily modify the exemplified
studies to
test the activity of polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of the
invention.
Example 42: An~io~enic Effect on Chick Chorioallantoic Membrane
Chick chorioallantoic membrane (CAM) is a well-established system to examine
angiogenesis. Blood vessel formation on CAM is easily visible and
quantifiable. The
ability of polypeptides of the invention to stimulate angiogenesis in CAM can
be
examined.
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Fertilized eggs of the White Leghorn chick (callus gallus) and the Japanese
qual
(Coturnix coturnix) are incubated at 37.8°C and 80% humidity.
Differentiated CAM of
16-day-old chick and 13-day-old qual embryos is studied with the following
methods.
On Day 4 of development, a window is made into the egg shell of chick eggs.
The
embryos are checked for normal development and the eggs sealed with cellotape.
They
are further incubated until Day 13. Thermanox coverslips (Nunc, Naperville,
IL) are cut
into disks of about 5 mm in diameter. Sterile and salt-free growth factors are
dissolved iri
distilled water and about 3.3 mg/ 5 ml are.pipetted on the disks. After air-
drying, the
inverted disks are applied on CAM. After 3 days, the specimens are fixed in 3%
glutaraldehyde and 2% formaldehyde and rinsed in 0.12 M sodium cacodylate
buffer.
They are photographed with a stereo microscope [Wild M8] and embedded for semi-
and
ultrathin sectioning as described above. Controls are performed with carrier
disks alone.
The studies described in this example tested activity of a polypeptide of the
invention. However, one skilled in the art could easily modify the exemplified
studies to
1 S test the activity of polynucleotides (e.g., gene therapy), agonists,
and/or antagonists of the
invention.
Example 43: An~io~enesis Assay Using a Matri~el Implant in Mouse
In vivo angiogenesis assay of a polypeptide of the invention measures the
ability of
an existing capillary network to form new vessels in an implanted capsule of
murine
extracellular matrix material (Matrigel). The protein is mixed with the liquid
Matrigel at 4
degree C and the mixture is then injected subcutaneously in mice where it
solidifies. After
7 days, the solid "plug" of Matrigel is removed and examined for the presence
of new
blood vessels. Matrigel is purchased from Becton Dickinson
Labware/Collaborative
Biomedical Products.
When thawed at 4 degree C the Matrigel material is a liquid. The Matrigel is
mixed with a polypeptide of the invention at 150 ng/ml at 4 degrees C and
drawn into cold
3 ml syringes. Female C57B1/6 mice approximately 8 weeks old are injected with
the
mixture of Matrigel and experimental protein at 2 sites at the midventral
aspect of the
abdomen (0.5 ml/site). A$er 7 days, the mice are sacrificed by cervical
dislocation, the
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Matrigel plugs are removed and cleaned (i.e., all clinging membranes and
fibrous tissue is
removed). Replicate whole plugs are fixed in neutral buffered 10%
formaldehyde,
embedded in paraffin and used to produce sections for histological examination
after
staining with Masson's Trichrome. Cross sections from 3 different regions of
each plug
are processed. Selected sections are stained for the presence of v,WF. The
positive control
for this assay is bovine basic FGF (150 ng/ml). Matrigel alone is used to
determine basal
levels of angiogenesis.
The studies described in this example tested activity of a polypeptide of the
invention. However, one skilled in the art could easily modify the exemplified
studies to
test the activity of polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of the
invention.
Example 44: Rescue of Ischemia in Rabbit Lower Limb Model
To study the in vivo effects of polynucleotides and polypeptides of the
invention
on ischemia, a rabbit hindlimb ischemia model is created by surgical removal
of one
femoral arteries as described previously (Takeshita et al., Am J. Pathol
147:1649-1660
(1995)). The excision of the femoral artery results in retrograde propagation
of thrombus
and occlusion of the external iliac artery. Consequently, blood flow to the
ischemic limb
is dependent upon collateral vessels originating from the internal iliac
artery (Takeshitaet
al. Am J. Pathol 147:1649-1660 (1995)). An interval of 10 days is allowed for
post-
operative recovery of rabbits and development of endogenous collateral
vessels. At 10
day post-operatively (day 0), after performing a baseline angiogram, the
internal i 1 iac
artery of the ischemic limb is transfected with 500 mg naked expression
plasmid
containing a polynucleotide of the invention by arterial gene transfer
technology using a
hydrogel-coated balloon catheter as described (Riessen et al. Hum Gene Ther.
4:749-758
(1993); Leclerc et al. J. Clin. Invest. 90: 936-944 (1992)). When a
polypeptide of the
invention is used in the treatment, a single bolus of 500 mg polypeptide of
the invention or
control is delivered into the internal iliac artery of the ischemic limb over
a period of 1
min. through an infusion catheter. On day 30, various parameters are measured
in these
rabbits: (a) BP ratio - The blood pressure ratio of systolic pressure of the
ischemic limb to
that of normal limb; (b) Blood Flow. and Flow Reserve - Resting FL: the blood
flow
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during undilated condition and Max FL: the blood flow during fully dilated
condition (also
an indirect measure of the blood vessel amount) and Flow Reserve is reflected
by the ratio
of max FL: resting FL; (c) Angiographic Score - This is measured by the
angiogram of
collateral vessels. A score is determined by the percentage of circles in an
overlaying grid
that with crossing opacified arteries divided by the total number m the rabbit
thigh; (d)
Capillary density - The number of collateral capillaries determined in light
microscopic .
sections taken from hindlimbs.
The studies described in this example tested activity of polynucleotides and
polypeptides of the invention. However, one skilled in the art could' easily
modify the
exemplified studies to test the agonists, and/or antagonists of the invention.
Example 45: Effect of Polypeptides of the Invention on Vasodilation
Since dilation of vascular endothelium is important in reducing blood
pressure, the
ability of polypeptides of the invention to affect the blood pressure in
spontaneously
hypertensive rats (SHR) is examined. Increasing doses (0, 10, 30, 100, 300,
and 900
mg/kg) of the polypeptides of the invention are administered to 13-14 week old
spontaneously hypertensive rats (SHR). Data are expressed as the mean +/- SEM.
Statistical analysis are performed with a paired t-test and statistical
significance is defined
as p<0.05 vs. the response to buffer alone.
The studies described in this example tested activity of a polypeptide of the
invention. However, one skilled in the art could easily modifythe exemplified
studies to
test the activity of polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of the
invention.
Example 46: Rat Ischemic Skin Flap Model
The evaluation parameters include skin blood flow, skin temperature, and
factor
VIII immunohistochemistry or endothelial alkaline phosphatase reaction.
Expression of
polypeptides of the invention, during the skin ischemia, is studied using in
situ
hybridization.
The study in this model is divided into three parts as follows:
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Ischemic skin
Ischemic skin wounds
Normal wounds
The experimental protocol includes:
Raising a 3x4 cm, single pedicle full-thickness random skin flap (myocutaneous
flap over the lower back of the animal).
An excisional wounding (4-6 mm in diameter) in the ischemic skin (skin-flap).
Topical treatment with a polypeptide of the invention of the excisional wounds
(day 0, 1, 2, 3, 4 post-wounding) at the following various dosage ranges: lmg
to 100 mg.
Harvesting the wound tissues at day 3, S, 7, 10, 14 and 21 post-wounding for
histological, immunohistochemical, and in situ studies.
The studies described in this example tested activity of a polypeptide of the
invention. However, one skilled in the art could easily modify the exemplified
studies to
test the activity of polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of the
invention.
Example 47: Peripheral Arterial Disease Model
Angiogenic therapy using a polypeptide of the invention is a novel therapeutic
strategy to obtain restoration of blood flow around the ischemia in case of
peripheral
arterial diseases. The experimentaj protocol includes:
One side of the femoral artery is ligated to create ischemic muscle of
the hindlimb, the other side of hindlimb serves as a control. _
a polypeptide of the invention, in a dosage range of 20 mg - 500 mg, is
delivered
intravenously and/or intramuscularly 3 times (perhaps more) per week for 2-3
weeks.
The ischemic muscle tissue is collected after ligation of the femoral
artery at 1, 2, and 3 weeks for the analysis of expression of a polypeptide of
the
invention and histology. Biopsy is also performed on the other side of normal
muscle of
the contralateral hindlimb.
The studies described in this example tested activity of a polypeptide of the
invention. However, one skilled in the art could easily modify the exemplified
studies to
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test the activity of polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of the
invention.
Example 48: Ischemic Myocardial Disease Model
A polypeptide of the invention is evaluated as a potent mitogen capable of
stimulating the development of collateral vessels, and restructuring new
vessels after
coronary artery occlusion. Alteration of expression of the polypeptide is
investigated in
situ. The experimental protocol includes:
The heart is exposed through a left-side thoracotomy in the rat. Immediately,
the
left coronary artery is occluded with a thin suture (6-0) and the thorax is
closed.
a polypeptide of the invention, in a dosage range of 20 mg - 500 mg, is
delivered
intravenously and/or intramuscularly 3 times (perhaps more) per week for 2-4
weeks.
Thirty days after the surgery, the heart is removed and cross-sectioned
for morphometric and in situ analyzes.
The studies described in this example tested activity of a polypeptide of the
invention. However, one skilled in the art could easily modify the exemplified
studies to
test the activity of polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of the
invention.
Example 49: Rat Corneal. Wound Healing Model
This animal model shows the effect of a polypeptide of the invention on
neovascularization. The eXperimental protocol includes:
Making a 1-1.5 mm long incision from the center of cornea into the stromal
layer.
Inserting a spatula below the lip of the incision facing the outer corner of
the eye.
Making a pocket (its base is 1-1.5 mm form the edge of the eye).
Positioning a pellet, containing SOng- Sug of a polypeptide of the invention,
within
the pocket.
Treatment with a polypeptide of the invention can also be applied topically to
the
corneal wounds in a dosage range of 20mg - SOOmg (daily treatment for five
days).
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The studies described in this example tested activity of a polypeptide of the
invention. However, one skilled in the art could easily modify the exemplified
studies to
test the activity of polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of the
invention.
Example 50: Diabetic Mouse and Glucocorticoid-Impaired Wound Healing
Models
Diabetic db+/db+ Mouse Model.
To demonstrate that a polypeptide of the invention accelerates the healing
process,
the genetically diabetic mouse model of wound healing is used. The full
thickness wound
healing model in the db+/db+ mouse is a well characterized, clinically
relevant and
reproducible model of impaired wound healing: Healing of the diabetic wound is
dependent on formation of granulation tissue and re-epithelialization rather
than
contraction (Gartner, M.H. et al., J. Surg. Res. 52:389 (1992); Greenhalgh,
D.G. et al.,
Am. J. Pathol. 136:1235 (1990)).
The diabetic animals have many of the characteristic features observed in Type
II
diabetes mellitus. Homozygous (db+/db+) mice are obese in comparison to their
normal
heterozygous (db+/+m) littermates. Mutant diabetic (db+/db+) mice have a
single
autosomal recessive mutation on chromosome 4 (db+) (Coleman et al. Proc. Natl.
Acad.
Sci. USA 77:283-293 (1982)). Animals show polyphagia, polydipsia and polyuria.
Mutant diabetic mice (db+/db+) have elevated blood glucose, increased or
normal insulin
levels, and suppressed cell-mediated immunity (Mandel et al., J. Immunol.
120:1375
(1978); Debray-Sachs, M. et al., Clin. Exp. Immunol. 51 (1):1-7 (1983); Leiter
et al., Am.
J. ofPathol. 114:46-55 (1985)). Peripheral neuropathy, myocardial
complications, and
microvascular lesions, basement membrane thickening and glomerular filtration
abnormalities have been described in these animals (Norido, F. et al., Exp.
Neurol.
83(2):221-232 (1984); Robertson et al., Diabetes 29(1):60-67 (1980);
Giacomelli et al.,
Lab Invest.~40(4):460-473 (1979); Coleman, D.L., Diabetes 31 (Suppl):1-6
(1982)). These
homozygous diabetic mice develop hyperglycemia that is resistant to insulin
analogous to
human type II diabetes (Mandel et al., J. Immunol. 120:1375-1377 (1978)).
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The characteristics observed in these animals suggests that healing in this
model
may be similar to the healing observed in human diabetes (Greenhalgh, et al.,
Am. J. of
Pathol. 136:1235-1246 (1990)).
Genetically diabetic female C57BL/KsJ (db+/db+) mice and their non-diabetic
(db+/+m) heterozygous littermates are used iri this study (Jackson
Laboratories). The
animals are purchased at 6 weeks of age and are 8 weeks old at the beginning
of the study.
Animals are individually housed and received food and water ad libitum. All
manipulations are performed using aseptic techniques. The experiments are
conducted
according to the rules and guidelines of Human Genome Sciences, Inc.
Institutional
Animal Care and Use Committee and the Guidelines for the Care and Use of
Laboratory
Animals.
Wounding protocol is performed according to previously reported methods
(Tsuboi, R. and Riflcin, D.B:, J. Exp. Med. 172:245-251 (1990)). Briefly, on
the day of
wounding, animals are anesthetized with an intraperitoneal injection of
Avertin (0.01
mg/mL), 2,2,2-tribromoethanol and 2-methyl-2-butanol dissolved in deionized
water. The
dorsal region of the animal is shaved and the skin washed with 70% ethanol
solution and
iodine. The surgical area is dried with sterile gauze prior to wounding. An 8
mm full-
thickness wound is then created using a Keyes tissue punch. Immediately
following
wounding, the surrounding skin is gently stretched to eliminate wound
expansion. The
~ wounds are left open for the duration of the experiment. Application of the
treatment is
given topically for 5 consecutive days commencing on the day of wounding.
Prior to
treatment, wounds are gently cleansed with sterile saline and gauze sponges.
' Wounds are visually examined and photographed at a fixed distance at the day
of
surgery and at two day intervals thereafter. Wound closure is determined by
daily
measurement on days 1-S and on day 8. Wounds are measured horizontally and
vertically
using a calibrated Jameson caliper. Wounds are considered healed if
granulation tissue is
no longer visible and the wound is covered by a continuous epithelium. .
A polypeptide of the invention is administered using at a range different
doses,
from 4mg to SOOmg per wound per day for 8 days in vehicle. Vehicle control
groups
received SOmL of vehicle solution.
Animals are euthanized on day 8 with an intraperitoneal injection of sodium
pentobarbital (300mg/kg). The wounds and surrounding skin are then harvested
for -.
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histology and immunohistochemistry. Tissue specimens are placed in 10% neutral
buffered formalin in tissue cassettes between biopsy sponges for further
processing.
Three groups of 10 animals each (5 diabetic and 5 non-diabetic controls) are
evaluated: 1) Vehicle placebo control, 2) untreated group, and 3) treated
group.
Wound closure is analyzed by measuring the area in the vertical and horizontal
axis and obtaining the total square area of the wound. Contraction is then
estimated by
establishing the differences between the initial wound area (day 0) and that
of post
treatment (day 8). The wound area on day 1 is 64mmz, the corresponding size of
the
dermal punch. Calculations are made using the following formula:
[Open area on day 8] - [Open area on day 1] / [Open area on day 1]
Specimens are fixed in 10% buffered formalin and paraffin embedded blocks are
sectioned perpendicular to the wound surface (Smm) and cut using a Reichert-
Jung
microtome. Routine hematoxylin-eosin (H&E) staining is performed on cross-
sections of
bisected wounds. Histologic examination of the wounds are used to assess
whether the
healing process and the morphologic appearance of the repaired skin is altered
by
treatment with a polypeptide of the invention. This assessment included
verification of
the presence of cell accumulation, inflammatory cells, capillaries,
fbroblasts, re-
epithelialization and epidermal maturity (Greenhalgh, D.G. et al., Am. J.
Pathol. 136:1235
( 1990)). A calibrated lens micrometer is used by a blinded observer.
Tissue sections are also stained immunohistochemically with a polyclonal
rabbit
anti-human keratin antibody using ABC Elite detection system. Human skin is
used as a
positive tissue control while non-immune IgG is used as a negative control.
Keratinocyte
growth is determined by evaluating the extent of reepithelialization of the
wound using a
calibrated lens micrometer.
Proliferating cell nuclear antigen/cyclin (PCNA) in skin specimens is
demonstrated
by using anti-PCNA antibody (1:50) with an ABC Elite detection system. Human
colon
cancer can serve as a positive tissue control and human brain tissue can.be
used as a
negative tissue control. Each specimen includes a section with omission of the
primary
antibody and substitution with non-immune mouse IgG. Ranking of these sections
is
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based on the extent of proliferation on a scale of 0-8, the lower side of the
scale reflecting
slight proliferation to the higher side reflecting intense proliferation.
Experimental data are analyzed using an unpaired t test. A p value of < 0.05
is
considered significant.
Steroid Impaired Rat Model
The inhibition of wound healing by steroids has been well documented in
various
in vitro and in vivo systems (Wahl, Glucocorticoids and Wound healing. In:
Anti-
Inflammatory Steroid Action: Basic and Clinical Aspects. 280-302 (1989);
Wahlet al., J.
Immunol. 1l S: 476-481 (1975); Werb et al., J. Exp. Med. 147:1684-1694
(1978)).
Glucocorticoids retard wound healing by inhibiting angiogenesis, decreasing
vascular
permeability (Ebert et al., An. Intern. Med. 37:701-705 (1952)), fibroblast
proliferation-,
and collagen synthesis (Beck et al.; Growth Factors. 5: 295-304 (1991); Haynes
et al.,
J. Clin. Invest. 61: 703-797 (1978)) and producing a transient reduction of
circulating
monocytes (Haynes et al., J. Clin. Invest. 61: 703-797 (1978); Wahl,
"Glucocorticoids and
wound healing", In: Antiinflammatory Steroid Action: Basic and Clinical
Aspects,
Academic Press, New York, pp. 280-302 (1989)). The systemic administration of
steroids
to impaired wound healing is a well establish phenomenon in rats (Beck et al.,
Growth
Factors. 5: 295-304 (1991); Haynes et al., J. Clin. Invest. 61: 703-797
(1978); Wahl,
"Glucocorticoids and wound healing", In: Antiinflammatory Steroid Action:
Basic and
Clinical Aspects, Academic Press, New York, pp. 280-302 (1989); Pierce et al.,
Proc.
Natl. Acad. Sci. USA 86: 2229-2233 (1989)).
To demonstrate that a polypeptide of the invention can accelerate the healing
process, the effects of multiple topical applications of the polypeptide on
full thickness
excisional skin wounds in rats in which healing has been impaired by the
systemic
administration of methylprednisolone is assessed.
Young adult male Sprague Dawley rats weighing 250-300 g (Charles River
Laboratories) are used in this example: The animals are purchased at 8 weeks
of age and
are 9 weeks old at the beginning of the study. The healing response of rats is
impaired by
the systemic administration of methylprednisolone (l7mg/kg/iat
intramuscularly) at the
time of wounding. Animals are individually housed and received food and water
ad
libitum. All manipulations are performed using aseptic techniques. This study
is
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conducted according to the rules and guidelines of Human Genome Sciences, Inc.
Institutional Animal Care and Use Committee and the Guidelines for the Care
and Use of
Laboratory Animals.
The wounding protocol is followed according to section A, above. On the day of
wounding, animals are anesthetized with an intramuscular injection of ketamine
(50
mg/kg) and xylazine (5 mg/kg). The dorsal region of the animal is shaved and
the skin
washed with 70% ethanol and iodine solutions. The surgical area is dried with
sterile
gauze prior to wounding. An 8 mm full-thickness wound is created using a Keyes
tissue
punch. The wounds are left open for the duration of the experiment.
Applications ofthe
testing~materials are given topically once a day for 7 consecutive days
commencing on the
day of wounding and subsequent to methylprednisolone administration. Prior to
treatment, wounds are gently cleansed with sterile saline and gauze sponges.
Wounds are visually examined and photographed at a fixed distance at the day
of
wounding and at the end of treatment. Wound closure, is determined by daily
measurement
on days 1-5 and on day 8. Wounds are measured horizontally and vertically
using a
calibrated Jameson caliper. Wounds are considered healed if granulation tissue
is no
longer visible and the wound is covered by a continuous epithelium.
The polypeptide of the invention is administered using at a range different
doses,
from 4mg to SOOmg per wound per day for 8 days in vehicle. Vehicle control
groups
received SOmL of vehicle solution.
Animals are euthanized on day 8 with an intraperitoneal injection of sodium
pentobarbital (300mg/kg). The wounds and surrounding skin are then harvested
for
histology. Tissue specimens are placed in 10% neutral buffered formalin in
tissue
cassettes between biopsy sponges for further processing.
Four groups of 10 animals each (5 with methylprednisolone and 5 without
glucocorticoid) are evaluated: 1) Untreated group 2) Vehicle placebo control
3) treated
groups.
Wound closure is analyzed by measuring the area in the vertical and horizontal
axis and obtaining the total area of the wound. Closure is then estimated by
establishing
the differences between the initial wound area (day 0) and that of post
treatment (day 8).
The wound area on day 1 is 64mm2, the corresponding size of the dermal punch.
Calculations are made using the following formula: --
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[Open area on day 8] - [Open area on day 1 ] / [Open area on day 1
Specimens are fixed in 10% buffered formalin and paraffin embedded blocks are
sectioned perpendicular to the wound surface (5mm) and cut using an Olympus
microtome. Routine hematoxylin-eosin (H&E) staining is performed on cross-
sections of
bisected wounds. Histologic examination of the wounds allows assessment of
whether the
healing process and the morphologic appearance of the repaired skin is
improved by
treatment with a polypeptide of the invention. A calibrated lens micrometer is
used by a
blinded observer to determine the distance of the wound gap.
Experimental data are analyzed using am unpaired t test. A p value of < 0.05
is
considered significant.
The studies described in this example tested activity of a polypeptide of the
invention. However, one skilled in the art could easily modify the exemplified
studies to
test the activity of polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of the
invention.
Example 51: Lymphadema Animal Model
The purpose of this experimental approach is to create an appropriate and
consistent lymphedema model for testing the therapeutic effects of a
polypeptide of the
invention in lymphangiogenesis and re-establishment of the lymphatic
circulatory system
in the rat hind limb. Effectiveness is measured by swelling volume of the
affected limb;
quantification of the amount of lymphatic vasculature, total blood plasma
protein, and
histopathology. Acute lymphedema is observed for 7-10 days. Perhaps more
importantly,
the chronic progress of the edema is followed for up to 3-4 weeks.
Prior to beginning surgery, blood sample is drawn for protein concentration
analysis. Male rats weighing approximately ~350g are dosed with Pentobarbital.
Subsequently, the right legs are shaved from knee to hip. The shaved area is
swabbed
with gauze soaked in 70% EtOH. Blood is drawn for serum total protein testing.
Circumference and volumetric measurements are made prior to injecting dye into
paws
after marking 2 measurement levels (0.5 cm above heel, at mid-pt of dorsal
paw). The
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intradermal dorsum ofboth right and left paws are injected with 0.05 ml of 1%
Evan's
Blue. Circumference and volumetric measurements are then made following
injection of
dye into paws.
Using the knee joint as a landmark, a mid-leg inguinal incision is made
circumferentially allowing the femoral vessels to be located. Forceps and
hemostats are
used to dissect and separate the skin flaps. After locating the femoral
vessels, the
lymphatic. vessel that runs along side and underneath the vessels) is located.
The main
lymphatic vessels in this area are then electrically coagulated suture
ligated.
Using a microscope, muscles in back of the leg (near the semitendinosis and
adductors) are bluntly dissected. The popliteal lymph node is then located.
The 2
proximal and 2 distal lymphatic vessels and distal blood supply of the
popliteal node are
then and ligated by suturing. The popliteal lymph node, and any accompanying
adipose
tissue, is then removed by cutting connective tissues.
Care is taken to control any mild bleeding resulting from this procedure.
After
lymphatics are occluded, the skin flaps are sealed by using liquid skin
(Vetbond) (AJ ,
Buck). The separated skin edges are sealed to the underlying muscle tissue
while leaving
a gap of ~0.5 cm around the leg. Skin also may be anchored by suturing to
underlying
muscle when necessary.
To avoid infection, animals are housed individually with mesh (no bedding).
' Recovering animals are checked daily through the optimal edematous peak,
which
typically occurred by day 5-7. The plateau edematous peak are then observed.
To
evaluate the intensity of the lymphedema, the circumference and volumes of 2
designated
places on each paw before operation and daily for 7 days are measured. ' The
effect plasma
proteins on lymphedema is determined and whether protein analysis is a useful
testing
perimeter is also investigated. The weights of both control and edematous
limbs are
evaluated at 2 places. Analysis is performed in a blind manner.
Circumference Measurements: Under brief gas anesthetic to prevent limb
movement, a cloth tape is used to measure limb circumference. Measurements are
done at
the ankle bone and dorsal paw by 2 different people then those 2 readings are
averaged.
Readings are taken from both control and edematous limbs.
Volumetric Measurements: On the day of surgery, animals are anesthetized with
Pentobarbital and are tested prior to surgery. For daily volumetrics animals
are under
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brief halothane anesthetic (rapid immobilization and quick recovery), both
legs are shaved
and equally marked using waterproof marker on legs. Legs are first dipped in
water, then
dipped into instrument to each marked level then measured by Buxco edema
software(Chen/Victor). Data is recorded by one person, while the other is
dipping the
limb to marked area.
Blood-plasma protein measurements: Blood is drawn, spun, and serum separated
prior to surgery and then at conclusion for total protein and Ca2+ comparison.
Limb Weight Comparison: After drawing blood, the animal is prepared for tissue
collection. The limbs are amputated using a quillitine, then both experimental
and control
legs are cut at the ligature and.weighed. A second weighing is done as the
tibio-cacaneal
joint is disarticulated and the foot is weighed.
Histological Preparations: The transverse muscle located behind the knee
(popliteal) area is dissected and arranged in a metal mold, filled with
freezeGel, dipped
into cold methylbutane, placed into labeled sample bags at - 80EC until
sectioning. Upon
sectioning, the muscle is observed under fluorescent microscopy for
lymphatics..
The studies described in this example tested activity of a polypeptide of the
invention. However, one skilled in the art could easily modify the exemplified
studies to
test the activity of polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of the
invention.
Example 52: Suppression of TNF alpha-induced adhesion molecule expression
by a Polypeptide of the Invention
The recruitment of lymphocytes to areas of inflammation and angiogenesis
involves specific receptor-ligand interactions between cell surface adhesion
molecules
(CAMS) on lymphocytes and the vascular endothelium.. The adhesion process, in
both
normal and pathological settings, follows a mufti-step cascade that involves
intercellular
adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and
endothelial leukocyte adhesion molecule-1 (E-selectin) expression on
endothelial cells
(EC). The expression of these molecules and others on the vascular endothelium
determines the efficiency with which leukocytes may adhere to the local
vasculature and
extravasate into the local tissue during the development of an inflammatory
response. The
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local concentration of cytokines and growth factor participate in the
modulation of the
expression of these CAMS.
Tumor necrosis factor alpha (TNF-a), a potent proinflammatory cytokine, is a
stimulator of all three CAMs on endothelial cells and may be involved iri a
wide variety of
inflammatory responses, often resulting in a pathological outcome.
The potential of a polypeptide of the invention to mediate a suppression of
TNF-a
induced CAM expression can be examined. A modified ELISA assay which uses ECs
as a
solid phase absorbent is employed to measure the amount of CAM expression on
TNF-a
treated ECs when co-stimulated with a member of the FGF family of proteins.
To perform the experiment, human umbilical vein endothelial cell (HUVEC)
cultures are obtained from pooled cord harvests and maintained in growth
medium (EGM-
2; Clonetics, San Diego, CA) supplemented with 10% FCS and 1%
penicillin/sfreptomycin in a 37 degree C humidified incubator containing 5%
C02.
HUVECs are seeded in 96-well plates at concentrations of 1 x 104 cells/well in
EGM
medium at 37 degree C for 18-24 hrs or until confluent. The monolayers are
subsequently
washed 3 times with a serum-free solution of RPMI-1640 supplemented with 100
U/ml
penicillin and 100 mg/ml streptomycin, and treated with a given cytokine
and/or growth
factors) for 24 h at 37 degree C. Following incubation, the cells are then
evaluated for
CAM expression.
Human Umbilical Vein Endothelial cells (HUVECs) are grown in a standard 96
well plate to confluence. Growth medium is removed from the cells and replaced
with 90
u1 of 199 Medium (10% FBS). Samples for testing and positive or negative
controls are
added to the plate in triplicate. (in 10 u1 volumes). Plates are incubated at
37 degree C for
either 5 h (selectin and integrin expression) or 24 h (integrin expression
only). Plates are
aspirated to remove medium and 100 ~l of 0.1% paraformaldehyde-PBS(with Ca++
and
Mg++) is added to each well. Plates are held at 4°C for 30 min.
Fixative is then removed from the wells and wells are washed 1X with
PBS(+Ca,Mg)+0.5% BSA and drained. Do not allow the wells to dry. Add 10 p1 of
diluted primary antibody to the test and control wells. Anti-ICAM-1-Biotin,
Anti-VCAM-
1-Biotin and Anti-E-selectin-Biotin are used at a concentration of 10 ~ g/ml (
1:10 dilution
of 0.1 mg/ml stock antibody). Cells are incubated at 37°C for 30 min.
in a humidified
environment. Wells are washed X3 with PBS(+Ca,Mg)+0.5% BSA.
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Then add 20 ~l of diluted ExtrAvidin-Alkaline Phosphotase (1:5,000 dilution)
to
each well and incubated at 37°C for 30 min. Wells are washed X3 with
PBS(+Ca,Mg)+0.5% BSA. 1 tablet of p-Nitrophenol Phosphate pNPP is dissolved in
5 ml
of glycine buffer (pH 10.4). 100 w1 of pNPP substrate in glycine buffer is
added to each
test well. Standard wells in triplicate are prepared from the working dilution
of the
ExtrAvidin-Alkaline Phosphotase in glycine buffer: 1:5,000 (10°) >
10'°'S > 10'~ > 10'~~5. 5
p1 of each dilution is added to triplicate wells and the resulting AP content
in each well is
5.50 ng, 1.74 ng, 0.55 ng, 0.18 ng. 100 ~l of pNNP reagent must then be added
to each of
the standard wells. The plate must be incubated at 37°C for 4h. A
volume of 50 p1 of 3M
NaOH is added to all wells. The results are quantified on a plate reader at
405 nm. The
background subtraction option is used on blank wells filled with glycine
buffer only. The
template is set up to indicate the concentration of AP-conjugate in each
standard well [
5.50 ng; 1.74 ng; 0.55 ng; 0.18 ng]. Results are indicated as amount of bound
AP-
conjugate in each sample.
The studies described in this example tested activity of a polypeptide of the
invention. However, one skilled in the art could easily modify the exemplified
studies to
test the activity of polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of the
'invention.
Example 53: Assay for the Stimulation of Bone Marrow CD34+ Cell
Proliferation
This assay is based on the ability of human CD34+ to proliferate in the
presence of hematopoietic growth factors and evaluates the ability of isolated
polypeptides expressed in mammalian cells to stimulate proliferation of CD34+
cells.
It has been previously shown that most mature precursors will respond to only
a single signal. More immature precursors require at least two signals to
respond.
Therefore, to test the effect of polypeptides on hematopoietic activity of a
wide range
of progenitor cells, the assay contains a given polypeptide in the presence or
absence
of other hematopoietic growth factors. Isolated cells are cultured for 5 days
in the
presence of Stem Cell Factor (SCF) in combination with tested sample. SCF
alone
has a very limited effect on the proliferation of bone marrow (BM) cells,
acting in
such conditions only as a "survival" factor. However; combined with any factor
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exhibiting stimulatory effect on these cells (e.g., IL-3), SCF will cause a
synergistic
effect. Therefore, if the tested polypeptide has a stimulatory effect on a
hematopoietic
progenitors, such activity can be easily detected. Since normal BM cells have
a low
level of cycling cells, it is likely that any inhibitory~effect of a given
polypeptide, or
agonists or antagonists thereof, might not be detected. Accordingly, assays
for an
inhibitory effect on progenitors is preferably tested in cells that are first
subjected to
in vitro stimulation with SCF+IL+3, and then contacted with the compound that
is
being evaluated for inhibition of such induced proliferation.
Briefly, CD34+ cells are isolated using methods known in the art. The cells
are thawed and resuspended in medium (QBSF 60 serum-free medium with 1% L-
glutamine (SOOmI) Quality Biological, Inc., Gaithersburg, MD Cat# 160-204-101
).
After several gentle centrifugation steps at 200 x g, cells are allowed to
rest for one
hour. The cell count is adjusted to 2.5 x 105 cells/ml. During this time, 100
p,1 of
sterile water is added to the peripheral wells of a 96-well plate. The
cytokines that
can be tested with a given polypeptide in this assay is rhSCF.(R&D Systems,
Minneapolis, MN, Cat# 255-SC) at 50 ng/ml alone and.in combination with rhSCF
and rhIL-3 (R&D Systems, Minneapolis, MN, Cat# 203-ML) at 30 ng/ml. After one
hour, 10 ~,l of prepared cytokines, SO p,1 SID (supernatants at 1:2 dilution =
50 p,1) and
~,l of diluted cells are added to the media which is already present in the
wells to
20 allow for a final total volume of 100 p,1. The plates are then placed in a
37°C/5% COZ
incubator for five days.
Eighteen hours before the assay is harvested, 0.5 ~,Ci/well of [3H] Thymidine
is added in a 10 ~1 volume to each well to determine the proliferation rate.
The
experiment is terminated by harvesting the cells from each 96-well plate to a
filtermat
using the Tomtec Harvester 96. After harvesting, the filfermats are dried,
trimmed
and placed into OmniFilter assemblies consisting of one OmniFilter plate and
one
OmniFilter Tray. 60 ~1 Microscint is added to each well and the plate sealed
with
TopSeal-A press-on sealing film A bar code 15 sticker is affixed to the first
plate for
counting. The sealed plates is then loaded and the level of radioactivity
determined
via the Packard Top Count and the printed data collected for analysis. The
level of
radioactivity reflects the amount of cell proliferation.
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The studies described in this example test the activity of a given polypeptide
to stimulate bone marrow CD34+ cell proliferation. One skilled in the art
could
easilymodify the exemplified studies to test the activity of polynucleotides
(e.g., gene
therapy), antibodies, agonists, and/or antagonists and fragments and variants
thereof.
As a nonlimiting example, potential antagonists tested in this assay would be
expected
to inhibit cell proliferation in the presence of cytokines and/or to increase
the
inhibition of cell proliferation in the presence of cytokines and a given
polypeptide.
In contrast, potential agonists tested in this assay would be expected to
enhance cell
proliferation and/or to decrease the inhibition of cell proliferation in the
presence of
cytokines and a given polypeptide.
The ability of a gene to stimulate the proliferation of bone marrow CD34+
cells indicates that polynucleotides and polypeptides corresponding to the
gene are
useful for the diagnosis and treatment of disorders affecting the immune
system and
hematopoiesis. Representative uses are described in the "Immune Activity" and
"Infectious Disease" sections above, and elsewhere herein.
Example 54: Assay for Extracellular Matrix Enhanced Cell.Resnonse (EMECR
The objective of the Extracellular Matrix Enhanced Cell Response (EMECR)
assay is to identify gene products (e.g., isolated polypeptides) that act on
the
hematopoietic stem cells in the context of the extracellular matrix (ECM)
induced
signal.
Cells respond to the regulatory factors in the context of signals) received .
from the surrounding microenvironment. For example, fibroblasts, and
endothelial
and epithelial stem cells fail to replicate in the absence of signals from the
ECM.
Hematopoietic stem cells can undergo self renewal in the bone marrow, but not
in in
vitro suspension culture. The ability of stem cells to undergo self renewal in
vitro is
dependent upon their interaction with the stromal cells and the ECM protein
fibronectin (fn). Adhesion of cells to fn is mediated by the a5.(3~ and a4.~31
integrin
receptors, which are expressed by human and mouse hematopoietic stem cells.
The
factors) which integrate with the ECM environment and responsible for
stimulating
stem cell self renewal has not yet been identified. Discovery of such factors
should
be of great interest in gene therapy and bone marrow transplant applications
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Briefly, polystyrene, non tissue culture treated, 96-well plates are coated
with
fn fragment at a coating concentration of 0.2 pg/ cm2. Mouse bone marrow cells
are
plated (1,000 cells/well ) in 0.2 ml of serum-free medium. Cells cultured in
the
presence of IL-3 ( 5 ng/ml ) + SCF ( 50 ng/ml ) would serve as the positive
control,
conditions under which little self renewal but pronounced differentiation of
the stem
cells is to be expected. Gene products are tested with appropriate negative
controls in
the presence and absence of SCF(5.0 ng/ml), where test factor supernates
represent
10% of the total assay volume. The plated cells are then allowed to grow by
incubating in a low oxygen environment ( 5% CO2, 7% O2, and 88% Nz ) tissue
culture incubator for 7 days. The number of proliferating cells within the
wells is
then quantitated by measuring thymidine incorporation into cellular DNA.
Verification of the positive hits in the assay will require phenotypic
characterization
of the cells, which can be accomplished by scaling up of the culture system
and using
appropriate antibody reagents against cell surface antigens and FACScan.
One skilled in the art could easily modify the exemplified studies to test the
activity of polynucleotides (e.g., gene therapy), antibodies, agonists, and/or
antagonists and fragments and variants thereof.
If a particular gene product is found to be a stimulator of hematopoietic
progenitors, polynucleotides and polypeptides corresponding to the gene may be
useful for the diagnosis and treatment of disorders affecting the immune
system and
hematopoiesis. Representative uses are described in the "Immune Activity" and
"Infectious Disease" sections above, and elsewhere herein. The gene product
may
also be useful in the expansion of stem cells and committed progenitors of
various
blood lineages, and in the differentiation and/or proliferation of various
cell types.
Additionally, the polynucleotides and/or polypeptides of the gene of interest
and/or agonists and/or antagonists thereof, may also be employed to inhibit
the
proliferation and differentiation of hematopoietic cells and therefore may be
employed to protect bone marrow stem cells from chemotherapeutic agents during
chemotherapy. This antiproliferative effect may allow administration of higher
doses
of chemotherapeutic agents and, therefore, more effective chemotherapeutic
treatment.
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Moreover, polynucleotides and polypeptides corresponding to the gene of
interest may also be useful for the treatment and diagnosis of hematopoietic
related
disorders such as, for example, anemia, pancytopenia, leukopenia,
thrombocytopenia
or leukemia since stromal cells are important in the production of cells of
hematopoietic lineages. The uses include bone marrow cell ex-vivo culture,
bone
marrow transplantation, bone marrow reconstitution, radiotherapy or
chemotherapy of
neoplasia.
Example 55: Human Dermal Fibroblast and Aortic Smooth Muscle Cell
Proliferation
The polypeptide of interest is added to cultures of normal human dermal
fibroblasts (NHDF) and human aortic smooth muscle cells (AoSMC) and two co-
assays are performed with each sample. The first assay examines the effect of
the
polypeptide of interest on the proliferation of normal human dermal
fibroblasts
(NHDF) or aortic smooth muscle cells (AoSMC). Aberrant growth of fibroblasts
or
smooth muscle cells is a part of several pathological processes, including
fibrosis, and
restenosis. The second assay examines IL6 production by both NHDF and SMC. IL6
production is an indication of functional activation. Activated cells will
have
increased production of a number of cytokines and other factors, which can
result in a
proinflammatory or immunomodulatory outcome. Assays are run with and without
co-TNFa stimulation, in order to check for costimulatory or. inhibitory
activity.
Briefly, on day 1, 96-well black plates are set up with 1000 cells/well (NHDF)
or 2000 cells/well (AoSMC) in 100 p,1 culture media. NHDF culture media
contains:
Clonetics FB basal media, lmg/ml hFGF, 5mg/ml insulin, 50mg/ml gentamycin,
2%FBS, while AoSMC culture media contains Clonetics SM basal media, 0.5 ~g/ml
hEGF, 5mg/ml insulin, 1 p,g/ml hFGF, 50mg/ml gentamycin, 50 p,g/ml
Amphotericin
B, 5%FBS. After incubation @ 37°C for at least 4-5 hours culture media
is aspirated
and replaced with growth arrest media. Growth arrest media for NHDF contains
~ fibroblast basal media, 50mg/ml gentamycin, 2% FBS, while growth arrest
media for
AoSMC contains SM basal media, 50mg/ml gentamycin, 50~.g/ml Amphotericin B,
0.4% FBS. Incubate at 37C until day 2.
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On day 2, serial dilutions and templates of the polypeptide of interest are
designed which should always include media controls and known-protein
controls.
For both stimulation and inhibition experiments, proteins are diluted in
growth arrest
media. For inhibition experiments, TNFa is added to a final concentration of
2ng/ml
(NHDF) or 5ng/ml (AoSMC). Then add 1/3 vol media containing controls or
supernatants and incubate at 37C/5% COZ until day 5.
Transfer 60p1 from each well to another labeled 96-well plate, cover with a
plate-sealer, and store at 4C until Day 6 (for IL6 ELISA). To the remaining
100 p1 in
the cell culture plate, aseptically add Alamar Blue in an amount equal to 10%
of the
culture volume (101). Return plates to incubator for 3 to 4 hours. Then
measure
fluorescence with excitation at 530nm and emission at 590nm using the
CytoFluor.
This yields the growth stimulation/inhibition data.
On day 5, the IL6 ELISA is performed by coating a 96 well plate with 50-100
ul/well of Anti-Human IL6 Monoclonal antibody diluted in PBS, pH 7.4, incubate
ON
at room temperature.
On day 6, empty the plates into the sink and blot on paper towels. Prepare
Assay Buffer containing PBS with 4% BSA. Block the plates with 200 p.l/well of
Pierce Super Block blocking buffer in PBS for 1-2 hr and then wash plates with
wash
buffer (PBS, 0.05% Tween-20).. Blot plates on paper towels. Then add 50
pl/well of
diluted Anti-Human IL-6 Monoclonal, Biotin-labeled antibody at 0.50 mg/ml.
Make
dilutions of IL-6 stock in media (30, 10, 3, 1, 0.3, 0 ng/ml). Add duplicate
samples to
top row of plate. Cover the plates and incubate for 2 hours at RT on shaker.
Wash plates with wash buffer and blot on paper towels. Dilute EU-labeled
Streptavidin 1:1000 in Assay buffer, and add 100 pl/well. Cover the plate and
incubate 1 h at RT. Wash plates with wash buffer. Blot on paper towels.
Add 100 ~ul/well of Enhancement Solution. Shake for 5 minutes. Read the
plate on the Wallac DELFIA Fluorometer. Readings from triplicate samples in
each
assay were tabulated and averaged.
A positive result in this assay suggests AoSMC cell proliferation and that the
gene product of interest may be involved in dermal fibroblast proliferation
and/or
smooth muscle cell proliferation. A positive result also suggests
many.potential uses-
of polypeptides, polynucleotides, agonists and/or antagonists of the gene/gene
product
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of interest. For example, inflammation and immune responses, wound healing,
and
angiogenesis, as detailed throughout this specification. Particularly,
polypeptides of
the gene product and polynucleotides of the gene may be used in wound healing
and
dermal regeneration, as well as the promotion of vasculargenesis, both of the
blood
S vessels and lymphatics. The growth of vessels can be used in the treatment
of, for
example, cardiovascular diseases. Additionally, antagonists of polypeptides of
the
gene product and polynucleotides of the gene may be useful in treating
diseases,
disorders, and/or conditions which involve angiogenesis by acting as an anti-
vascular
(e.g., anti-angiogenesis). These diseases, disorders, and/or conditions are
known in
the art and/or are described herein, such as, for example, malignancies, solid
tumors,
benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas,
trachomas, and pyogenic granulomas; artheroscleric plaques; ocular angiogenic
diseases, for example, diabetic retinopathy, retinopathy of prematurity,
macular
degeneration, corneal graft rejection, neovascular glaucoma, retrolental
fibroplasia,
rubeosis, retinoblastoma, .uvietis and Pterygia (abnormal blood vessel growth)
of the
eye; rheumatoid arthritis; psoriasis; delayed wound healing; endometriosis;
vasculogenesis; granulations; hypertrophic scars (keloids); nonunion
fractures; .
scleroderma; trachoma; vascular adhesions; myocardial angiogenesis; coronary
collaterals; cerebral'collaterals; arteriovenous malformations; ischemic limb
angiogenesis; Osler-Webber Syndrome; plaque neovascularization;
telangiectasia;
hemophiliac joints; angiofibroma; fibromuscular dysplasia; wound granulation;
Crohn's disease; and atherosclerosis. Moreover, antagonists of polypeptides of
the
gene product and polynucleotides of the gene may be useful in treating anti-
hyperproliferative diseases and/or anti-inflammatory known in the art and/or
described herein.
One skilled in the art could easily modify the exemplified studies to test the
activity of polynucleotides (e.g., gene therapy), antibodies, agonists, and/or
antagonists and fragments and variants thereof.
Example 56: Cellular Adhesion Molecule (CAM) Expression on Endothelial
Cells
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The recruitment of lymphocytes to areas of inflammation and angiogenesis
involves specific receptor-ligand interactions between cell surface adhesion
molecules
(CAMS) on lymphocytes and the vascular endothelium. The adhesion process, in
both normal and pathological settings, follows a mufti-step cascade that
involves
intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1
(VCAM-1), and endothelial leukocyte adhesion molecule-1 (E-selectin)
expression on
endothelial cells (EC). The expression of these molecules and others on the
vascular
endothelium determines the efficiency with which leukocytes may adhere to the
local
vasculature and extravasate into the local tissue during the development of an
inflammatory response. The local concentration of cytokines and growth factor
.
participate in the modulation of the expression of these CAMS.
Briefly, endothelial cells (e.g., Human Umbilical Vein Endothelial cells
(HUVECs)) are grown in a standard 96 well plate to confluence, growth medium
is
removed from the cells and replaced with 100-~l of 199 Medium (10% fetal
bovine
serum (FBS)). Samples for testing and positive or negative controls are added
to the
plate in triplicate (in 10 ~l volumes). Plates are then incubated at
37°C for either 5 h
(selectin and integrin expression) or 24 h (integrin expression only). Plates
are
aspirated to remove medium and 100 ~l of 0.1% paraformaldehyde-PBS(with Ca++
and Mg++) is added to each well. Plates are held at 4°C for 30 min.
Fixative is
removed from the wells and wells are washed 1X with PBS(+Ca,Mg) + 0.5% BSA
and drained. 10 ~1 of diluted primary antibody is added to the test and
control wells.
Anti-ICAM-1-Biotin, Anti-VCAM-l-Biotin and Anti-E-selectin-Biotin are used at
a .
concentration of 10 ~g/ml (1:10 dilution of 0.1 mg/ml stock antibody). Cells
are
incubated at 37°C for 30 min. in a humidified environment. Wells are
washed three
times with PBS(+Ca,Mg) + 0.5% BSA. 20 ~1 of diluted ExtrAvidin-Alkaline
Phosphotase (1:5,000 dilution, refered to herein as the working dilution) are
added to
each well and incubated at 37°C for 30 min. Wells are washed three
times with
PBS(+Ca,Mg)+0.5% BSA. Dissolve 1 tablet of p-Nitrophenol Phosphate pNPP per 5
ml of glycine buffer (pH 10.4). 100 ~ l of pNPP substrate in glycine buffer is
added to
each~test well. Standard wells in triplicate are prepared from the working
dilution of
the ExtrAvidin-Alkaline Phosphotase in glycine buffer: 1:5,000 ( 10°) >
10-°'S > 10-' >
10-15. 5 ~l of each dilution is added to triplicate wells and the resulting AP
content in
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each, well_is 5.50 ng, 1.74 ng, 0.55 ng, 0.18 ng. 100 p1 of pNNP reagent is
then
added to each of the standard wells. The plate is incubated at 37°C for
4h. A volume
of 50 p1 of 3M NaOH is added to all wells. The plate is read on a plate reader
at 405
nm using the background subtraction option on blank wells filled with glycine
buffer
only. Additionally, the template is set up to indicate the concentration of AP-
conjugate in each standard well [ 5.50 ng; 1.74 ng; 0.55 ng; 0.18 ng]. Results
are
indicated as amount of bound AP-conjugate in each sample.
Example 57: Alamar Blue Endothelial Cells Proliferation Assay
This assay may be used to quantitatively determine protein mediated
inhibition of bFGF-induced proliferation of Bovine Lymphatic Endothelial Cells
(LECs), Bovine Aortic Endothelial Cells (BAECs) or Human Microvascular Uterine
Myometrial Cells (UTMECs). This assay incorporates a fluorometric growth
indicator based on detection of metabolic activity. A standard Alamar Blue
Proliferation Assay is prepared in EGM-2MV with 10 ng /ml of bFGF added as a
source of endothelial cell stimulation. This assay may be used with a variety
of
endothelial cells with slight changes in growth medium and cell concentration.
Dilutions of the protein batches to be tested are diluted as appropriate.
Serum-free
medium (GIBCO SFM) without bFGF is used as a non-stimulated control and
Angiostatin or TSP-1 are included as a known inhibitory controls.
Briefly, LEC, BAECs or UTMECs are seeded in growth media at a density of
5000 to 2000 cells/well in a 96 well plate and placed at 37-C overnight.
After.the
overnight incubation of the cells, the growth media is removed and replaced
with
GIBCO EC-SFM. The cells are treated with the appropriate dilutions of the
protein ~of
interest or control protein samples) (prepared in SFM ) in triplicate wells
with
additional bFGF to a concentration of 10 ng/ ml. Once the cells have been
treated
with the samples, the plates) is/are placed back in the 37° C incubator
for three days.
After three days 10 ml of stock alamar blue (Biosource Cat# DAL 1100) is added
to
each well and the plates) is/are placed back in the 37°C incubator for
four hours. The
plates) are then read at 530nm excitation and 590nm emission using the
CytoFluor
fluorescence reader. Direct output is, recorded in relative fluorescence
units.
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Alamar blue is an oxidation-reduction indicator that both fluoresces and
changes color in response to chemical reduction of growth medium resulting
from cell
growth. As cells grow in culture, innate metabolic activity results in a
chemical
reduction of the immediate surrounding environment. Reduction related to
growth
. causes the indicator to change from oxidized (non-fluorescent blue) form to
reduced
(fluorescent red) form. i.e. stimulated proliferation will produce a stronger
signal and
inhibited proliferation will produce a weaker signal and the total signal is
proportional
to the total number of cells as well as their metabolic activity. The
background level .
of activity is observed with the starvation medium alone. This is compared to
the
output observed from the positive control samples (bFGF in growth medium) and
protein dilutions.
Example 58: Detection of Inhibition of a Mixed Lymphocyte Reaction
This assay can be used to detect and evaluate inhibition of a Mixed
Lymphocyte Reaction (MLR) by gene products (e.g., isolated polypeptides).
Inhibition of a MLR may be due to a direct effect on cell proliferation and
viability,
modulation of costimulatory molecules on interacting cells, modulation of
adhesiveness between lymphocytes and accessory cells, or modulation of
cytokine
production by accessory cells. Multiple cells may be targeted by these
polypeptides
since the peripheral blood mononuclear fraction used in this assay includes T,
B and
natural killer lymphocytes, as well as monocytes and dendritic cells.
Polypeptides of interest found to inhibit the MLR may find application in
diseases associated with lymphocyte and monocyte activation or proliferation.
These
include, but are not limited to, diseases such as asthma, arthritis, diabetes,
inflammatory skin conditions, psoriasis, eczema, systemic lupus erythematosus,
multiple sclerosis, glomerulonephritis, inflammatory bowel disease, crohn's
disease,
ulcerative colitis, arteriosclerosis, cirrhosis, graft vs. host disease, host
vs. graft
disease, hepatitis, leukemia and lymphoma.
Briefly, PBMCs from human donors are purified by density gradient
centrifugation using Lymphocyte Separation Medium (LSM~, density 1.0770 g/ml,
l
Organon Teknika Corporation, West Chester, PA). PBMCs from two donors are
adjusted to 2 x 106 cells/ml in RPMI-1640 (Life Technologies, Grand Island,
NY)
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supplemented with 10% FCS and 2 mM glutamine. PBMCs from a third donor is
adjusted to 2 x 105 cells/ml. Fifty microliters of PBMCs from each donor is
added to
wells of a 96-well round bottom microtiter plate. Dilutions of test materials
(50 ~l) is
added in triplicate to microtiter wells. Test samples (of the protein of
interest) are
added for final dilution of 1:4; rhuIL-2 (R&D Systems, Minneapolis, MN,
catalog
number 202-IL) is added to a final concentration of 1 ~g/ml; anti-CD4 mAb (R&D
Systems, clone 34930.11, catalog number MAB379) is added to a final
concentration
of 10 pg/ml. Cells are cultured for 7-8 days at 37°C in 5% COZ, and 1
~C of [3H]
thymidine is added to wells for the last 16 hrs of culture. Cells are
harvested and
thymidine incorporation determined using a Packard TopCount. Data is expressed
as
the mean and standard deviation of triplicate determinations.
Samples of the protein of interest are screened in separate experiments and
compared to the negative control treatment, anti-CD4 mAb, which inhibits
proliferation of lymphocytes and the positive control treatment, IL-2 (either
as
recombinant material or supernatant), which enhances proliferation of
lymphocytes.
One skilled in the art could easily modify the exemplified studies to test the
activity of polynucleotides (e.g., gene therapy), antibodies, agonists, and/or
antagonists and fragments and variants thereof.
It will be clear that the invention may be practiced otherwise than as
particularly described in the foregoing description and examples. Numerous
modifications and variations of the present invention are possible in light of
the above
teachings and, therefore, are within the scope of the appended claims.
The entire disclosure of each document cited (including patents, patent
applications, journal articles, abstracts, laboratory manuals, books, or other
disclosures) in the Background of the Invention, Detailed Description, and
Examples
is hereby incorporated herein by reference. Further, the hard copy of the
sequence
listing submitted herewith and the corresponding computer readable form are
both
incorporated herein by reference in their entireties. Additionally, the
contents of U.S.
provisional applications Serial Nos. 60/179,065, 60/180,628, 60/251,988,
60/226,279,
and Serial No. unknown (Atty. Docket PS730PP2) filed January 5, 2001 are all
hereby incorporated by reference in their entirety.
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INDICATIONS
RELATING
TO A
DEPOSITED
MICROORGANISM
OR OTHER
BIOLOGICAL
MATERIAL
(PCT
Rule
136is)
A. The
indications
made
below
relate
to the
deposited
microorganism
or other
biological
material
referred
to in
the
description
in Table
I on
page
67.
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OF DEPOSIT
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deposits
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Address
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Date Accession
of deposit Number
09 June PTA-2079
2000
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In respect
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to an
expert
nominated
by the
person
requesting
the
sample
(Rule
28(4)
EPC).
Continued
on additional
sheets
E. SEPARATE
FURNISHING
OF INDICATIONS
(!care
blank
iJeioi
apptieabte~
The indications
listed
below
will
be submitted
to the
international
Bureau
later
(specify
the
general
nature
ojthe
indications
e.g..
'Accession
Number
ojDeposit')
For receiving For International Bureau
Office use only use only
0 This ~ This
sheet sheet
was was
received received
with by the
the International
international Bureau
application on:
Authorized Authorized
officer officer
Revised Forth PCT/RO/134 (January 2001) Pctro134ep.sollist
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
435
ATCC Deposit No. PTA-2079
CANADA
The applicant requests that, until either a Canadian patent has been issued on
the basis of an
application or the application has been refused, or is abandoned and no longer
subject to
reinstatement, or is withdrawn, the Commissioner of Patents only authorizes
the furnishing of
a sample of the deposited biological material referred to in the application
to an independent
expert nominated by the Commissioner, the applicant must, by a written
statement, inform
the International Bureau accordingly before completion of technical
preparations for
publication of the international application. .
NORWAY
The applicant hereby requests that the application has been laid open to
public inspection (by
the Norwegian Patent Office), or has been finally decided upon by the
Norwegian Patent
Office without having been laid open inspection, the furnishing of a sample
shall only be
effected to an expert in the art. The request to this effect shall be filed by
the applicant with
the Norwegian Patent Office not later than at the time when the application is
made available
to the public under Sections 22 and 33(3) of the Norwegian Patents Act. If
such a request has
been filed by the applicant; any request made by a third party for the
furnishing of a sample
shall indicate the expert to be used. That expert may be any person entered on
the list of
recognized experts drawn up by the Norwegian Patent Office or any person
approved by the
applicant in the individual case.
AUSTRALIA
The applicant hereby gives notice that the furnishing of a sample of a
microorganism shall
only be effected prior to the grant of a patent, or prior to the lapsing,
refusal or withdrawal of
the application, to a person who is a skilled addressee without an interest~in
the invention
(Regulation 3.25(3) of the Australian Patents Regulations).
FINLAND
The applicant hereby requests that, until the application has been laid open
to public
inspection (by the National Board of Patents and Regulations), or has been
finally decided
upon by the National~$oard of Patents and Registration without having been
laid open to
public inspection, the furnishing of a sample shall only be effected to an
expert in the art.
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
436
ATCC Deposit No.: PTA-2079
UNITED KINGDOM
The applicant hereby requests that the furnishing of a sample of a
microorganism shall only
be made available to an expert. The request to this effect must be filed by
the applicant with
the International Bureau before the. completion of the technical preparations
for the
international publication of the application.
DENMARK
The applicant hereby requests that, until the application has been laid open
to public
inspection (by the Danish Patent Office), or has been finally decided upon by
the Danish
Patent office without having been laid open to public inspection, the
furnishing of a sample
shall only be effected to an expert in the art. The request to this effect
shall be filed by the
applicant with the Danish Patent Office not later that at the time when the
application is made
available to the public under Sections 22 and 33(3) of the Danish Patents Act.
If such a
request has been filed by the applicant, any request made by a third party for
the furnishing of
a sample shall indicate the expert to be used. That expert may be any person
entered on a list
of recognized experts drawn up by the Danish Patent Office or any person by
the applicant in
the individual case.
SWEDEN
The applicant hereby requests that, until the application has been laid open
to public
inspection (by the Swedish Patent Office), or has been finally decided upon by
the Swedish
Patent Office without having been laid open to public inspection, the
furnishing of a sample
shall only be effected to an expert in the art. The request to this effect
shall be filed by the
applicant with the International Bureau before the expiration of 16 months
from the priority
date (preferably on the Form PCT/RO/134 reproduced in annex Z of Volume I of
the PCT
Applicant's Guide). If such a request has been filed by the applicant any
request made by a
third party for the furnishing of a sample shall indicate the expert to be
used. That expert
may be any person entered on a list of recognized experts drawn up by the
Swedish Patent
Office or any person approved by a applicant in the individual case.
NETHERLANDS
The applicant hereby requests that until the date of a grant of a Netherlands
patent or until the
date on which the application is refused or withdrawn or lapsed, the
microorganism shall be
made available as provided in the 31 F( 1 ) of the Patent Rules only by the
issue of a sample to
an expert. The request to this effect must be furnished by the applicant with
the Netherlands
Industrial Property Office before the date on which the application is made
available to the
public under Section 22C or Section 25 of the Patents Act of the Kingdom of
the Netherlands,
whichever of the two dates occurs earlier.
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
1
<110> Human Genome Sciences, Inc.
<120> 22 Human Secreted Proteins
<130> PS730PCT
<150> 60/179,065
<151> 2000-O1-31
<150> 60/180,628
<151> 2000-02-04
<150> 60/251,988
<151> 2000-12-05
<150> 60/226,279
<151> 2000-OB-18
<150> Unassigned
<151> 2001-O1-05
<160> 146
<170> PatentIn Ver. 2.0
<210> 1
<211> 733
<212> DNA
<213> Homo Sapiens
<400>
1
gggatccggagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctg 60
aattcgagggtgcaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatga 120
tctcccggactcctgaggtcacatgcgtggtggtggacgtaagccacgaagaccctgagg 180
tcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcggg 240
aggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggact 300
ggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccaacccccatcg 360
agaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccc 420
catcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttct 480
atccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaaga 540
ccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtgg 600
acaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgc 660
acaaccactacacgcagaagagcctctccctgtctccgggtaaatgagtgcgacggccgc 720
gactctagaggat 733
<210> 2
<211> 5
<212> PRT
<213> Homo Sapiens
<220>
<221> Site
<222> (3)
<223> Xaa equals any of the twenty naturally ocurring L-amino acids
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
2
<400> 2
Trp Ser Xaa Trp Ser
1 5
<210> 3
<211> 86
<212> DNA
<213> Artificial Sequence
<220>
<221> Primer_Bind
<223> Synthetic sequence with 4 tandem copies of the GAS binding site found
in the IRF1 promoter (Rothman et al., Immunity 1:457-468 (1994)), 18
nucleotides complementary to the SV40 early promoter, and a Xho I
restriction site.
<400> 3
gcgcctcgag atttccccga aatctagatt tccccgaaat gatttccccg aaatgatttc 60
cccgaaatat ctgccatctc aattag 86
<210> 4
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<221> Primer_Bind
<223> Synthetic sequence complementary to the SV40 promter; includes a Hind
III restriction site.
<400> 4
gcggcaagct ttttgcaaag cctaggc 27
<210> 5
<211> 271
<212> DNA
<213> Artificial Sequence
<220>
<221> Protein Bind
<223> Synthetic promoter for use in biological assays; includes GAS binding
sites found in the IRF1 promoter (Rothman et al., Immunity 1:457-468
(1994) ) .
<400> 5
ctcgagattt ccccgaaatc tagatttccc cgaaatgatt tccccgaaat gatttccccg 60
aaatatctgc catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc 120
gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat 180
ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt 240
ttttggaggc ctaggctttt gcaaaaagct t 271
<210> 6
<211> 32
<212> DNA
<213> Artificial Sequence
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
3
<220>
<221> Primer_Bind
<223> Synthetic primer complementary to human genomic EGR-1 promoter
sequence (Sakamoto et al., Oncogene 6:867-871 (1991)); includes a Xho I
restriction site.
<400> 6
gcgctcgagg gatgacagcg atagaacccc gg 32
<210> 7
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<221> Primer_Bind
<223> Synthetic primer complementary to human genomic EGR-1 promoter
sequence (Sakamoto et al., Oncogene 6:867-871 (1991)); includes a Hind III
restriction site.
<400> 7
gcgaagcttc gcgactcccc ggatccgcct c 31
<210> 8
<211> 12
<212> DNA
<213> Homo sapiens
<400> 8
ggggactttc cc 12
<210> 9
<211> 73
<212> DNA
<213> Artificial Sequence
<220>
<221> Primer_Bind
<223> Synthetic primer with 4 tandem copies of the NF-KB binding site
(GGGGACTTTCCC), 18 nucleotides complementary to the 5' end of the SV40
early promoter sequence, and a XhoI restriction site.
<400> 9
gcggcctcga ggggactttc ccggggactt tccggggact ttccgggact ttccatcctg 60
ccatctcaat tag 73
<210> 10
<211> 256
<212> DNA
<213> Artificial Sequence
<220>
<221> Protein Bind
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
4
<223> Synthetic promoter for use in biological assays; includes NF-KB
binding sites.
<400> 10
ctcgagggga ctttcccggg gactttccgg ggactttccg ggactttcca tctgccatct 60
caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc 120
cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga 180
ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg 240
cttttgcaaa aagctt 256
<210> 11
<211> 1631
<212> DNA
<213> Homo Sapiens
<400>
11
ggcacgagcttgtcgtgtggctctgtgtcgacactgtgcgccaccatggccgtgactgcc 60
tgtcagggcttggggttcgtggtttcactgattgggattgcgggcatcattgctgccacc 120
tgcatggaccagtggagcacccaagacttgtacaacaaccccgtaacagctgttttcaac 180
taccaggggctgtggcgctcctgtgtccgagagagctctggcttcaccgagtgccggggc 240
tacttcaccctgctggggctgccagccatgctgcaggcagtgcgagccctgatgatcgta 300
ggcatcgtcctgggtgccattggcctcctggtatccatctttgccctgaaatgcatccgc 360
attggcagcatggaggactctgccaaagccaacatgacactgacctccgggatcatgttc 420
attgtctcaggtctttgtgcaattgctggagtgtctgtgtttgccaacatgctggtgact 480
aacttctggatgtccacagctaacatgtacaccggcatgggtgggatggtgcagactgtt 540
cagaccaggtacacatttggtgcggctctgttcgtgggctgggtcgctggaggcctcaca 600
ctaattgggggtgtgatgatgtgcatcgcctgccggggcctggcaccagaagaaaccaac 660
tacaaagccgtttcttatcatgcctcaggccacagtgttgcctacaagcctggaggcttc 720
aaggccagcactggctttgggtccaacaccaaaaacaagaagagatacgatggaggtgcc 780
cgcacagaggacgaggtacaatcttatccttccaagcacgactatgtgtaatgctctaag 840
acctctcagcacgggcggaagaaactcccggagagctcacccaaaaaacaaggagatccc 900
atctagatttcttcttgcttttgactcacagctggaagttagaaaagcctcgatttcatc 960
tttggagaggccaaatggtcttagcctcagtctctgtctctaaatattccaccataaaac 1020
agctgagttatttatgaattagaggctatagctcacattttcaatcctctatttcttttt 1080
ttaaatataactttctactctgatgagagaatgtggttttaatctctctctcacattttg 1140
atgatttagacagactccccctcttcctcctagtcaataaacccattgatgatctatttc 1200
ccagcttatccccaagaaaacttttgaaaggaaagagtagacccaaagatgttattttct 1260
gctgtttgaattttgtctccccacccccaacttggctagtaataaacacttactgaagaa 1320
gaagcaataagagaaagatatttgtaatctctccagcccatgatctcggttttcttacac 1380
tgtgatcttaaaagttaccaaaccaaagtcattttcagtttgaggcaaccaaacctttct 1440
actgctgttgacatcttcttattacagcaacaccattctaggagtttcctgagctctcca 1500
ctggagtcctctttctgtcgcgggtcagaaattgtccctagatgaatgagaaaattattt 1560
tttttaatttaagtcctaaatatagttaaaataaataatgttttagtaaaatgaaaaaaa 1620
aaaaaaaaaaa 1631
<210> 12
<211> 1699
<212> DNA
<213> Homo Sapiens
<220>
<221> SITE
<222> (1692)
<223> n equals a,t,g, or c
<220>
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
<221> SITE
<222> (1693)
<223> n equals a,t,g, or c
<220>
<221> SITE
<222> (1696)
<223> n equals a,t,g, or c
<400>
12
gaacggggsatcaccagcgacacctgckawttggactacytctgaascattctttccctt 60
yctagggctttgctggatcccgccsgccccgagatccaacagaacaaascagaraggaga 120
cttaatcccgtattctccagatgtacagatacacgcagagcgattcatcaactataacca 180
gacagtttctcgaatgcgaggtatctacacagctccctcgggtctggagtccacttgttt 240
ggttgtggcctatggtttggacatttaccaaactcgagtctacccatccaagcagtttga 300
cgttctgaaggatgactatgactacgtgttaatcagcagcgtcctctttggcctggtttt 360
tgccaccatgatcactaagagactggcacaggtgaagctcctgaatcgggcctggcgata 420
aagaacaaagactgtgcctaaaagtggagagccaggggagtgtgggtcagataagcagct 480
acagctgcagtttggtggattggtggagtatgtgtgtgtgtcagtgctcagctaagaact 540
gtagggaagatggatgaccttcacgcagaactccttttgggatatacatgatgcagaaag 600
gatcctacatggagagagacagaactctctcagctgacactctcagagattcctgatggg 660
ctttctcttgaagtccaaaggcgtctgcattgtttcctttctttgcccatccatgaatgt 720
tctgttttgttttgttttttttaataagaattccggctgatttttgtgaggcctgtttaa 780
attgactttactttgccttttgtgtttctcaattttatctagaaatctttctgacttttt 840
ccatctcttgcttcaaagtaagaggggaactctccttgccgactccaccttataggtaca 900
tttggtgttttgcactgggaagaaataggatccatccttagctgaggcttgaggactgat 960
ccagcctctcatggcttccctccaaagtaacttagggttgagggatctatatgtgatgtc 1020
aaaacttactttaaacctctagtttcgtgctgtcatttattaggctgggccaccaaatct 1080
ttgtttcaatttatcagaagccaagtgcatactagcgtcttgtttgttgcccattgccta 1140
tacttttcacctgagatgtgtgagttggggccttttaaaaactactgaattgtctgagcc 1200
ttgaagacatttccagggagaagagataatctctcatttcacccacaggctggtctaatc 1260
ataacctagttaaagatgtccttgtttaagaaccccattatttatttttagtttttaata 1320
taaattaacatgtgggtcattatatttctccttaaatgaggaaattttaaattttattga 1380
tctaacctttgaagctttaaaaaaggagaaagagggtaggggtgggaaactggcatactg 1440
tgtgtatagcactgccgattggctaggccactgtgtctctgctacaaattaaagaaatcc 1500
taaaagttttccttggtcatagagttggggaatgacagaatttttctttgttgtgaaatg 1560
tatgtacagagtagaccatctctagccctgtggtgaaagaggtacactcgaatgtttgca 1620
taaagcaagtgacaaatgaaaaaaaaaaaaaaaaaaaaaccccgggggggggcccggtaa 1680
cccatttggccnnaanggg 1699
<210> 13
<211> 950
<212> DNA
<213> Homo Sapiens
<400>
13
cccacgcgtccggggtcaaccacgagagctggcgtgacagtggccagactgacggcctga 60
cctttggccacctgaagatggtgctgctgtgggcctctgtgctcttcccggcgcccgagg 120
actgggcagagctgcagggcgccgtgtaccgcctgctggtggtgctgctctgttgcctgg 180
ccacgcggaagctgccccacttcctccacccgcagcgcaacctgctgcagggcagcggcc 240
tggaccttggtgccatctaccagcgcgtggagggcttcgccagccagcccgaggcggccc 300
tgcgcatccacgccacccacctgggccgcagccccccgccgcgcatcggctccgggctca 360
aggcgctcctgcagctgccagccagtgaccccacttactgggccactgcctacttcgacg 420
tcctgctggacaagttccaggtcttcaacatccaggataaggaccggatctctgccatgc 480
agagcatcttccagaagaccaggactctgggaggcgaggagagctgagctgggccacctg 540
gtctcagccacctgttcttggctccccaacagactctgcactgcaccatgggaggctcct 600
gggatgtttggaagaagaaacgggcttctccttgagggggtagtggagggattttgtccc 660
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
6
cagcagtggcctctgagagtctttcagtgcctggtggggcagggcaggcctcttggagca 720
cctcctccctgggtcagggcctggatgcaggtgccaagctctccatgtggtgcatgttga 780
cccagccacgtggtgttgtcaagcaaacagcatcggcaggagacctggagctgaggactt 840
ggccctgcctgcactgtatgccattccttggtgatgaaatgctgtatatttggttttgaa 900
aaaatgaatgtgctgggtatacacagcagaaagggaaaaaaaaaaaaaaa 950
<210> 14
<211> 2624
<212> DNA
<213> Homo sapiens
<220>
<221> SITE
<222> (2504)
<223> n equals a,t,g, or c
<220>
<221> SITE
<222> (2549)
<223> n equals a,t,g, or c
<220>
<221> SITE
<222> (2616)
<223> n equals a,t,g, or c
<400>
14
gctatttaggtgacactatagaaggtacgcctgcaggtaccggtccggaattcccgggtc,60
gacccac.gcgtccggcgggggcatcgccgcccgcgcccctctaagtgccgggccgcaagc 120
tccaccgcagccgcctgcaagcagcggcgcctcggccctcgacctgcgcgcaaagcctgt 180
gctggagccgtcctcccgcggcggggaccgggaccggggacccaagccaatcgaaagctc 240
caaccatggccatggggctcttccgcgtgtgtctggtggtggtgacggccatcatcaacc 300
acccgctgctgttcccgcgggagaacgccacagtccccgagaacgaggaggagatcatcc 360
gcaagatgcaggcgcaccaggagaagctgcagctggagcagttgcgcctggaggaggagg 420
tggctcggctggcggccgaaaaggaggcactggagcaggtggcggaggagggcaggcagc 480
agaacgagacacgcgtggcctgggacctctggagcaccctctgcatgatcctcttcctga 540
tgatcgaggtgtggcggcaggaccaccaggaggggccctcacctgagtgcctgggcggtg 600
aggaggatgagctgcctgggctggggggcgcccccttgcagggcctcaccctgcccaaca 660
aggccacgcttggccacttttatgagcgctgcatccggggggccacggccgatgcagccc 720
gtacccgggagttcctggaaggcttcgtggatgacttgctggaagccctgaggagcctct 780
gcaaccgggacaccgacatggaggtggaggacttcattggcgtggacagcatgtacgaga 840
actggcaggtggacaggccactgctgtgccaccttttcgtgcccttcacaccccccgagc 900
cctaccgcttccacccagagctctggtgctccggccgctcagtgcccctggatcgccagg 960
gctacggccagatcaaggtggtccgcgccgatggggacacattgagctgcatctgcggca 1020
agaccaagctcggggaagacatgctgtgtctcctgcacggcaggaacagcatggcgcctc 1080
cctgcggygacatggagaacctgctgtgtgccacagattccctgtacctggacacgatgc 1140
aggtcatgaagtggttccagacggccctcaccagagcctggaagggcatcgcccacaagt 1200
acgagttcgacctggcctttggccagctggacagcccggggtccctgaagatcaagttcc 1260
gttcagggaagttcatgcccttcaacctgattcctgtgatccagtgtgatgactcggacc 1320
tgtactttgtctcccaccttcccagggagccctctgagggcaccccagcctccagcacag 1380
actggctcctgtcctttgctgtctatgagcgacacttcctcaggacgacactaaaggcac 1440
tgcccgagggcgcctgccacctcagctgcctgcagatagcatccttcctgctctccaagc 1500
agagccgcctgaccggtcccagcgggctcagcagctaccacctgaagacggccctactgc 1560
acctcctactcctccggcaggccgccgactggaaggcggggcagctggacgctcgtctgc 1620
acgagttgctgtgcttcctggagaagagcttgctccagaagaagctccaccacttcttca 1680
tcggcaaccgcaaggtgcctgaggccatgggactccctgaggccgtgctcagggccgagc 1740
ccctcaacctcttccggcccttcgtcctgcagcgaagcctttaccgtaagacactggact 1800
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
ccttctatgagatgctcaagaatgccccagcgctcattagcgagtattccctacatgtcc 1860
cctcagaccagcctaccccaaaaagctgacgtcttttacagaatgtgggatcctcgagct 1920
aagatgagggcatccctcacgttcacacccctggtggcatctgccagccctgttctgggg 1980
acaaggcgggctttcgtgggagccgtgctcagcctgccaggaagccaagccctacagtgc 2040
agaggaaacagaatttcaacgggaagctggtttgcttcataccattgggatctgctggta 2100
aagctgttatttgggtttagggactgatcccttgcagtttacttctggatcaccatgaat 2160
ggccaagatggtggcagaacacgctgtggaccctgagttagagacaatgcaaatgttgga 2220
ttgggtgtaattctttttgaatcccagatccagtctgtacttgaatatgagcagaggatc 2280
tacaagaatgctgacagggaaccgtgttaagacccagcacccctattcccaggagcttct 2340
ggcctgaccatctgcagccaaagcactaacagggacagatatgggaatgtccacctttga 2400
tccgcatcctgcacaatagtggtcccaccatggctgccacttttttatactatttggaga 2460
aaagaccttgtataaattcgaggcccgaatgactaacgtctctntcacacggaaatgggt 2520
acttggtgggcatagagacaatgcaaatntttggattgggtgtaattcttttttgaatcc 2580
cagatccagtctgtacttggaatatgagccagaggntctacaag 2624
<210> 15
<211> 1959
<212> DNA
<213> Homo Sapiens
<400> 15
ggcacgagcgtgatcatcgggatgctcgtgctcctgctggactttcttggcttggtgcac 60
ctgggccagctgctcatcttccacatctacctgaaggccaagaagatgaccacctttgag 120
tatctcattaataaccgcaaagaagagagttcaaaacatcaagcagtgaggaaagatcca 180
tacgtgcaaatggacaaaggagttctccagcaaggagctggcgccctgggctcatctgca 240
cagggagtcaaagccaagagctccctgctgattcacaagcacttatgtcacttctgcact 300
tcagtaaaccaggatggggattcgacggcacgggtgcatttgtgagtcccctcagtgtct 360
ctgcagcttctatgtgtggatgaatagtgaagccacatgaggcctggtctgaagcagaga 420
agatccgctcagcatcatgttgaatcccaagcccgcgtctccgtgggctccaggacaatc 480
ctatgaaagtgacaccgtcgttcattgttcacatcggggaggagaattccatctgaaaat 540
gcacgtgacttcactgacacccgcgtccgtggcacagccctgtgctgagctccacagacc 600
tacagtccatcgcctccccttggagtgggcccacagctgcagacagtgttgtccaaagaa 660
gatgctctgagtagatgaatggaacaaactggaaactccagaaacaagcccttctttaca 720
tttctaatgtttggcttaaatggaaggtgccaagagccaaggccaggaactcccggctga 780
tttgcaggcgcctgtgtcagttctccactcgtgtacacccagacgggggctcgatggcac 840
aggaagcagatgatgccccgagtatatctacacttgggctgcaacaagaaacaacagagc 900
ccatgaaaactgacagtgctgaaagtgaagactgagattcaggagctcaggtgcccctgt 960
gatccaggtcttctaccctgaaaccccaccctccatcaaggtcctgcctgtagagtctac 1020
cttgcaaagcctcctgctcctacccatgctacaggccaggaaccagagcccatcatctca 1080
gaggcccctggatgtccttcgaaggaaccaggaccctcagagcccagcatccatctctgt 1140
catcatcttcatcacacccaaagaagagccagccttgcaggagggtttacatctccagga 1200
agatgggctgccagcaactgcagaggatgcagccacctgcttaactgtgctgtccagcca 1260
gccagccagctgcagggcctcttgctgcttaagagctgatgggccgggcatgttggctca 1320
cacctgtgagcacagtactgggaaatgggagcacagtactaggaaatgggagcacagtac 1380
tgggaaatgggagcacagtactgggaaatgggggctcacagcactgcaaaatgggagcac 1440
agtattgggaaatgggagcacagtactgggaagtgggagcacagtactgagaagtgggag 1500
cacagtactgagaaatgggagtacactactgagaaatgggagcacagtactgggaaatgg 1560
gcatacagtactgggaaatgggcatacagtactgagaaatgggagcacagtactgggaaa 1620
tgggagcacagtactgggaaatgggagcccacagtactgggaaaggggagttcacagtac 1680
tcggaaatgggagcatagtactgggaaatgggagcacagtactgggaaatgggagcatag 1740
tactgggaaaccccagacctggattctgagtttttcagcctagcccagacttcttatctt 1800
agtagacaaaaagagtcaataccagagaaccagaggcatcctctgtattttaatgaactc 1860
tgcattttaatctgtttagtagtcattttttaaaagataatcagttttccaaatatatct 1920
ataagttactacgtgcaaaaaaaaaaaaaaaaaaaaaaa 1959
<210> 16
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
<211> 3024
<212> DNA
<213> Homo sapiens
<400> 16
ccacgcgtccggagcgaggctgccctttcttcgcagcgtgatttattttcttcttttttt 60
ctgaactcttcttccagggagaggctagtggtaacaggccgagctggatggatgggtatg 120
gggagaggggcaggacgttcagccctgggattctggccgaccctcgccttccttctctgc 180
agcttccccgcagccacctccccgtgcaagatcctcaagtgcaactctgagttctggagc 240
gccacgtcgggcagccacgccccagcctcagacgacacccccgagttctgtgcagccttg 300
cgcagctacgccctgtgcacgcggcggacggcccgcacctgccggggtgacctggcctac 360
cactcggccgtccatggcatagaggacctcatgagccagcacaactgctccaaggatggc 420
cccacctcgcagccacgcctgcgcacgctcccaccggccggagacagccaggagcgctcg 480
gacagccccgagatctgccattacgagaagagctttcacaagcactcggccacccccaac 540
tacacgcactgtggcctcttcggggacccacacctcaggactttcaccgaccgcttccag 600
acctgcaaggtgcagggcgcctggccgctcatcgacaataattacctgaacgtgcaggtc 660
accaacacgcctgtgctgcccggctcagcggccactgccaccagcaagctcaccatcatc 720
ttcaagaacttccaggagtgtgtggaccagaaggtgtaccaggctgagatggacgagctc 780
ccggccgccttcgtggatggctctaagaacggtggggacaagcacggggccaacagcctg 840
aagatcactgagaaggtgtcaggccagcacgtggagatccaggccaagtacatcggcacc 900
accatcgtggtgcgccaggtgggccgctacctgacctttgccgtccgcatgccagaggaa 960
gtggtcaatgctgtggaggactgggacagccagggtctctacctctgcctgcggggctgc 1020
cccctcaaccagcagatcgacttccaggccttccacaccaatgctgagggcaccggtgcc 1080
cgcaggctggcagccgccagccctgcacccacagcccccgagaccttcccatacgagaca 1140
gccgtggccaagtgcaaggagaagctgccggtggaggacctgtactaccaggcctgcgtc 1200
ttcgacctcctcaccacgggcgacgtgaacttcacactggccgcctactacgcgttggag 1260
gatgtcaagatgctccactccaacaaagacaaactgcacctgtatgagaggactcgggac 1320
ctgccaggcagggcggctgcggggctgcccctggccccccggcccctcctgggcgccctg 1380
gtcccgctcctggccctgctccctgtgttctgctagacgcgtagatgtggagggaggcgc 1440
gggctccgtcctctcggcttccccatgtgtgggctgggaccgcccacggggtgcagatct 1500
cctggcgtgtccaccatggccccgcagaacgccagggaccgcctgctgccaagggctcag 1560
gcatggacccctccccttctagtgcacgtgacaaggttgtggtgactggtgccgtgatgt 1620
ttgacagtagagctgtgtgagagggagagcagctcccctcgccccgcccctgcagtgtga 1680
atgtgtgaaacatcccctcaggctgaagccccccacccccaccagagacacactgggaac 1740
cgtcagagtcagctccttccccctcgcaatgcactgaaaggcccggccgactgctgctcg 1800
ctgatccgtggggccccctgtgcccgccacacgcacgcacacactcttacacgagagcac 1860
actcgatccccctaggccagcggggacaccccagccacacagggaggcatccttggggct 1920
tggccccaggcagggcaaccccggggcgctgcttggcaccttagcagactgctggaacct 1980
tttggccagtaggtcgtgcccgcctggtgccttctggcctgtggcctccctgcccatgtt 2040
cacctggctgctgtgggtaccagtgcaggtcccggttttcaggcacctgctcagctgccc 2100
gtctctggcctgggcccctgccccttccaccctgtgcttagaaagtcgaagtgcttggtt 2160
ctaaatgtctaaacagagaagagatccttgacttctgttcctctccctcctgcagatgca 2220
agagctcctgggcaggggtgcctgggccccagggtgtggcaggagacccagtggatgggg 2280
ccagctggcctgccctgatcctctgcttcctcctcacaaccccaagagcccccagcccgg 2340
tccatccacgtctggagtctggggagaggagcagggtcttaggactctcagctctgagca 2400
tccctggcagggtcttcaacctctaatctcttcccttaagccctgtggccacacagccag 2460
gagagacttgccgctggctcccgcctcatttcagcccagggtgctcatccaggggcccag 2520
aacagtcccacctgtgctgctatgcccacagcacaaagccaggcttcactcccaaaagtg 2580
cagccaggccctggagggtgatcctgccagcagccctacagctccacaccctacccaccc 2640
atcggcagcccctctgctgttccccagggacctctcatacactggccaggaggctgcaga 2700
acgtgtgtctccccctccctccaagaggtcctgctccctctgccagaaccgtgtgtgggc 2760
gggtgggagggcgctcggggcccggcccctccctctccctgctggttttagttggtccct 2820
atgttggaagtaaaaagtgaagcactttattttggttgtgtttgctcacgttctgcttgg 2880
aagtggggacccctcactgcgtccacgtgtctgcgacctgtgtggagtgtcaccgcgtgt 2940
acatactgtaaattatttattaatggctaaatgcaagtaaagtttggtttttttgttatt 3000
ttcttttaaaaaaaaaaaaaaaaa 3024
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
9
<210> 17
<211> 1151
<212> DNA
<213> Homo Sapiens
<400> ,
17
ggcacgaggagaacaggggcaatgacacatgctccttggcttcctagtcctgatcccttg60
gggttccttgatcctagggtcatctgacttagatccatcctcccttcctctaggcaccag120
gggccatggctggaggtggccacccctcagtcctgtacagattctgtacccactggctgg180
agacccccatgcagctgtctcctgctcttgctgcggggagactgaactcagggctctgct240
cactggctccctcccaatggaagccttctctgggcttcacagcattgaatattcctccag300
aacagcttgttaaatcaaattttttttttttttagatggagtcttgctctgtcgcccagg360
ctggagtgcagtggcacgatcttggctcactgcaaactccgcctcccgggtttaagcgat420
tctcctgcctcggcctcccgaatagctgggactacaggtgctaccacacccagtaatttt480
tgtatttttagtagaaacggggattcactatgttggccaggctagtctcgaactcctgac540
ctcatgatctgtaatcccagcactttgggaggccgaggcaggtggatcacctaagttcgg600
gagtttgagaccagcctgaccaacatggagaaaccccatctctactaaatatacaaaatt660
aggctgggcgcagtggctcacgcctgtaatcccatcactttgggaggctgagacaggtgg720
gtccaaggtcaggagttcaagaccagcctggccaagatggtgaaaccccatctctactaa780
aaacacacacacacacacacaaaaattagccgggtgtggtggtgggcacctgtaatccca840
gctactagggaggctgaggcagagaactgcttgaacccggaaggcggaggttgcagtgag900
ccaagatcgcgccactgcactccaacctggacaacagggcaagactccctcttgaaaata960
taaaaaataaatatacaaaattagccaggtatggtggcgcatgcctgtggtcccggctgc1020
tcgggaggctgaggcaggagaatcacttgaatccgggagggagaggttgcggtgggctga1080
gattgcgccattgcactccagcctaggcagcaagagggaaactccgtctcaaaaaaaaaa1140
aaaaaaaaaaa 1151
<210> 18
<211> 961
<212> DNA
<213> Homo Sapiens
<400>
18
actagtggatccaaagaattttggcacgagcacaaactgggtggcttaaaccacagaaat 60
gtattgtcttacagttctgggggccagaagtccaaaatcaaggtgtcagcaggcccatgc 120
tccatttgaaggcactggggaggggtctgctccaggcctgtctcctggctactggtggct 180
ccatggcttgtgacagcatcactccaatcttcacacagcattctccccgtgtgcctctga 240
gccccagttttcccttacttcaagagactggggcttttgctccatcacctagactggagg 300
gcagtggcatgatcttggctcactgctgccttgaactcctgtgctcaagtgatcctcctg 360
cctcggccatccagaattgctgggactatagatgtgagccacgacacctggcttcaattt 420
cctctttctataaggatatcggtcgtattggattagggcccgtcctactccagtataacc 480
tcatcttagtgaattacatctgccacaactctgtttccaaataaactcacattctgaggt 540
acagggagttaggacctcaacacaggaattttgggggctaaggtaaggagaaaacaagaa 600
gagtttggtgtcacagaagtgagaatgtccggccaggtatggtggctcaagcctgtgatc 660
ctagcactttgggaggccgaggtgggcagatcacctgaggtgagcagttcaagaccagcc 720
tggccaacatggtgaaactctgtctctactaaaaatacaaaaattagctgggcgtggtgg 780
cagacacctataatcccagctatccgggaa.gctgaggcaggagaatcacttgaacccagg 840
agatggaggctgcagttgcagtgagccaagatcacaccactgcactccagcttgggtgac 900
agagggagatgccgtctcaaaaaaaaaaaaaaaaaaaaaaaaactcgagagtacttctag 960
a 961
<210> 19
<211> 1609
<212> DNA
<213> Homo Sapiens
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
<220>
<221> SITE
<222> (1468)
<223> n equals a,t,g, or c
<220>
<221> SITE
<222> (1567)
<223> n equals a,t,g, or c
<400>
19
cggacgcgtgggggccgagtctggactagtgtactcctctttgcctttgccactggggta 60
cgctccaacgggctggtcagtgttggcttcctcatgcattctcaatgccaaggctttttc 120
tcttctctaacgatgctgaatcctctgagacagctctttaagctgatggcctctctgttt 180
ctgtcggtgttcacacttggccttccctttgccctctttcagtattatgcctacacccaa 240
ttctgtctgccaggctcagcccgccccattcctgagcctttggtacagttagctgtagac 300
aagggctaccggattgcagagggaaatgaaccgccttggtgcttctgggatgttccacta 360
atatacagctatawcyaggatgtctactggaatgttggctttttgaaatactatgagctc 420
aagcaggtgcccaattttctactggctgcaccagtggctatactggttgcctgggcaact 480
tggacatacgtgaccactcacccttggctctgccttacacttgggctgcaaaggagcaag 540
aacaataagaccctagagaagcccgatcttggattcctcagtcctcaggtgtttgtgtac 600
gtggtccacgctgcagtgctgctgctgtttggaggtctgtgcatgcatgttcaggttctc 660
accaggtttttgggctcctccactcctattatgtactggtttccagctcacttgcttcag 720
gatcaagagccgctgttgagatccttaaagactgtgccttggaagcctcttgcagaggac 780
tccccaccaggacaaaaggtccccagaaatcctatcatgggacttttgtatcactggaaa 840
acctgttctccagtcacacgatacattctaggctacttcctgacttactggctcctggga 900
ctactcctacattgcaacttcctgccttggacatgacctggactctccagggacaggttg 960
gaagccaacttaacccaggggtctgaaagtaaaaatacacattggaactgcctctgctgc 1020
cctgggatcattactgtgtccattataatctttctctttctctttgaaagctggtcagga 1080
atgggagaagtgtcagacactagagagccccttctggtcctggctagggcaaattttaga 1140
caactattttctctgtaagtgaagattgtcgtattccaagtctaaaatacacctggatct 1200
gtctagtcaatcaacatagcagagacagtcttaaacctaccattgacctgtgtgtaaatt 1260
taaatgtcaatttattgaagtgtaaatttcatcaaaggcattagctgacaggctggtaac 1320
agtccacacaagatggtataggcctgaacagtgtagtggcagtaataaagtgggaccatt 1380
ttttccaaatgcgcatgttctgatgtttttatgatgtgaggatttattgaaaactcaagt 1440
tcgatactctcagtacatgaaaaaaaanaagaagaaaaaaaaaaagtttttgccgggtcc 1500
tttggttcttcctgggtctgggctcgcctagggacctcgggatggccgcctatcgagggc 1560
tcgcggnggcggtccggtgtttgcccagggtccaaggtctgtccgcttg 1609
<210> 20
<211> 1088
<212> DNA
<213> Homo sapiens
<400>
ctactatagggaamgctkktacgcctgcaggtmccggtccggaattcccgggtcgaccca 60
cgcgtccggaggtggggggcagagtccccaggcagttgggattccggggagggaccacag 120
tgctgggaaccacagcactgacgccatacacaccaggggatgtttgctcttgcatggaaa 180
gtgattttcagtgtcatgctgcagaatcccatacgttatccttcagtccttggtataaag 240
tcctccctgctcagtagtcttgtgttggtgatggtttggggaaatgaaaagagtgggccc 300
tgccccacaccaaagtccaggaagggaagaaggagctgtccagcacaggtggggagggga 360
gaagaggggtcctattgggacccagaattcagactgagcaggaaatccaaccaggggttg 420
aggagggattacctgagcttgtatcacttcaacctgcattttagagacacattctaatct 480
cacagatgcagaatttggggatccctggacagctaacacttggggtgcaaatgtttcttg 540
ttgaatgagaggaactgggtgtagtaaatgaaattcttgcatggaagtctgaatacaact 600
ggtggccgcatcccgaccaacagacaagaggacaaactttcaaaaatgctgtgtcttctt 660
ctcatccttcttttagagtctgtctaccagaattccaccatctgaagtccccggggtgca 720
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
11
aatcctgtttgctgttaattcttccagctctcacgtatggtggcttgttgtgtcacgtgt 780
tttgcaattttgcattgcgcgttccatctttgtcagagctttatccgtgggaatcggtgt 840
gtctaggctgggagtccgtctctccctggggggtgagcttatacttccacgaggcaccag 900
aagacactccaaacccaagactgcttgaagtttagttcccatcttgcatgkagaataaat 960
tcagatccaaaatattcatgagaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagggcggc 1020
cgctctagaggatccaagcttacgtacgcgtgcatgcgacgtcatagctcttctatagtg 1080
tcacctaa 1088
<210> 21
<211> 866
<212> DNA
<213> Homo Sapiens
<220>
<221> SITE
<222> (833)
<223> n equals a,t,g, or c
<400>
21
gaatacgccagctctaatacgactcactatagggaaagctggtacgcctgcaggtaccgg 60
tccggaattcccgggctcttgatctttcctggtcctggtacggggatgacctctctttta 120
gaggggcgaatggttctctgtgtctcctgcctcctccttcccctgctgttgttgctgaaa 180
cactttaatgggttgatgacaccatatttagctcataatgtatattgtcctatagaatat 240
atttcttttttcccatttcatgaaaaaaatatagaatatatttctatttggttcatcttt 300
gacagttttaagtttatctattcgaggctt.ttgtgtatttcccaaatttatgtgctttat 360
agagcatatactttgccccattagatgcagtggtattgcaatagttatcctcagggaggt 420
aggctgtgtgtgtgtgtgggtgtgagtgtatgtatttgtataaaagaaaatccaatatta 480
catgcatttggccgggcacggtggctcacatttgtaatctcagtgctttgggaggctgag 540
gtggacaggtcatttgaggtcaggagttcgagaccagcctggccaacatggtgaaactct 600
gtctctactaaaaattaaaaaattagccaggcgtggtggtgtacatctgtaatcccagct 660
acctgggaggctgaggcaggagaatctcttgaacctgggaggtggaagctgcagtgagcc 720
aagattgcaccactgcactccaggctgggggacagagcaagactccggtctcttcaaaaa 780
aaaaaaaaaaaaaaaaaaaaaaaaaaaaagggcggccgctctagaggatccangcttacg 840
tacgcgtgcatgcgacgtcatactct 866
<210> 22
<211> 1395
<212> DNA
<213> Homo Sapiens
<220>
<221> SITE
<222> (922)
<223> n equals a,t,g, or c
<220>
<221> SITE
<222> (935)
<223> n equals a,t,g, or c
<220>
<221> SITE
<222> (1385)
<223> n equals a,t,g, or c
<400> 22
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
12
gaattcggcacgaggcggcgcgggacctgcagtcgccagggattccctccaggtgacgat 60
gctctggttctccggcgtcggggctctggctgagcgttactgccgccgctcgcctgggat 120
tacgtgctgcgtcttgctgctactcaattgctcgggggtccccatgtctctggcttcctc 180
cttcttgacaggttctgttgcaaaatgtgaaaatgaaggtgaagtcctccagattccatt 240
tatcacagacaacccttgcataatgtgtgtctgcttgaacaaggaagtgacatgtaagag 300
agagaagtgccccgtgctgtcccgagactgtgccctggccatcaagcagaggggagcctg 360
ttgtgaacagtgcaaaggttgcacctatgaaggaaatacctataacagctccttcaaatg 420
gcagagcccggctgagccttgtgttctacgccagtgccaggagggtgttgtcacagagtc 480
tggggtgcgctgtgttgktcattgtaaaaaccctttggagcatctgggaatgtgctgccc 540
cacatgtccaggctgtgtgtttgagggtgtgcagtatcaagaaggggaggaatttcagcc 600
agaaggaagcaaatgtacca.agtgttcctgcactggaggcaggacacaatgtgtgagaga 660
agtctgtcccattctctcctgtccccagcaccttagtcacatacccccaggacagtgctg 720
ccccaaatgtttgggtcagaggaaagtgtttgacctcccttttgggagctgcctctttcg 780
aagtgatgtttatgacaatggatcctcatttctgtacgataactgcacagcttgtacctg 840
cagggactctactgtggtttgcaagaggaagtgctcccaccctggtggctgtgaccaagg 900
ccaggagggctgttgtgaarantgcctcctacgantgcccccagaagacatcaaagtatg 960
caaatttggcaacaagattttccaggatggagagatgtggtcctctatcaattgtaccat 1020
ctgtgcttgtgtgaaaggcaggacggagtgtcscaataagcagtgcattcccatcagtag 1080
ctgcccacagggcaaaattctcaacagaaaaggatgctgtcctatttgcactgaaaagcc 1140
cggcgtttgcacggtgtttggagatccccactacaacacttttgacggtcggacatttaa 1200
ctttcaggggacgtgtcagtacgttttgacaaaagactgctcctcccctgcctcgccctt 1260
ccaggtgctggtgaagaacgacgcccgccggacacgctccttctcgtggaccaagtcggt 1320
ggarctggtgctgggcgaracagggtcagcctgcagcagcacctcaccgtgcgctggtac 1380
ggctngcgcatcgct 1395
<210> 23
<211> 3140
<212> DNA
<213> Homo sapiens
<220>
<221> SITE
<222> (3)
<223> n equals a,t,g, or c
<220>
<221> SITE
<222> (5)
<223> n equals a,t,g, or c
<220>
<221> SITE
<222> (21)
<223> n equals a,t,g, or c
<220>
<221> SITE
<222> (1628)
<223> n equals a,t,g, or c
<220>
<221> SITE
<222> (1642)
<223> n equals a~,t,g, or c
<220>
<221> SITE
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
13
<222> (1656)
<223> n equals a,t,g, or c
<220>
<221> SITE
<222> (2094)
<223> n equals a,t,g, or c
<400>
23
tgntncgagtcacccaggtgnagaatgaggagaaactgaaggagctagagcagatttgat 60
cccaaactctttctkgttttccttcttggacttatgctatttttttgtggagacttgctg 120
agcagaagtcaaattttctactactctactgggatgactgtgggaattgtggcctctctg 180
ctaatcatcatttttatactatctaagtttatgcctaagaaaagtcccatttacgtcatc 240
ctggtgggaggctggtctttttctctgtacctcattcaactagtttttaaaaatttacaa 300
gagatctggaggtgttactggcagtatcttttaagttatgtcctcacagttggattcatg 360
agttttgcagtatgttacaagtatgggcccttggagaatgaacgaagtatcaacctgctg 420
acctggaccttgcagctgatgggcctgtgtttcatgtattctggcatccagataccacat 480
attgcccttgccattatcatcattgctctttgtactaagaacctggaacaccctattcag 540
tggctgtacatcacctgcagaaaggtgtgtaagggagcagaaaagcctgttccccctcgt 600
ctcctgacagaagaagaatatcggatacaaggagaggtagaaacccgaaaggctttagag 660
gagctccgagaattttgtaacagtccagactgctctgcttggaagactgtttctcgaatc 720
cagtctccaaaaagatttgctgactttgtggaaggctcttcccacctcacgccaaatgaa 780
gtttctgtccatgagcaggagtatggattagggagcattattgcccaggatgaaatctat 840
gaggaagcatcctctgaggaggaggactcatattctcggtgtcctgctatcacacagaac 900
aactttctaacctaggtagtggtcagttatctttacgtggactggcttggtgccttggtc 960
catgttgcatgtgttgtgcaattgctttcaaccctttgaaacagagtgagatagataggg 1020
tagaaattctcctactgaaataagaggcctaaaaaggcctccctttggaaatgggaggtc 1080
tctatgggatccctgaggaaggagagtggataaagtagtgaatgctgggtagttcacttc 1140
ccattggttaagctaacagcccacttttatgtttccagagaaattggatggccacagcta 1200
gcatggcattctagctccttcttgaaagttgattcaatcatggcatttctgtcactggct 1260
ggctctccaaagtaagaactgttgttaagtgcaggaatgcttttagactataggctgcaa 1320
cttccagagagaaatccacaaatctgagcctccttcactccagcttttatttcrgtgact 1380
ttagaataattattgatttaactgttttgggaggaaaatagatttttattgttttgtttt 1440
ttaaatgaatgtcttttaaaaamcwtamcaaactcatgttccagaaccagcaagtgctcc 1500
agagtgacacaccccctaggcccctacatatttattaatatggattatccattaaagccc 1560
caggagctgttgttttaagctttgatttagttctcatacatatgatagaaagtcctattt 1620
gcctttangaacatgcctgtangctcttctgcaggntgagatgttctgggctttttatta 1680
tattcaactttcaattccatcttaaaaaacatttgtwttcttctcttcccattcttcctt 1740
accctgcctttgccctttcaggaagggtcagttcccttacctgtgaactatgtatgttca 1800
gagtagcattattcctgctagctaggagaagtcatcttgtttaggggatttggatgcttt 1860
ttatacgttctccattttcctgtcattgggtcatgttatctttgagttgctatgaaatca 1920
ggaaactgtctccttttcctttcccttcctttgtctacatgctctgtccattcctttcag 1980
ccttttctcaccacccatactcccccaaatctgggtaatttttaagccttgaaactatgt 2040
agtttcttgatacacaatttgtagttatgcagcagccacaatttgcattgccanggaaat 2100
argctccaggttatcttcatgcctctgggtgctcattcagctgtcaagtttccatgaact 2160
tacacttatttatgattgcgtttctgacctgagatgtatgctgcctgttattgcagtagc 2220
attagtttcagattcttttgccattgcaaagtaccccttataaaccagcaatgtcatctg 2280
tgaggaagcaaattctcaagtgtctgtcatttacttggttctttttctttgtggtcttca 2340
cccttataccctggaaaagtctgtaattaccttagccaggaagatagatggtcatggcaa 2400
gcgcacagcaccagacttactggctcaccaagatgatggaaaaaggcagatgatttttta 2460
aaaagccgtaatgactcctttagaccagccatttagcgtggtaattttgaaaggcctagc 2520
tccattgcagacttccaaagggtcagctctgagactgccctccaggtgggcagttgatta 2580
tttccaccagtgttttccagagccttaaactgtcctaagtgacaactacctcagttggca 2640
ggaaagagacatatagtagaaagtgaaaaatgagcagtatttgggcagatgctatgggtt 2700
acagttgaagggtaaaaggaactttacattgggaaacctttatacccttgtgaattatgt 2760
acatggtaaaatgttctctctctacaaagaactattaaaacttctgaaatatactatttt 2820
ttaccttatttatagaaattgagacctagcatatttaagcataagtttattttaaaaaat 2880
aattcaactcgtgcaagtggtctcaggattctctggagattttggtgcctcccctactta 2940
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
14
gggaggtgat agcttgccta waagggtgac ttttccygat catgtcttta tttcaatgag 3000
aaagcactgt gaaattgtga aagawtctcc tctttctctg tttaataaac ccccatgaaa 3060
tataaaaaaa aaaaaaaagg gcggccgctc tagaggatcc ctcgaggggc ccaagcttac 3120
gcgtgcatgc gacgtcatag 3140
<210> 24
<211> 2316
<212> DNA
<213> Homo sapiens
<220>
<221> SITE
<222> (39)
<223> n equals a,t,g, or c
<400>
24
cgggggactctcaacgggaaagacttcctatagggaatnctggtacgcctgcaggtaccg 60
gtccggaattcccgggtcgacccacgcgtccggkgagagaagggggccttatttctcgga 120
aaccagccctgctgccctcttcccatggccacaggagcctccccagtgacggagttgtga 180
gattgggggtttctggtgtcctgacttccgtcccagggatcaccttgggtttcccacgtt 240
gcagagactaactgaaaggacatgagggctttaccctgggaatgctctgctggggcaggt 300
gggtgttagctgcgattctgtgttattttcccatcctcagaggtctgcggggtttccgag 360
aggctcgccgggatttctggcggggggctgagagcctggaggctgccctgacccacaacg 420
cagaggttcccaggcgccgggcccaggaggcagaagaggcaggagctgctttgaggacgg 480
ctcgagctgggtaccggggacgggcactggattatgccctgcagatcaacgtgattgagg 540
acaagaggaagtttgacatcatggartttgtgctgcgtttggtggaggcccagsctaccc 600
atttcagcaggccatgaggagctgagccggctgtcccagtatcgaaaggagctgggcgcc 660
cagttgcaccagctggtcttgaattcagcacgagagaagagggacatggagcagagacac 720
gtgctgctgaaacagaaggagctgggtggggaggagccagaaccaagcttaagagagggg 780
cctggtggcctggtgatggaaggacatctcttcaaacgggccagcaacgcatttaagacc 840
tggagcagacgctggttcaccattcagagcaaccaactggtttaccagaagaagtacaag 900
gaccctgtgactgtggtggtggatgaccttcgtctctgcacagtgaaactctgccctgac 960
tcagaaaggcggttctgctttgaggtggtgtccaccagcaagtcctgcctcctccaggct 1020
gactcagagcgcctcctgcagctgtgggtcagtgctgtgcagagcagcattgcttctgcc 1080
ttcagtcaggctcgccttgatgacagcccccggggtccaggccagggctcaggacacctg 1140
gccataggctctgctgccaccctgggctctggtggaatggccaggggaagggagcctggg 1200
ggagtcgggcacgtggtggcccaggtccagagtgtggatggcaatgcccagtgctgcgac 1260
tgccgggagccagccccggagtgggccagcatcaaccttggtgtcaccctctgcattcag 1320
tgttccggcatccacaggagccttggtgttcacttctccaaagtccggtctctgaccctt 1380
gactcatgggagccagaactagtgaagctcatgtgtgagctgggaaatgtcatcatcaac 1440
cagatctatgaggcccgcgtggaggccatggcagtgaagaaaccagggcccagctgctcc 1500
cggcaggagaaggaggcctggattcacgctaaatacgtggagaagaagttcctgaccaag 1560
ctgcctgagattcgagggcgaagaggtggccgggggcgcccaagggggcagcctcctgtg 1620
cccccaaagccttccatcaggccccggccagggagcttgagatccaagccagagcccccc 1680
tctgaggacctgggaagcctgcaccctggggccctactgtttcgagcgtctgggcatcct 1740
ccatctcttcccaccatggctgatgcccttgcccatggagctgatgtcaactgggtcaat 1800
gggggccaagataatgccacaccgctgatccaggccacagctgctaattctcttctggcc 1860
tgtgagtttctcctccagaacggggcgaacgtgaaccaagcggacagtgcgggccggggc 1920
ccgctgcaccacgcaaccattcttggccacacggggctcgcctgcctgttcctgaaacgg 1980
ggagctgatctgggggctcgagactctgaaggcagggaccctctgaccatcgccatggaa 2040
acagccaacgctgacatcgtcaccctgctacgactggcaaagatgagggaggctgaagcg 2100
gcccaggggcaggcaggagatgagacgtatcttgacatcttccgcgacttctccctcatg 2160
gcgtcagacgacccggagaagctgagccgtcgcagtcatgacctccacacgctgtgaccc 2220
gaggcccacggggcccgcgcctgcctcccttccccgccaccgggccctctgccattaaag 2280
cctccgtgcttcgctcttcaaaaaaaaaaaaaaaaa 2316
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
IS
<210> 25
<211> 2304
<212> DNA
<213> Homo Sapiens
<400>
25
ctcgaggtcgacggtatcgataagcttgatatcgaattcggcacgagctggtactatcaa 60
aactcaacagtataaggtatggatttagtatacttattaaatgaaagatatcctataata 120
tgacctgtgaatggattaaataaaaggtatgtacagttataaaattccataayaacagac 180
atgcaatttatttttttattttttaacttcttgccttttaataatatctttgaatataga 240
tggtttcaattctccaataacaagatgtagagtggccaaatggtttttcatacatgtttc 300
cctagtccagacacccaagagtatgtccctctcttgggcataagaaaacctagagttata 360
tttagggcatcatgtgtggtgaatagttgaccaagctggcatgctcacaatgaccaaatt 420
atgtttgtcatattttaatccaatacattgatctactatgaactkgtaaataatctattt 480
gcyttctagacacattccmggaagaccagagaaactgacttttatccatatggaataatc 540
cactggcaaatttcttttaggacagaccaaagctgatctataacataatctttacatact 600
trgatccttcccatcagacgagaaagaacatctggggtgaaaggctccattagaagagca 660
atgtgggatggtsctgtsctgacaactggtagttcctgccctacgtctttgtagaggact 720
ggagcccagagcatgccatggtcaactcgtatgttgaagtgaagacctccttcgaggtgt 780
gcatgtgcttattacagatatgccacggtgctaagtaccccaggactmcatgaaaaaata 840
agaatagatgctgtatccatgatcatgaggcacacaggccagagcgccagctgggaatcg 900
cacagcctgggttcaaagcctggctgggccatgaccacctgaatgacctgaggaatggtc 960
tcaggcaaatttgtaaaaagtggagaccctgcctgccagggaggcatgtggtaagaggtg 1020
catccagtcaggtcagggcactgcgtcctctctttggaacctgcggagcaaggctggtgg 1080
cccttgaggccacggcagccatggagaaggcgggcctggctccaggtggcacagaggcac 1140
tggagaggccccggggaagcctggcgcgatctggctggtcctgcgctctgcttccaggtt 1200
ctggccctgtaacctgggggacagggccggccaaaacagggccactgggtgctagccagc 1260
acytgggccaggcgccaggtggaagggctctggcggatcagcytcgcacccccaacagcc 1320
ccacaggggggcccatccagggccacacacttgcccccaggagcaggatatccctgaggc 1380
tagagtccagctggaccggtggaagggtctcaccctttgccctttgactcctcttgtagg 1440
caccctcgctgggctcctaagcactcctccacacactggctctgtcaccagccccatagt 1500
gatgtcataaactcccagatgcccagtgtgcacccagccacagagaagtgggtgacttag 1560
gagyatcctctccgcttctgacccttagtttcgtctgtgcacaactcactcaaaatgggc 1620
aactcgctaagcgtattttgttcctggttctgccgcaggtcctggccatgccatcggcaa 1680
cctgctcgtcttgtccgtgaggccttcccagctggccgggctcaccctgcagctcctgca 1740
cctgtgcctgcccggggaattgtgggccgtttcccactcctcttcaaccgtcagcgacat 1800
yttgggccttyttttccagtcaggtgggacggcgcccctatgaggctgtgtcttatccct 1860
cggaacacgggcaccccacagagggtcctgcgtcctgtggtctggagccccccctcaagg 1920
aagaaacccgtgctgtctcctcacaactccataatgtttggacacctcagccccgtaagg 1980
atcccttgtctcagaggcaagtttaaccttcaacttccttcattagatgatcaggtgatc 2040
ccagccaggctcccgaagacggaggtgagcgcagaagagcccaaagaagcaacagaggtg 2100
aaagaccaggtagagacccaggggcaggaggacaataaaaggggcccctgtagcaatggg 2160
gaagcagcctccacctctaggcccctggagactcagggaaacctcacttcttcctggtac 2220
aatcccaggcccttggagggaaatgtccacctcaagagcttgacagaaaagaatcagact 2280
gacaaggcccaggtgcatgcagtc 2304
<210> 26
<211> 2346
<212> DNA
<213> Homo Sapiens
<400> 26
gcccacgcgt ccgcccacgc gtccggtgcg ggtttcggtt ggaggactcg ttggggaggt 60
ggcctgcgct tgtagagact gcatccccga gacgatggcg gagggagata atcgcagcac 120
caacctgctg gctgcagaga ctgcaagtct ggaagaacag ctgcaaggat ggggagaagt 180
gatgctgatg gctgataaag tcctccgatg ggaaagagcc tggtttccac ctgccatcat 240
gggtgtggtt tctttggtgt ttctgattat ctactatcta gatccatctg t'tctgtccgg 300
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
16
cgtttcctgttttgttatgtttttgtgcttggctgactaccttgttcccattctagcgcc360
tagaatttttggctccaataaatggaccactgaacaacagcaaagattccatgaaatttg420
cagcaatctagtaaaaactcgacgcagagctgtgggttggtggaaacgcctcttcacact480
aaaggaagaaaaacctaagatgtacttcatgaccatgatcgtttcccttgctgcggttgc540
ttgggtgggacaacaagtccacaacctgcttctcacctacctgatagtgacttccttact600
attgcttcctggactaaaccaacatggaatcattttgaagtacattggaatggccaagag660
ggagataaacaaacttctcaaacaaaaagaaaagaaaaacgaatgattcatctgctttaa720
tcagtgtgattaatgcagcacccattgccccgggaaccgtttctgctgtactatctggat780
actaaaatgttacggaagtagctctttgttctccctcactctgcccttagttaatagaaa840
ttcagactcgccaagtaaggcttcgtgcatagtgtcttcatgtcgcgtatagttgagcgc900
gttcttagcagttggcttcatggacaactcattagtgttttgacttttcttacccagcgt960
taattgaattcttgcttttagacaacttcctttttgtagtggtgaaccttgccctttagt1020
acagttcaagtgaatctggataattgttcatctttgctttagcttagataccatgtagtg1080
gtctgtggctacaggaagctggttctgtctgcttccacagtctgcttaaaaaactgtctg1140
acttcgtgaatatagagaccaagtttaccacttctgatgaagagaccaattaagattcat1200
tcctcattctgtttctttccagtgggagaagagtccccatgaaataagatgaaactgatt1260
ccatgcactagtacatgtaggcttctcccttgtgcaaagcttagcaatttgtaggaaact1320
ttgatctttttgtccaagaaaaggaatgtctgacaggcttaagctttcgtccccttgcac1380
ttagactcgaagttagtaaatccttaaaggctttttaatagcagacttccaaaagattgc1440
atttaggatttctagcatgcttttaatttcagattttcagctgacattagctatagtata1500
cagtaggttaagactcatgtctatgactttcactctaagactggcaaaaggacagcagtc1560
ttctatgtttagtcaatattcatttcagtagaagataatcttatctaatttttgagacca1620
gaataagccttttaaggtaaacctcaaaattatcattttatggtaatactgaccatttta1680
gtcccctaggtttgacatgggagatagtgactacactggtgtctgacttttttcctagag1740
atttctccctgaaaaatacaagggctgttggtgagagcagacttgaggtgatgatagttg1800
gcctctggtctacaaagatttcataactccttggaaagcttcttataatcattcttaact1860
tcttggtagctagaaatttagagtagttgaaatctttaggaatgaacttctgagggccaa1920
aaaatgtgactgacgggaacaattcttaaactgattaactagctgtaatatagttttgtg1980
aatttattgcactgatgttgtaccttgtggtatatctgtccctattaaataagtgttgtt2040
ttctcctctttaatattgctgtgaacagtggtgcccattgtagcatatgtttgatttttt2100
tttattatttcataagaaaactacgttaattttaccttactttcattgtaaataagcctg2160
tcttcctatctggattttttgtgtgcatacatattctactgattaactacttttgcagtt2220
ttaatcctgtattatttcttctactttgttttgtgtaaaaggggaaaaaataaaaaaagc2280
tggaatcttaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagggc2340
ggccgc 2346
<210> 27
<211> 1672
<212> DNA
<213> Homo Sapiens
<400>
27
gaattcggcacgagtgaacctgtggggcaagggggtcttccctgggccccaagagtgcag 60
agatgcctgggtccgcagcctggcagggcagctgcagctttccccagggagcttccgccc 120
tgccaactctgagagggtggggctcctacttgttgctggctcccactggttctgtggagc 180
atgcagcaccagctgcacctcctcatatgctggggcaagggctctaagtccaacacatca 240
tgcctgggcccagtgctaagctgctccaacatgtggagtctggctctgctggtagtggct 300
ggctctatgggggtagcatacagctctgtggtgatgtacgttttgctctgggtgccgctc 360
ccacttccttcccacttccttccttcaggggcccctgaagcacagcccacaacttgggcc 420
cagtccccacactccgtgtgcaagtgtggcaccamcttgggcccagctaaacctcagggc 480
ccctctttgcctamtcctccatgcctgatcatgttgctgtcctgccgcaggcagctaggt 540
ctggccccatcawggtggctgccggggktgggttcccatggtggcgaactccggggatgc 600
tcccaggggtgggctccagggattgcccacctcaatatctgcacctgatgacccttggtc 660
tgtccctgttccccactgcagctgacatcatggtastgatcacttcamacaggctgccgc 720
tgtcctcacatacaatttataatttaaattagaaagaaacataaataaacgacataagat 780
tttgaatataatctatgtaagtaatttcaaaatatgttttattatcttctccatgctaga 840
gaacattttattagtttatattttaactaattttaaaaacgtaaacataaaattacatgt 900
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
17
gttgtcaagctaactcaatcgatgtttgagataaaaattaacctaagaccaaagtatatt 960
attttgatttccttagaggaaaagttatgcagtaattacttgaagagtaattctctctac 1020
mattagcattgcagatgcatccattataccactaaagtacacttaataaataatacttaa 1080
garryyaccagacaccatakggaaaaataaaaataaaaagtcattttgaaaaaagtttag 1140
aaagtaaatcattcctagaatgcttggaaaagtatgtgcctgtgtgccagtatttctgat 1200
cctaagagttatgaatatcttatgaatagttatgaatatcactgatagtgaacctgagtg 1260
taaatgcttgggcttgggktgcagatggactacagatacaaatctaaagtagtgtwccta 1320
gtagcactgtaactcagttaagtacttactttttcatgccctcagtatttttatctgcaa 1380
aataggaataaaatagctaatcatgttatcataagggttaagttatgagatataataact 1440
atgaagtacttaatgggtaaaggaaaaaataagcttagcagttgttgttgttgttgttgt 1500
tgacgaccttatttcaccatatacacttgttagtaactcaaaggggttctccaaagtgaa 1560
atatttaaaaaacgaatgagataaattattattttgttattagaaaatatatttagcaat 1620
ttgtctcactggatcataaaactaattgtggtatgagaacatttataaaaca 1672
<210> 28
<211> 1539
<212> DNA
<213> Homo Sapiens
<400>
28
aaaaacaatgggatgtcatrggataaataatatgagtcaaattttgccagaaagttctgg 60
aaatgtaggacttggttctttttataagtgcttttaaccttttcagactcattaatttca 120
taaaccaatttaagcaattatttctatcagctacagatattttcttcacaaatataaagg 180
gtttgctcccctcccsatcctccagcctccaccaatttgtgcctgtaactgccacttgat 240
agctgctcagtaagactttgtttaaatatcaggttctagtgccaacaaatccagaagtca 300
gaaaaattctaagttacaatcgccagaatatctttcatgcattctattttactttattgt 360
tacagtttaaacaaattcagaaacgtggggtgtttctggctacttgagtaaaacgcactg 420
caaacgcaaagggaacaacgctgtaaacttaaatgatttctgaacagctcaggatcctcc 480
tggaggggatccttttcatctcacatattgattctgaacactatgttagcacctgctccc 540
agaaggtcccttgacactggagcaagtcagaatatcttaatatcatagcttatttaacag 600
caggaaakcaactttaacatagacttgaggtttaggtagatctggtattatctagaaccc 660
aggcttaaattttgcaaaaagtttccaaagagcataagaaagtaaaacagaggtaccaga 720
actgaagacatcattaacaaacaaaatatttccactcctaaccaaactgcaaccctttca 780
gtatagcctgaggtatttttaaacatttgtacaagctaaagatcattcacagtttccaaa 840
gctgctttttactgagatagcttcaaattttcacaatatgtgactttcagacatgttttc 900
atataccagagtctctgccctcagactcatggaaacatcagagatatttagtggcagaaa 960
atggttcttctagtgataagggttctatttcccaattattgtgctattcctaggatgtga 1020
ttaaaaaaatatgaaatttgcaatttgtataaaaggaaaggaaacttgcttaaaattcaa 1080
aaagcaagtccagtctggtggaggtatgtaagatattaaataatagagtcagattctttc 1140
ttgaacattccagatgaacttgacagcagagagttctggttttccaacattttctccatc 1200
ttttcttttccagtggttttactgatctctctttgcttcttactatggatccacgggata 1260
aaacaaagcacagcacctccaataaggggagggactccagcgaggtagaatgccacatca 1320
taggagcccagtttgtcacgaagtaaccctgcaatgggtgggccaacagtcatgggtata 1380
gacatgaatccgagcagaaatccaattgcttgggagacatcctgggcaccaactaactca 1440
aaggctatgggagccataatggaaatgaagcatccatcgaagagacccatgatgaggcac 1500
acagcaatgagggccccaaagatgctacacagaggaatc 1539
<210> 29
<211> 1077
<212> DNA
<213> Homo Sapiens
<400> 29
ggcacgagcg gcacgagccc acagcctgca ggaccgaggc ccctgtgtga gggactgggg 60
gtgtgaggag ttgccaggag aagcagaccc tgccaggtgc ccatctcctc tcagcacccc 120
tccccccaac acacttcctg taatggcgtc tccaatgggg agcccattag tcctgcttag 180
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
18
ggcagcacctaagtactgtggctgataaatataacatgagcccatcgattagaatcctcc 240
tagtcctacagcagctcggctccctaatggctcccctcccctcagcacactgactaccca 300
cctctcaccccccaccccaggctgattttctggtttacttgaggctgtggggcaaacatg 360
agaggagcagaagtgaggtgatgggtgcaacacccagagcccagtcaaaggggccactgc 420
ttggagacaggctggactggtctgggccctgaggcagcctggaagcccctcggtcagtta 480
ggggcagctaggggaggagccgcctgtgagttcccggggccccggccctgcggtaggaca 540
tggagcaaggaaggcagagggcccctactttctgtgaggcgcaagacacagcacctgccc 600
ttgggggcccttgggggctctcctggtaactcctcagagaaacctgcagtgatgccagct 660
ctggccctgtcaccacgccctcttctccctggcatctcctgcccaagcccccgctgcacc 720
tgtcccagcccagccaaccctctctgtcttcatttcacccgtctccagggcgtccacatt 780
tcctcatcagatcagggttctgagggcaggggctgtgtctcctgcatcagcttagggccc 840
ctaaggggtagggactgcttccccagggtgggggctctggaagtcatgaggcttttcccc 900
ccaaaccaccaaatttggactgagaactgcatagggtagggtctgccgcaccccccgccc 960
ccaccataaggtaatcaaaccaattaggccttcagacttgttaaccatcagtgctgggtg 1020
gaagctgagacagcagagtgagacagctctgctacacctccccccagcctgaaaccc 1077
<210> 30
.
<211>
2902
<212> DNA
<213> Homo Sapiens
<220>
<221> SITE
<222> (11)
<223> n equals or c
a,t,g,
<220>
<221> SITE
<222> (12)
<223> n equals or c
a,t,g,
<220>
<221> SITE
<222> (15)
<223> n equals or c
a,t,g,
<220>
<221> SITE
<222> (2782)
<223> n equals or c
a,t,g,
<220>
<221> SITE
<222> (2848)
<223> n equals or c
a,t,g,
<400> 30
ccttttagct nntgnaaaaggctatttaggtgacactatagaaggtacgcctgcaggtac 60
cggtccggaa ttcccgggtcgacccacgcgtccgcccacgcgtccgyyyacgcgtccgcc 120
cacgcgtccg ggctggccctgggcatgggccagggcctcaaagcctggccacggtaccgg 180
gttgtgggct ccgcagacgctgggcagtacaacctggagatcacagatgctgagctctct 240
gacgacgcct cttacgagtgccaggccacggaggccgccctgcgctctcggcgggccaaa 300
ctcaccgtgc tcatccccccagaggacaccaggattgacggaggccctgtgattctactg 360
caggcaggca ccccccacaacctcacatgccgggccttcaatgcgaagcctgctgccacc 420
atcatctggt tccgggacgggacgcagcaggagggcgctgtggccagcacggaattgctg 480
aaggatggga agagggagaccaccgtgagccaactgcttattaaccccacggacctggac 540
atagggcgtg tcttcacttgccgaagcatgaacgaagccatccctagtggcaaggagact 600
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
19
tccatcgagctggatgtgcaccaccctcctacagtgaccctgtccattgagccacagacg660
gtgcaggagggtgagcgtgttgtctttacctgccaggccacagccaaccccgagatcttg720
ggctacaggtgggccaaagggggtttcttgattgaagacgcccacgagagtcgctatgag780
acaaatgtggattattcctttttcacggagcctgtgtcttgtgaggttcacaacaaagtg840
ggaagcaccaatgtcagcactttagtaaatgtccactttgctccccggattgtagttgac900
cccaaacccacaaccacagacattggctctgatgtgacccttacctgtgtctgggttggg960
aatccccccctcactctcacctggaccaaaaaggactcaaatatggggcccaggcctcct1020
ggctccccacccgaggctgctctctctgcccaggtcctgagtaacagcaaccagctgctg1080
ctgaagtcggtgactcaggcagacgctggcacctacacctgccgggccatcgtgcctcga1140
atcggagtggctgagcgggaggtgccgctctatgtgaacgggccccccatcatctccagt1200
gaggcagtgcagtatgctgtgaggggtgacggtggcaaggtggagtgtttcattgggagc1260
acaccacccccagaccgcatagcatgggcctggaaggagaacttcttggaggtggggacc1320
ctggaacgctatacagtggagaggaccaactcaggcagtggggtgctatccacgctcacc1380
atcaacaatgtcatggaggccgactttcagactcactacaactgcaccgcctggaacagc1440
ttcgggccaggcacagccatcatccagctggaagagcgagaggtgttacctgtgggcatc1500
atagctggggccaccatcggcgcgagcatcctgctcatcttcttcttcatcgccttggta1560
ttcttcctctaccggcgccgcaaaggcagtcgcaaagacgtgaccctgaggaagctggat1620
atcaaggtggagacagtgaaccgagagccacttacgatgcattctgaccgggaggatgac1680
accgccagcgtctccacagcaacccgggtcatgaaggccatctactcgtcgtttaaggat1740
gatgtggatctgaagcaggacctgcgctgcgacaccatcgacacccgggaggagtatgag1800
atgaaggaccccaccaatggctactacaacgtgcgtgcccatgaagaccgcccgtcttcc1860
agggcagtgctctatgctgactaccgtgcccctggccctgcccgcttcgacggccgcccc1920
tcatcccgtctctcccactccagcggctatgcccagctcaacacctatagccggggccct1980
gcctctgactatggccctgagcccacaccccctggccctgctgccccagctggcactgac2040
acaaccagccagctgtcctacgagaactatgagaagttcaactcccatcccttccctggg2100
gcagctgggtaccccacctaccgactgggctacccccaggccccaccctctggcctggag2160
cggaccccatatgaggcgtatgaccccattggcaagtacgccacagccactcgattctcc2220
tacacctcccagcactcggactacggccagcgattccagcagcgcatgcagactcacgtg2280
taggggccagagcctggctggggcatctctgcggggcagaggagaaggctttcacagctg2340
ttccctgatattcaggggcattgctcattgctcccttctcggaccagccttcttcctccc2400
accatggcaggtggggagcaggtctcccagaaacaccccgtcccgaggatggtgctctgt2460
gcatgccccagcctcctgggcctgcccttccctcttcttcgggaggatgtgtctcttctg2520
acctgcactcttgcctgaccctagaatggggacagggaaagtgaaggttagggaaagcag2580
aggggggcactttttagcattccctttctatcccacccctctgatctcccataagtggaa2640
atgggggtacccagggatgggcaggctttggcctagggacatgaagtatgggagtgggtg2700
gctgwggcacagacaggtggaaaacgggatagcctggcagtccctctgttgctgcattcg2760
tgcctgggtgcctctcttcttnctcaggtactgcagaagggagcgaacagggtactgtcg2820
ctcttgctacagacaagcctggcactgnattcaaatcagcttcattactggatcaaatgc2880
agtaccttgctaccatgggaca 2902
<210> 31
<211> 2508
<212> DNA
<213> Homo sapiens
<220>
<221> SITE
<222> (1074)
<223> n equals a,t,g, or c
<400> 31
ggcacgagca gcgcgcactt accaccatgc agatccaggt cgcggggctg ctgcagtttg 60
ccgtgcccct cttctccact gcagaggaag acctgcttgc aatccagctc ctgctgaact 120
cctcagagtc cagccttcac cagctgaccg ccatggtgga ctgccgaggg ctgcacaagg 180
attatctgga cgctcttgct ggcatctgct acgacggcct ccagggcttg ctgtaccttg 240
gcctcttctc cttcctggcc gccctcgcct tctccaccat gatctgtgca gggccaaggg 300
cctggaagca cttcaccacc agaaacagag actacgatga cattgatgat gatgacccct 360
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
ttaacccccaagcctggcgcatggcggctcacagtcccccgaggggacagcttcacagct 420
tctgcagctacagcagtggcctgggaagtcagaccagcctgcagcccccggcccagacca 480
tctccaacgcccctgtctccgagtacatgaaccaagccatgctctttggtaggaacccac 540
gctacgagaacgtgccactaatcgggagagcctcccctccgcctacgtactctcccagca 600
tgagagccacctacctgtctgtggcggatgagcacctgaggcactacgggaatcagtttc 660
cagcctaacagactttcgggggttcctgcctcctttttccgttctggtttttaattagtg 720
caaatacaagctgcgtttctttaatagaaaccaaaggcatctggagcccgagaggcctcc 780
tgctgtggcagaggagcagctgggattcccgaccaaagccccagggggtgcagaagactc 840
accacgcgggccagcctctctcttttgccctgctctccacaccagaaatgcccccaggtg 900
cttggctgcctcagaggtaccatccctgagctggctgcctggccctgctcacccctacgc 960
ctcgcccttgccaggaggggagtggcagtgaggagggggccaggtcaggcaccaccatca 1020
agagagctgtgtgttctctctggtcccacaacgatgactctgcctctttgggtncaggcc 1080
ccaggcccaaagagcccagacgacccctctgtcctcgttccctgtcctcgttccctgcag 1140
gtaacatgagaagggctgatcaggagatgctctttaagaagttcgcacccctgctgacac 1200
cagaacagcccaaatcagagttcccagggccagacaggctcttcctgggccacagagggg 1260
aggcatcaggaaagctctgcagtggggggctggtggctccggggctgggggatcacaggc 1320
tggtgaaccccggtgggaacagaggtgaaagcctgccacattccgcctgtctccctaacc 1380
ctccattgcc~tcgcctctattccagaatcaatgctgcagaatgtgttagctgcagatagg 1440
catggtctcaggtatgaacagacactttgaaacgactttaggtctttcttttctccagtg 1500
ttttaaacatgttgattatccaaagaattgaaactcctagcacatccagtttttacaaca 1560
gatttgcagctcattccttaccctggttaggtcactacttttgcagattttgctggcact 1620
gatctggagatctgcagatctggaggagacgggaaggagtcgattcttaaataaggatca 1680
gtgaggcatcctgtcccaagctactgtttggtggggatctgggttcatctcacccacaga 1740
gggaggatctttaagaggagaaaaaagccaagagggaaagccagagttccctgttctagg 1800
ggactagccaaatgcctacatcagctgtcccctccctgttgtctccaagtaagtttgcca 1860
gaaaaggttttagcaaagtgctacaactgtgtctttataggaggataggcctctgccctg 1920
ccccacccccaccacctgtccccacccagtgtcccaggccacaggagcttattggccagg 1980
agggaataatgtcccccaatactgcctgttgagggaccagagttggggtctttggtgctt 2040
ccaacctcctgccaacctggagttcacaacaccagagccccacggcctcgcacactgaag 2100
caggggcgtgcggtgactcggtgcttctgttttggaagaaccacctgtcatcaaaacatg 2160
gacagcagggtgttctcagctcccagcgaagcctccacaacagaatggggccacagggca 2220
gccgggactccctgtctcacctacattaacccatgcatactgtatgccataaactcactt 2280
tggtatatccgcgtcacatgcagagaggaactctgcgacgtcaaagtgttgcttcttaaa 2340
gtttcattattggcaactagagggttgtttttaatgcatggaaactaaacagattcctcg 2400
gggagttcctgaaggaaccaggtgggcaaacctttgcttatatacatgcggcctcacctg 2460
gaagagaaataaaccacttgtactaaaaaaaaaaaaaaaaaaaaaaaa 2508
<210> 32
<211> 2114
<212> DNA
<213> Homo sapiens
<220>
<221> SITE
<222> (2108)
<223> n equals a,t,g, or c
<400>
32
ggctaggtggcgcgcgggtccggcgggcggttggcttgagcgggaccggagctgaggcag 60
gaagagccggcgccatggtggagaaggaggaggctggcggcggcattagcgaggaggagg 120
cggcacagtatgaccggcagatccgcctgtggggactggaggcccagaaacggctgcggg 180
cctctcgggtgcttcttgtcggcttgaaaggacttggggctgaaattgccaagaatctca 240
tcttggcaggagtgaaaggactgaccatgctggatcacgaacaggtaactccagaagatc 300
ccggagctcagttcttgattcgtactgggtctgttggccgaaatagggctgaagcctctt 360
tggagcgagctcagaatctcaaccccatggtggatgtgaaggtggacactgaggatatag 420
agaagaaaccagagtcatttttcactcaattcgatgctgtgtgtctgacttgctgctcca 480
gggatgtcatagttaaagttgaccagatctgtcacaaaaatagcatcaagttctttacag 540
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
21
gagatgtttttggctaccatggatacacatttgccaatctaggagagcatgagtttgtag 600
aggagaaaactaaagttgccaaagttagccaaggagtagaagatgggcccgacaccaaga 660
gagcaaaacttgattcttctgagacaacgatggtcaaaaagaaggtggtcttctgccctg 720
ttaaagaagccctggaggtggactggagcagtgagaaagcaaaggctgctctgaagcgca 780
cgacctccgactactttctccttcaagtgctcttaaagttccgtacagataaaggaagag 840
atcccagttctgatacatatgaggaagattctgagttgttgctccagatacgaaatgatg 900
tgcttgactcactgggtattagtcctgacctgcttcctgaggactttgtcaggtactgct 960
tctccgagatggccccagtgtgtgcggtggttggagggattttggcacaggaaattgtga 1020
aggccctgtctcagcgggaccctcctcacaacaacttcttcttcttcgatggcatgaagg 1080
ggaatgggattgtggagtgccttggccccaagtgaactcaagatttggcagccccagaga 1140
tgccaactgcagcatgcccacctgtattccctgtccccttccttcatgaaggcatctcca 1200
ggcaaggaaaactgaagtcattggcccgatacaaaacatttcctgcaacgaaggaggtgg 1260
tgccgacgtgctgcttcccatcaccagcagctgctcgacaaggggcgcagggtggctgtc 1320
tttgttccagcactgttcaggctgcctgtcatcccgggcctgccagctcccctgagtgat 1380
gagcacttccaagcacccctctgccctttctctgtccttatgctgtcccggcctcgccag 1440
ccctctggggcattgtgggagatgcctgccaggaatgagcaagctctgttgctcgggagc 1500
ctcttgtcaccttcttggacttattccccacctgataccttatagagaaaagtgtgaatt 1560
caggtggagagtaggcccaggccccatgaggcaccagtggaagcacagctccaagttcag 1620
acaggtgcccttagagaggaaaaccatgacaggcaaatgcatttcctctggagtttgaga 1680
ccctgacaaacaacaggtggcatctggtgtgctgttcttgagttttcgtttaggattagt 1740
tgagttccagctgggttttgggagaaaggagatgctaccaagtcttggatgttagggcga 1800
gaccctgcaagttgagtattagagagcttgtctttcaaggcaggttcctggggcttcagg 1860
gctaggagggaggagcctgcccttttaacagaaccccagtcacatgcggctcaagtcact 1920
cagaggctgttgcatttcagggctatgttggtcctttgtttacctcctaaaccacagctg 1980
tttgtgtttcacatatgttgtgaattttccttggttctttttaaaggaatgataataaag 2040
ttacttgctttaggaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 2100
aaaaaaanaaaggg 2114
<210> 33
<211> 2304
<212> DNA
<213> Homoapiens
S
<220>
<221> SITE
<222> (2273)
<223> n ls a,t,g,
equa or c
<220>
<221> SITE
<222> (2278)
<223> n ls a,t,g,
equa or c
<400> 33
atgaatacgccagctcgaaattaaccctcactaaagggaacaaaagctggagctccaccg 60
cggtggcggccgctctagaactagtggatcccccgggctgcaggaattcggcacgagctt 120
gtcgtgtggctctgtgtcgacactgtgcgccaccatggccgtgactgcctgtcagggctt 180
ggggttcgtggtttcactgattgggattgcgggcatcattgctgccacctgcatggacca 240
gtggagcacccaagacttgtacaacaaccccgtaacagctgttttcaactaccaggggct 300
gtggcgctcctgtgtccgagagagctctggcttcaccgagtgccggggctacttcaccct 360
gctggggctgccagccatgctgcaggcagtgcgagccctgatgatcgtaggcatcgtcct 420
gggtgccattggcctcctggtatccatctttgccctgaaatgcatccgcattggcagcat 480
ggaggactctgccaaagccaacatgacactgacctccgggatcatgktcattgkctcagg 540
tctttgtgcaattgctggagtgtctgtgtttgccaacatgctggtgactaacttctggat 600
gtccacagctaacatgtacaccggcatgggkgggatggtgcagactgttcagaccaggta 660
cacatttggtgcggctctgttcgtgggctgggtcgctggaggcctcacactaattggggg 720
tgtgatgatgtgcatcgcctgccggggcctggcaccagaagaaaccaactacaaagccgt 780
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
22
ttcttatcatgcctcaggccacagtgttgcctacaagcctggaggcttcaaggccagcac840
tggctttgggtccaacaccaaaaacaagaagagatacgatggaggtgcccgcacagagga900
cgaggtacaatcttatccttccaagcacgactatgtgtaatgctctaagacctctcagca960
cgggcggaagaaactcccggagagctcacccaaaaaacaaggagatcccatctagatttc1020
ttcttgcttttgactcacagctggaagttagaaaagcctcgatttcatctttggagaggc1080
caaatggtcttagcctcagtctctgtctctaaatattccaccataaaacagctgagttat1140
ttatgaattagaggctatagctcacattttcaatcctctatttctttttttaaatataac1200
tttctactctgatgagagaatgtggttttaatctctctctcacattttgatgatttagac1260
agactccccctcttcctcctagtcaataaacccattgatgatctatttcccagcttatcc1320
ccaagaaaacttttgaaaggaaagagtagacccaaagatgttattttctgctgtttgaat1380
tttgtctccccacccccaacttggctagtaataaacacttactgaagaagaagcaataag1440
agaaagatatttgtaatctctccagcccatgatctcggttttcttacactgtgatcttaa1500
aagttaccaaaccaaagtcattttcagtttgaggcaaccaaacctttctactgctgttga1560
catcttcttattacagcaacaccattctaggagtttcctgagctctccactggagtcctc1620
tttctgtcgcgggtcagaaattgtccctagatgaatgagaaaattattttttttaattta1680
agtcctaaatatagttaaaataaataatgttttagtaaaatgaaaaaaaaaaaaaaaaaa1740
ctcgagggggggcccggtacccaattcgccctatagtgagtcgtattacaattcactggc1800
cgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgc1860
agcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttc1920
ccaacagttgcgcagcctgaatggcgaatggcaaattgtaagcgttaatattttgttaaa1980
attcgcgttaaatttttgttaaatcagctcattttttaaccaataggccgaaatcggcaa2040
aatcccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaa2100
caagagtccactattaaagaacgkggactccaacgtcaaagggcgaaaaaccgtctatca2160
gggcgatggcccactacgtgaaccattaccctaatcaaggttttttggggtcgaggtgcc2220
gtaaagcactaaatcggaaccctaaagggagccccgattaaagcttgacgggnaaagncg2280
gcaacgtgcgagaaggaaggagaa 2304
<210>34
<211>1323
<212>DNA
<213>Homo
Sapiens
<220>
<221>SITE
<222>(1133)
<223>n equalsa,t,g,or
c
<220>
<221>SITE
<222>(1183)
<223>n equalsa,t,g,or
c
<220>
<221>SITE
<222>(1202)
<223>n equalsa,t,g,or
c
<220>
<221>SITE
<222>(1282)
<223>n equalsa,t,g,or
c
<220>
<221>SITE
<222>(1302)
<223>n equalsa,t,g,or
c
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
23
<220>
<221> SITE
<222> (1321)
<223> n equals a,t,g, or c
<400>
34
gtgcatgcgctcgcatcatggcggctgagtgggcttctcgtttctggctttgggctacgc 60
tgctgattcctgcggccgcggtctacgaagaccaagtgggcaagtttgattggagacagc 120
aatatgttgggaaggtcaagtttgcctccttggaattttcccctggatccaagaagttgg 180
ttgtagccacagagaagaatgtgattgcagcattaaattcccgaactggggagatcttgt 240
ggcgccatgttgacaagggcacggcagaaggggctgtggatgccatgctgctgcacggac 300
aggatgtgatcactgtgtccaatggaggccgaatcatgcgttcctgggagactaacatcg 360
ggggcctgaactgggagataaccctggaca.gtggcagtttccaggcacttgggctggttg 420
gcctgcaggagtctgtaaggtacatcgcagtcctgaagaagactacacttgccctccatc 480
acctctccagtgggcacctcaagtgggtggaacatctcccagaaagtgacagcatccact 540
accagatggtgtattcttacggctctggggtggtgtgggccctcggagttgttcccttca 600
gccatgtgaacattgtcaagtttaatgtggaagatggagagattgttcagcaggttaggg 660
tttcaactccgtggctgcagcacctgtctggagcctgtggtgtggtggatgargctgtcc 720
tggtgtgtcctgacccgagctcacgttccctccaaactttggctctggagacggaatggg 780
agttgagacagatcccactgcagtctctcgacttagaatttggaagtggattccaacccc 840
gggtcctgcctacccagcccaacccagtggacgcttcccgggcccagttcttcctgcact 900
tgtccccaagccactatgctctgctgcagtaccattatggaacgctgagtttgcttaaaa 960
acttcccacagactgccctagtgagctttgccaccactggggagaagacggtggctgcag 1020
tcatggcctgtcggaatgaagtgcagaaaactagcagttctgaagatgggtcaatggggg 1080
arcttttcggagaagtctaagttcaaaggactcttctggcttgcttcaatcanacctaca 1140
ccatttaacctaatacctcgtggaagacaggtcggcgggctgnttggacaccacgataac 1200
cnttaagcctggaacagaaccgggacttcggcctgaaccggctggattttccagggggtt 1260
cttgaaaaaaggatgacttcangggcttaccgggcttttggnccaaaccagaaggatcat 1320
ngg 1323
<210> 35
<211> 2737
<212> DNA
<213> Homo Sapiens
<220>
<221> SITE
<222> (2730)
<223> n equals or c
a,t,g,
<220>
<221> SITE
<222> (2731)
<223> n equals or c
a,t,g,
<220>
<221> SITE
<222> (2734)
<223> n equals or c
a,t,g,
<400> 35
ggctcggaag ccccggagtcagattaagaatgagatcaacattgacaccctggccagaga 60
tgaattcaac ctccagaagatgatggtgatggtaacagcctcaggcaagctttttggcat 120
tgagagcagc tctggcaccatcctgtggaaacagtatctacccaatgtcaagccagactc 180
ctcctttaaa ctgatggtccagagaactactgctcatttcccccatcccccacagtgcac 240
cctgctggtg aaggacaaggagtcgggaatgagttctctgtatgtcttcaatcccatttt 300
tgggaagtgg agtcaggtagctcccccagtgctgaagcgccccatcttgcagtccttgct 360
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
24
tctcccagtcatggatcaagactacgccaaggtgttgctgttgatagatgatgaatacaa 420
ggtcacagcttttccagccactcggaatgtcttgcgacagctacatgagcttgccccttc 480
catcttcttctatttggtggatgcagagcagggacggctgtgtggatatcggcttcgaaa 540
ggatctcaccactgagctgagttgggagctgaccattcccccagaagtacagcggatcgt 600
caaggtgaaggggaaacgcagcagtgagcacgttcattcccagggccgtgtgatggggga 660
ccgcagtgtgctctacaagagcctgaaccccaacctgctggccgtggtgacagagagcac 720
agacgcgcaccatgagcgcacctttattggcatcttcctcattgatggcgtcactgggcg 780
tatcattcactcctctgtgcagaagaaagccaaaggccctgtccatatcgtgcattcaga 840
gaactgggtggtgtaccagtactggaacaccaaggctcggcgcaacgagtttaccgtact 900
ggagctctatgagggcactgagcaatacaacgccaccgccttcagctccctggaccgccc 960
ccagctgccccaggtcctccagcagtcctatatcttcccgtcctccatcagtgccatgga 1020
ggccaccatcaccgaacggggcatcaccagccgacacctgctgattggactaccttctgg 1080
agcaattctttcccttcctaaggctttgctggatccccgccgccccgagatcccaacaga 1140
acaaagcagagaggagaacttaatcccgtattctccagatgtacagatacacgcagagcg 1200
attcatcaactataaccagacagtttctcgaatgcgaggtatctacacagctccctcggg 1260
tctggagtccacttgtttggttgtggcctatggtttggacatttaccaaactcgagtcta 1320
cccatccaagcagtttgacgttctgaaggatgactatgactacgtgttaatcagcagcgt 1380
cctctttggcctggtttttgccaccatgatcactaagagactggcacaggtgaagctcct 1440
gaatcgggcctggcgataaagaacaaagactgtgcctaaaagtggagagccaggggagtg 1500
tgggtcagataagcagctacagctgcagtttggtggattggtggagtatgtgtgtgtgtc 1560
agtgctcagctaagaactgtagggaagatggatgaccttcacgcagaactccttttggga 1620
tatacatgatgcagaaaggatcctacatggagagagacagaactctctcagctgacactc 1680
tcagagattcctgatgggctttctcttgaagtccaaaggcgtctgcattgtttcctttct 1740
ttgcccatccatgaatgttctgttttgttttgttttttttaataagaattccggctgatt 1800
tttgtgaggcctgtttaaattgactttactttgccttttgtgtttctcaattttatctag 1860
aaatctttctgactttttccatctcttgcttcaaagtaagaggggaactctccttgccga 1920
ctccaccttataggtacatttggtgttttgcactgggaagaaataggatccatccttagc 1980
tgaggcttgaggactgatccagcctctcatggcttccctccaaagtaacttagggttgag 2040
ggatctatatgtgatgtcaaaacttactttaaacctctagtttcgtgctgtcatttatta 2100
ggctgggccaccaaatctttgtttcaatttatcagaagccaagtgcatactagcgtcttg 2160
tttgttgcccattgcctatacttttcacctgagatgtgtgagttggggccttttaaaaac 2220
tactgaattgtctgagccttgaagacatttccagggagaagagataatctctcatttcac 2280
ccacaggctggtctaatcataacctagttaaagatgtccttgtttaagaaccccattatt 2340
tatttttagtttttaatataaattaacatgtgggtcattatatttctccttaaatgagga 2400
aattttaaattttattgatctaacctttgaagctttaaaaaaggagaaagagggtagggg 2460
tgggaaactggcatactgtgtgtatagcactgccgattggctaggccactgtgtctctgc 2520
tacaaattaaagaaatcctaaaagttttccttggtcatagagttggggaatgacagaatt 2580
tttctttgttgtgaaatgtatgtacagagtagaccatctctagccctgtggtgaaagagg 2640
tacactcgaatgtttgcataaagcaagtgacaaatgaaaaaaaaaaaaaaaaaaaaaccc 2700
cgggggggggcccggtaacccatttggccnnaanggg 2737
<210> 36
<211> 1507
<212> DNA
<213> Homo sapiens
<220>
<221> SITE
<222> (486)
<223> n equals a,t,g, or c
<220>
<221> SITE
<222> (1237)
<223> n equals a,t,g, or c
<400> 36
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
ggggtcaaccacgagagctggcgtgacagtggccagactgacggcctgacctttggccac60
ctgaagatggtgctgctgtgggcctctgtgctcttcccggcgcccgaggactgggcagag120
ctgcagggcgccgtgtaccgcctgctggtggtgctgctctgttgcctggccacgcggaag180
ctgccccact.tcctccacccgcagcgcaacctgctgcagggcagcggcctggaccttggt240
gccatctaccagcgcgtggagggcttcgccagccagcccgaggcggccctgcgcatccac300
gccacccacctgggccgcagccccccgccgcgcatcggctccgggctcaaggcgctcctg360
cagctgccagccagtgaccccacttactgggccactgcctacttcgacgtcctgctggac420
aagttccaggtcttyaacatccaggataaggaccggatctctgccatgcagagcatcttc480
cagaanaccaggactctgggaggcgaggagagctgagctgggccacctggtctcagccac540
ctgttcttggctccccaacagactctgcactgcaccatgggaggctcctgggatgtttgg600
aagaagaaacgggcttctccttgagggggtagtggagggattttgtccccagcagtggcc660
tctgagagtctttcagtgcctggtggggcagggcaggcctcttggagcacctcctccctg720
ggtcagggcctggatgcaggtgccaagctctccatgtggtgcatgttgacccagccacgt780
ggtgttgtcaagcaaacagcatcggcaggagacctggagctgaggacttggccctgcctg840
cactgtatgccattccttggtgacraaatgctgtatatttggttttgaaaaaatgaatgt900
gctgggtatacacagcagaaagggtactgtccactttttgtatatcagtgtggaaaatat960
ttcccctagaagtagaaaaggctcatgtgctgatggataattttgagtcttctccattct1020
ctgtgaatgacccccttccccaggcatccccacctcctacctcgttcttagagcaaacta1080
aagccaactgagggtgcacacacagccatgagcccacctgcccaggactactcccatctg1140
cttcttcccgtccccgtggaagtggcccctgatatggattgtatctgtatccccacccaa1200
atctcaggtagaattggatctgtgtccccacccaagntytcaggtagacttgtaatcccc1260
agtgttggaggaggggcccagtggaggtgattggatcatgggggtggatttctcccttac1320
tggtctcatgatagtgagtgagttctcagagatctggktttttgagtgtgtggccatccc1380
gtctcttttgcctcttctctttctcggcatgtagaatgctcatctcttcacttcaccaga1440
tgcaagtccgagctccagcaagttccgatagctcggatggagcaataaccctgaggataa1500
aaaagaa 1507
<210>
37
<211>
872
<212>
DNA
<213> sapiens
Homo
<220>
<221>
SITE
<222>
(651)
<223>
n equals
a,t,g,
or c
<220>
<221>
SITE
<222>
(750)
<223>
n equals
a,t,g,
or c
<400>
37
tatttaggtgacactatagaaggtacgcctgcaggtaccggtccggaattcccgggtcga 60
cccacgcgtccggcgggggcatcgccgcccgcgcccctctaagtgccgggccgcaagctc 120
caccgcagccgcctgcaagcagcggcgcctcggccctcgacctgcgcgcaaagcctgtgc 180
tggagccgtcctcccgcggcggggaccgggaccggggacccaagccaatcgaaagctcca 240
accatggccatggggctcttccgcgtgtgtctggtggtggtgacggccatcatcaaccac 300
ccgctgctgttcccgcgggagaacgccacagtccccgagaacgaggaggagatcatccgc 360
aagatgcaggcgcaccaggagaagctgcagctggagcagttgcgcctggaggaggaggtg 420
gctcggctggcggccgaaaaggaggcactggagcaggtggcggaggagggcaggcagcag 480
aacgagacacgcgtggcctgggacctctggagcaccctctgcatgatcctcttcctgatg 540
atcgaggtgtggcggcaggaccaccaggaggggccctcacctgagtgcctgggcggtgag 600
gaggatgagctgcctggctggggggcgcccccttgcagggcctcaccctgnccaacaagg 660
cacgcttgccacttttatgagcgctgcatccggggggccacggccgatgcagcccgtacc 720
cggragttcctggaaggcttcgtggatganttgctggaagccctgaggagcctctgcaac 780
cgggacaccgacatggaggtggaggacttcattggcgtggacagcatgtacsagaactgg 840
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
26
caggtggaca ggccactgct gtgccacctt tt 872
<210> 38
<211> 1816
<212> DNA
<213> Homo Sapiens
<220>
<221> SITE
<222> (1807)
<223> n equals a,t,g, or c
<400>
38
ggcggccgctctagaactagtggatcccccgggcaggaattcggcacgagcgtgatcatc 60
gggatgctcgtgctcctgctggactttcttggcttggtgcacctgggccagctgctcatc 120
ttccacatctacctgaaggccaagaagatgaccacctttgagtatctcattaataaccgc 180
aaagaagagagttcaaaacatcaagcagtgaggaaagatccatacgtgcaaatggacaaa 240
ggagttctccagcaaggagctggcgccctgggctcatctgcacagggagtcaaagccaag 300
agctccctgctgattcacaagcacttatgtcacttctgcacttcagtaaaccaggatggg 360
gattcgacggcacgggtgcatttgtgagtcccctcagtgtctctgcagcttctatgtgtg 420
gatgaatagtgaagccacatgaggcctggtctgaagcagagaagatccgctcagcatcat 480
gttgaatcccaagcccgcgtctccgtgggctccaggacaatcctatgaaagtgacaccgt 540
cgttcattgttcacatcggggaggagaattccatctgaaaatgcacgtgacttcactgac 600
acccgcgtccgtggcacagccctgtgctgagctccacagacctacagtccatcgcctccc 660
cttggagtgggcccacagctgcagacagtgttgtccaaagaagatgctctgagtagatga 720
atggaacaaactggaaactccagaaacaagcccttctttacattyctaatgtttggctta 780
aatggaaggtgccaagagccaaggccaggaactcccggctgatttgcaggcgcctgtgtc 840
agttctccactcgtgtacacccagacgggggctcgatggcacaggaagcagatgatgccc 900
cgagtatatctacacttgggctgcaacaagaaacaacagagcccatgaaaactgacagtg 960
ctgaaagtgaagactgagattcaggagctsaggtgcccctgtgatccaggtcttcyaccc 1020
tgaaaccccaycctccatcaaggtcctgcctgtagagtctaccttgcaaagcctcctgct 1080
cctacccatgctacaggccaggaaccagagcccatcatctcagaggcccctggatgtcct 1140
tcgaaggaaccaggaccctcagagcccagcatccatctctgtcatcatcttcatcacacc 1200
caaagaagagccagccttgcaggagggtttacatctccaggaagatgggctgccagcaac 1260
tgcagaggatgcagccacctgcttaactgtgctgtccagccagccagccagctgcagggc 1320
ctcttgctgcttaagagctgatgggccgggcatgttggctcacacctgtgagcacagtac 1380
tgggaaatgggagcacagtactaggaaatgggagcacagtactgggaaatgggagcacag 1440
tactgggaaatgggggctcacagcactgcaaaatgggagcacagtattgggaaatgggag 1500
cacagtactgggaagtgggagcacagtactgagaagtgggagcacagtactgagaaatgg 1560
gagtacactactgagaaatgggagcacagtactgggaaatgggcatacagtactggggaa 1620
atgggcatacagtactgaraaatgggarcacagtactgggaaatgggagcacagtactgg 1680
ggaaatgggarcccacagtwctgggaaaagggartttcacattactccggaaattgggar 1740
cmtaktactggggaawtgggagcacrttactggggaaatgggaaacattattactgggga 1800
aaccccngaacctgga 1816
<210> 39
<211> 631
<212> DNA
<213> Homo Sapiens
<220>
<221> SITE
<222> (337) '
<223> n equals a,t,g, or c
<220>
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
27
<221> SITE
<222> (338)
<223> n equals a,t,g, or c
<400> 39
gggaaatgggagctcacagcactgggaaatgggagcayagcaytgggaaatgggagcaca 60
gtactgggaattgggagcacagtactgggaaatgggagctcacagtactgggaaatggga 120
gctcacagtactgggaaatgggagcacagtattcggaaatgggagcacagtactgggaaa 180
tgggagctcacagcactgggaaatggagcatagcattgggaaatgggagcacagtactgg 240
gaaatggaagctcacagtcctgrgaaatgggagcayagtrctgggaaatgggagcacagt 300
actgrgaaatgggagcacagtactgggaaakgggagnncmcagtactgmggaaatgggag 360
cayagtactgggaaatgggagcacagtactgggaaatgggagcatagtactgggaaaccc 420
cagacctggattctgagtttttcagcctagcccagacttcttatcttagtagacaaaaag 480
agtcaataccagagaaccagaggcatcctctgtattttaatgaactctgcattttaatct 540
gtttagtagtcattttttaaaagataatcagttttccaaatatatctataagttactacg 600
tgcaaaaaaaaaaaaaaaaaaaaaaactcga 631
<210>
40
<211>
1186
<212>
DNA
<213> sapiens
Homo
<220>
<221>
SITE
<222>
(2)
<223> als a,t,g,or c
n equ
<220>
<221>
SITE
<222>
(31)
<223> als a,t,g,or c
n equ
<220>
<221>
SITE
<222>
(87)
<223> als a,t,g,or c
n equ
<220>
<221>
SITE
<222>
(92)
<223> als a,t,g,or c
n equ
<400>
40
cnaaacgcttcatttttaggcgtttgggtcngttctccactcgtgtacacccagacgggg 60
gctcgatggcacaggaagcagatgatnccccntgtatatctacacttgggctgcaacaag 120
aaacaacagagcccatgaaaactgacagtgctgaaagtgaagactgagattcaggagctc 180
aggtgcccctgtgatccaggtcttctaccctgaaaccccaccctccatcaaggtcctgcc 240
tgtagagtctaccttgcaaagcctcctgctcctacccatgctacaggccaggaaccagag 300
cccatcatctcagaggcccctggatgtccttcgaaggaaccaggaccctcagagcccagc 360
atccatctctgtcatcatcttcatcacacccaaagaagagccagccttgcaggagggttt 420
acatctccaggaagatgggctgccagcaactgcagaggatgcagccacctgcttaactgt 480
gctgtccagccagccagccagctgcagggcctcttgctgcttaagagctgatgggccggg 540
catgttggctcacacctgtgagcacagtactgggaaatgggagcacagtactaggaaatg 600
ggagcacagtactgggaaatgggagcacagtactgggaaatgggggctcacaggactgca 660
aaaatgggagccagtattgggaaaatgggaagcacagtacttggggaagtggggagcaca 720
gtactgagaagtgggagcacagwactgagaaatgggagtacactactgagaaatgggagc 780
acagtactgggaaatgggcatacagtactgggaaatgggcatacagtactgagaaatggg 840
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
28
agcacagtactgggaaatgggagcacagtactgggaaatgggagcccacagtactgggaa 900
aggggagttcacagtactcggaaatgggagcatagtactgggaaatgggagcacagtact 960
gggaaatgggagcatagtactgggaaaccccagacctggattctgagtttttcagcctag 1020
cccagacttcttatcttagtagacaaaaagagtcaataccagagaaccagaggcatcctc 1080
tgtattttaatgaactctgcattttaatctgtttagtagtcattttttaaaagataatca 1140
gttttccaaatatatctataagttactacgtgaaaaaaaaaaaaaa 1186
<210> 41
<211> 1516
<212> DNA
<213> Homo Sapiens
<220>
<221> SITE
<222> (7)
<223> n equals a,t,g, or c
<400> 41
ttgggcnccaaaaatcaacgggacttttccaaaatgtcgtaccaactccgccccattgac60
gcaaatggccggtaggcgtgtacggtggaaggtctatataagcagagctcgtttagtgaa120
ccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccggga180
ccgatccagcctccggactctagcctaggccgcgggacggataacaatttcacacaggaa240
acagctatgaccactagscttttgcaaaaagctatttaggtgacactatagaaggtacgc300
ctgcaggtaccggtccggaattcccgggtcgacccacgcgtccggagcgaggctgccctt360
tcttcgcagcgtgatttattttcttctttttttctgaactcttcttccagggagaggcta420
gtggtaacaggccgagctggatggatgggtatggggagaggggcaggacgttcagccctg480
ggattctggccgaccctcgccttccttctctgcagcttccccgcagccacctccccgtgc540
aagatcctcaagtgcaactctgagttctggagcgccacgtcgggcagccacgccccagcc600
tcagacgacacccccgagttctgtgcagccttgcgcagctacgccctgtgcacgcggcgg660
acggcccgcacctgccggggtgacctggcctaccactcggccgtccatggcatagaggac720
ctcatgagccagcacaactgctccaaggatggccccacctcgcagccacgcctgcgcacg780
ctcccaccggccggagacagccaggagcgctcggacagccccgagatctgccattacgag840
aagagctttcacaagcactcggscacccccaactacacgcactgtggcctcttcggggac900
ccacacctcaggactttcaccgaccgcttycagacctgcaaggtgcagggcgcctggccg960
ctcatcgacaataattacctgaacgtgcaggtcaccaacacgcctgtgctgcccggctca1020
gcggccactgccaccagcaagctcaccatcatcttcaagaacttccaggagtgtgtggac.1080
cagaaggtgtaccaggctgagatggacgagctcccggccgccttcgtggatggctctaag1140
aacggtggggacaagcacggggccaacagcctgaagatcactgagaaggtgtcaggccag1200
cacgtggagatccaggccaagtacatcggcaccaccatcgtggtgcgccaggtgggccgc1260
tacctgacctttgccgtccgcatgccagaggaagtggtcaatgctgtggaggactgggac1320
agccagggtctctacctctgcctgcggggctgccccctcaaccagcagatcgacttycag1380
gccttccacaccaatgctgarggcaccggtgcccgcargctggcagccgscagcctggac1440
ccacagsccccgagamcttyccatacsagacaggccgtggccaagtgcaaggagaagctg1500
ccggtggaggacctgt 1516
<210> 42
<211> 803
<212> DNA
<213> Homo Sapiens
<220>
<221> SITE
<222> (462)
<223> n equals a,t,g, or c
<220>
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
29
<221> SITE
<222> (696)
<223> n equals a,t,g,or c
<220>
<221> SITE
<222> (746)
<223> n equals a,t,g,or c
<220>
<221> SITE
<222> (761)
<223> n equals a,t,g,or c
<220>
<221> SITE
<222> (770)
<223> n equals a,t,g,or c
<220>
<221> SITE
<222> (780)
<223> n equals a,t,g,or c
<220>
<221> SITE
<222> (788)
<223> n equals a,t,g,or c
<400> 42
gactaccttt tgcawaagctatttaggtgacactatagaa ggtacgcctg caggtaccgg60
tccggaattc ccgggtcgacccacgcgtccggagcgaggc tgccctttct tcgcagcgtg120
atttattttc ttctttttttctgaactcttcttccaggga gaggctagtg gtaacaggcc180
gagctggatg gatgggtatggggagaggggcaggacgttc agccctggga ttctggccga240
ccctcgcctt ccttctctgcagcttccccgcagccacctc cccgtgcaag atcctcaagt300
gcaactctga gttctggagcgccacgtcgggcagccacgc cccagcctca gacgacaccc360
ccgagttctg tgcagccttgcgcagctacgccctgtgcac gcggcggacg gcccgcacct420
gccggggtga cctggcctaccactcggccgtccatggcat anaggacctc atgagccagc480
acaactgctc caaggatggccccacctcgcagccacgcct gcgcacgctc ccaccggcgg540
agacagccag gagcgctcggacagccccgagatctgccat tacgagaaga gctttcacaa600
gcactcggsc acccccaactacacgcactgtggcctcttc ggggacccac accttcagga660
ctttcaccga ccgctttcagacctgcaaggtgcaanggcg cctggccgct catcgacaat720
taattacctg aacgtgcaggtcaccnacacgcctgtgctt nccggctcan cggccactgn780
caaccaanca aggcttaccatca 803
<210> 43
<211> 1553
<212> DNA
<213> Homo Sapiens
<220>
<221> SITE
<222> (23)
<223> n equals a,t,g, or c
<220>
<221> SITE
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
<222> (41)
<223> n equals a,t,g, or c
<220>
<221> SITE
<222> (42)
<223> n equals a,t,.g, or c
<400>
43
tgcacgcgtaagcttgggccccncgagggatcctctagagnngtcgcscttttttttttt 60
ttttttaaaagaaaataacaaaaaaaccaaactttacttgcatttagccattaataaata 120
atttacagtatgtacacgcggtgacactccacacaggtcgcagacacgtggacgcagtga 180
ggggtccccacttccaagcagaacgtgagcaaacacaaccaaaataaagtgcttcacttt 240
ttacttccaacatagggaccaactaaaaccagcagggagagggaggggccgggccccgag 300
cgccctcccacccgcccacacacggttctggcagagggagcaggacctcttggagggagg 360
gggagacacacgttctgcagcctcctggccagtgtatgagaggtccctggggaacagcag 420
aggggctgccgatgggtgggtagggtgtggagctgtagggctgctggcaggatcaccctc 480
cagggcctggctgcacttttgggagtgaagcctggctttgtgctgtgggcatagcagcac 540
aggtgggactgttctgggcccctggatgagcaccctgggctgaaatgaggcgggagccag 600
cggcaagtctctcctggctgtgtggccacagggcttaagggaagagattagaggttgaag 660
accctgccagggatgctcagagctgagagtcctaagaccctgctcctctccccagactcc 720
agacgtggatggaccgggctgggggctcttggggttgtgaggaggaagcagaggatcagg 780
gcaggccagctggccccatccactgggtctcctgccacaccctggggcccaggcacccct 840
gcccaggagctcttgcatctgcaggagggagaggaacagaagtcaaggatctcttctctg 900
tttagacatttagaaccaagcacttcgactttctaagcacagggtggaaggggcaggggc 960
ccaggccagagacgggcagctgagcaggtgcctgaaaaccgggacctgcactggtaccca 1020
cagcagccaggtgaacatgggcagggcagccacaggccagaaggcaccaggcgggcacga 1080
cctactggccaaaaggttccagcagtctgctaaggtgccaagcagcgccccggggttgcc 1140
ctgcctsgggccaagccccaaggatgcctccctgtgtggctggggtgtccccgctggcct 1200
agggggatcgagtgtgctctcgtgtaagagtgtgtgcgtgcgtgtggcgggcacaggggg 1260
ccccacggatcggcgagcagcagtcggccgggcctttcagtgcattgcgagggggaagga 1320
gctgactctgacggttcccagtgtgtctctggtgggggtggggggcttcagcctgagggg 1380
atgtttcacacattcacactgcaggggcggggcgaggggagctgctctccctctcacaca 1440
gctctactgtcaaacatcacggcaccagtcaccacaaccttgtcacgtgcactagaaggg 1500
gaggggtccatgcctgagcccttggcagcaggcggtccctggcgttctgcggg 1553
<210> 44
<211> 368
<212> DNA
<213> Homo sapiens
<220>
<221> SITE
<222> (365)
<223> n equals a,t,g, or c
<400>
44
ggagaacaggggcaatgacacatgctccttggcttcctagtcctgatcccttggggttcc 60
ttgatcctagggtcatctgacttagatccatcctcccttcctctaggcaccaggggccat 120
ggctggaggtggccacccctcagtcctgtacagattctgtacccactggctggagacccc 180
catgcagctgtctcctgctcttgctgcggggagactgaactcagggctctgctcactggc 240
tccctcccaatggaagccttctctgggcttcacagcattgaatattcctccagaacagct 300
tgttaaatcaaattttttttttttttagatggagycttgctytgkcgcccaggctggaat 360
gcacnggc 368
<210> 45
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
31
<211> 1447
<212> DNA
<213> Homoapiens
S
<220>
<221> SITE
<222> (4)
<223> n ls a,t,g,
equa or c
<220>
<221> SITE
<222> (887)
<223> n ls a,t,g,
equa or c
<220>
<221> SITE
<222> (903)
<223> n ls a,t,g,
equa or c
<220>
<221> SITE
<222> (1033)
<223> n ls a,t,g,
equa or c
<220>
<221> SITE
<222> (1137)
<223> n ls a,t,g,
equa or c
<220>
<221> SITE
<222> (1371)
<223> n
equals
a-,t,g,
or c
<220>
<221> SITE
<222> (1413)
<223> n ls a,t,g,
equa or c
<400> 45
tccnttgggaataaccctcactaaagggaacaaaagctggagctcgcgcgcctgcaggtc 60
gacactagtggatccaaagaattttggcacgagcacaaactgggtggcttaaaccacaga 120
aatgtattgtcttacagttctgggggccagaagtccaaaatcaaggtgtcagcaggccca 180
tgctccatttgaaggcactggggaggggtctgctccaggcctgtctcctggctactggtg 240
gctccatggcttgtgacagcatcactccaatcttcacacagcattctccccgtgtgcctc 300
tgagccccagttttcccttacttcaagagactggggcttttgctccatcacctagactgg 360
agggcagtggcatgatcttggctcactgctgccttgaactcctgtgctcaagtgatcctc 420
ctgcctcggccatccagaattgctgggactatagatgtgagccacgacacctggcttcaa 480
tttcctctttctataaggatatcggtcgtattggattagggcccgtcctactccagtata 540
acctcatcttagygaattacatctgccacaactctgtttccaaataaactcacattctga 600
ggtacagggagttaggacctcaacacaggaattttgggggctaaggtaaggagaaaacaa 660
gaagagtttggtgtcacagaagtgagaatgtccggccaggtatggtggctcaagcctgtg 720
atcctagcactttgggaggccgaggtgggcagatcacctgaggtgagcagttcaagacca 780
gcctggccaacatggtgaaactctgtctctactaaaaatacaaaaattagctgggccgtg 840
gtggcagacacctataatcccagctatccgggaagctgaggcagganaatcacttgaacc 900
cangagatggaggctgcagttgcagtgagccaagatcacaccactgcactccagcttggg 960
tgacagagggagatgccgtctcaaaaaaaaaaaaaaaaaaaaaaaaactcgagagtactt 1020
ctagagcggccgngggcccatcgattttccacccgggtggggtaccaggtaagtgtaccc 1080
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
32
aattcgccctatagtgagtcgtattacaattcactggccgtcgttttacaacgtcgngac 1140
tgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagc 1200
tggcgtaatagcgaaaaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaat 1260
ggcgaatggagatccaatttttaagtgtataatgtgttaaactactgattctaattgttt 1320
gtgtattttagattcacagtcccaaggctcatttcaggcccctcagtcctnacagtctgt 1380
tcatgatcataatcagccataccacatttgtanaggttttacttgctttaaaaaaccttc 1440
cacacct 1447
<210> 46
<211> 425
<212> DNA
<213> Homo Sapiens
<220>
<221> SITE
<222> (352)
<223> n equals a,t,g,or c
<220>
<221> SITE
<222> (362)
<223> n equals a,t,g,or c
<220>
<221> SITE
<222> (369)
<223> n equals a,t,g,or c
<220>
<221> SITE
<222> (415)
<223> n equals a,t,g,or c
<400> 46
ggcacgagca caaactgggtggcttaaaccacagaaatgtattgtcttac agttctgggg60
gccagaagtc caaaatcaaggtgtcagcaggcccatgctccatttgaagg cactggggag120
gggtctgctc caggcctgtctcctggctactggtggctccatggcttgtg acagcatcac180
tccaatcttc acacagcattctccccgtgtgcctctgagccccagttttc ccttacttca240
agagactggg gcttttgctccatcacctagactggagggcagtggcatga tcttggytca300
ctgctgcctt gaactcctgtgctcaagtgatcctcctgcctcggccatcc anaattgctg360
gnactatana tgatgagccacgacacccttggcttcaatttcctctttct ataangatat420
cgctc 425
<210> 47
<211> 1530
<212> DNA
<213> Homo Sapiens
<220>
<221> SITE
<222> (1389)
<223> n equals a,t,g, or c
<220>
<221> SITE
<222> (1488)
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
33
<223> n equals a,t,g, or c
<400> 47
ggtgttggcttcctcatgcattctcaatgccaaggctttttctcttctctaacgatgctg60
aatcctctgagacagctctttaagctgatggcctctctgtttctgtcggtgttcacactt120
ggccttccctttgccctctttcagtattatgcctacacccaattctgtctgccaggctca180
gcccgccccattcctgagcctttggtacagttagctgtagacaagggctaccggattgca240
gagggaaatgaaccgcttggtgcttctgggatgttccactaatatacagctatawcyagg300
atgtctactggaatgttggctttttgaaatactatgagctcaagcaggtgcccaattttc360
tactggctgcaccagtggctatactggttgcctgggcaacttggacatacgtgaccactc420
acccttggctctgccttacacttgggctgcaaaggagcaagaacaataagaccctagaga480
agcccgatcttggattcctcagtcctcaggtgtttgtgtacgtggtccacgctgcagtgc540
tgctgctgtttggaggtctgtgcatgcatgttcaggttctcaccaggtttttgggctcct600
ccactcctattatgtactggtttccagctcacttgcttcaggatcaagagccgctgttga660
gatccttaaagactgtgccttggaagcctcttgcagaggactccccaccaggacaaaagg720
tccccagaaatcctatcatgggacttttgtatcactggaaaacctgttctccagtcacac780
gatacattctaggctacttcctgacttactggctcctgggactactcctacattgcaact840
tcctgccttggacatgacctggactctccagggacaggttggaagccaacttaacccagg900
ggtctgaaagtaaaaatacacattggaactgcctctgctgccctgggatcattactgtgt960
ccattataatctttctctttctctttgaaagctggtcaggaatgggagaagtgtcagaca1020
ctagagagccccttctggtcctggctagggcaaattttagacaactattttctctgtaag1080
tgaagattgtcgtattccaagtctaaaatacacctggatctgtctagtcaatcaacatag1140
cagagacagtcttaaacctaccattgacctgtgtgtaaatttaaatgtcaatttattgaa1200
gtgtaaatttcatcaaaggcattagctgacaggctggtaacagtccacacaagatggtat1260
aggcctgaacagtgtagtggcagtaataaagtgggaccattttttccaaatgcgcatgtt1320
ctgatgtttttatgatgtgaggatttattgaaaactcaagttcgatactctcagtacatg1380
aaaaaaaana.agaagaaaaaaaaaaagtttttgccgggtcctttggttcttcctgggtct1440
gggctcgcctagggacctcgggatggccgcctatcgagggctcgcggnggcggtccggtg1500
tttgcccagggtccaaggtctgtccgcttg 1530
<210> 48
<211> 100
<212> DNA
<213> Homo sapiens
<220>
<221> SITE
<222> (80)
<223> n equals a,t,g, or c
<400> 48
gccaccgcgg tggcggccgc tctagaacta gtggatcccc cgggctgcag gaattcggca 60
cgaggtgttg gcttcctcan gcattctcaa tgccaaggct 100
<210> 49
<211> 550
<212> DNA
<213> Homo sapiens
<220>
<221> SITE
<222> (18)
<223> n equals a,t,g, or c
<220>
<221> SITE
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
34
<222> (528)
<223> n equals a,t,g, or c
<400> 49
gggaacgcttttacgccngcaggtcccggtccggaattcccgggtcgacccacgcgtccg 60
gaggtgkkgggcagagtccccakkcagttgggattccggggagggaccacagtgctggga 120
accacagcactgacgccatacacaccaggggatgtttgctcttgcatggaaagtgatttt 180
cagtgtcatgctgcagaatcccatacgttatccttcagtccttggtataaagtcctccct 240
gctcagtagtcttgtgttggtgatggtttggggaaatgaaaagagtgggccctgccccac 300
accaaagtccaggaagggaagaaggagctgtccagcacaggtggggaggggagaaraggg 360
gtcctattgggacccagaattcagactgagcaggaaatccaaccaggggttgaggaggga' 420
ttacctgagcttgtatcacttcaacctgcattttagagacacattctaatctcacagatg 480
cagaatttggggatccctggacagctaacacttgggggtgcaaatgtncttgttgaatga 540
gaggaactgg 550
<210> 50
<211> 866
<212> DNA
<213> Homo Sapiens
<220>
<221> SITE
<222> (833)
<223> n equals a,t,g, or c
<400> 50
gaatacgccagctctaatacgactcactatagggaaagctggtacgcctgcaggtaccgg 60
tccggaattcccgggctcttgatctttcctggtcctggtacggggatgacctctctttta 120
gaggggcgaatggttctctgtgtctcctgcctcctccttcccctgctgttgttgctgaaa 180
cactttaatgggttgatgacaccatatttagctcataatgtatattgtcctatagaatat 240
atttcttttttcccatttcatgaaaaaaatatagaatatatttctatttggttcatcttt 300
gacagttttaagtttatctattcgaggcttttgtgtatttcccaaatttatgtgctttat 360
agagcatatactttgccccattagatgcagtggtattgcaatagttatcctcagggaggt 420
aggctgtgtgtgtgtgtgggtgtgagtgtatgtatttgtataaaagaaaatccaatatta 480
catgcatttggccgggcacggtggctcacatttgtaatctcagtgctttgggaggctgag 540
gtggacaggtcatttgaggtcaggagttcgagaccagcctggccaacatggtgaaactct 600
gtctctactaaaaattaaaaaattagccaggcgtggtggtgtacatc.tgtaatcccagct 660
acctgggaggctgaggcaggagaatctcttgaacctgggaggtggaagctgcagtgagcc 720
aagattgcaccactgcactccaggctgggggacagagcaagactccggtctcttcaaaaa 780
aaaaaaaaaaaaaamaaamaaraaaaaaagggcggccgctctagaggatccangcttacg 840
tacgcgtgcatgcgacgtcatactct 866
<210> 51
<211> 732
<212> DNA
<213> Homo Sapiens
<400> 51
cttgatctttcctggtcctggtacggggatgacctctcttttagaggggcgaatggttct 60
ctgtgtctcctgcctcctccttcccctgctgttgttgctgaaacactttaatgggttgat 120
gacaccatatttagctcataatgtatattgtcctatagaatatatttcttttttcccatt 180
tcatgaaaaaaatatagaatatatttctatttggttcatctttgacagttttaagtttat 240
ctattcgaggcttttgtgtatttcccaaatttatgtgctttatagagcatatactttgcc 300
ccattagatgcagtggtattgcaatagttatcctcagggaggtaggctgtgtgtgtgtgt 360
gggtgtgagtgtatgtatttgtataaaagaaaatccaatattacatgcatttggccgggc 420
acggtggctcacatttgtaatctcagtgctttgggaggctgaggtggacaggtcatttga 480
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
ggtcaggagttcgagaccagcctggccaacatggtgaaactctgtctcta ctaaaaatta540
aaaaattagccaggcgtggtggtgtacatctgtaatcccagctacctggg aggctgaggc600
aggagaatctcttgaacctgggaggtggaagctgcagtgagccaagattg caccactgca660
ctccaggctgggggacagagcaagactccggtctcttcaaaaaaaaaaaa aaaaaaaaaa720
aaaaaaaaaaas 732
<210>
52
<211>
604
<212>
DNA
<213> Sapiens
Homo
<220>
<221>
SITE
<222>
(584)
<223> als a,t,g,or c
n equ
<220>
<221>
SITE
<222>
(595)
<223> als a,t,g,or c
n equ
<400>
52
gaattcggcacgaggcggcgcgggacctgcagtcgccagggattccctccaggtgacgat 60
gctctggttctccggcgtcggggctctggctgagcgttactgccgccgctcgcctgggat 120
tacgtgctgcgtcttgctgctactcaattgctcgggggtccccatgtctctggcttcctc 180
cttcttgacaggttctgttgcaaaatgtgaaaatgaaggtgaagtcctccagattccatt 240
tatcacagacaacccttgcataatgtgtgtctgcttgaacaaggaagtgacatgtaagag 300
agagaagtgccccgtgctgtcccgagactgtgccctggccatcaagcagaggggagcctg 360
ttgtgaacaktgcaaaggttgcacctatgaaggaaatacctataacagctccttcaaatg 420
gcagagcccggctgagccttgtgttctacgccagtgccaggarggtgttgtcacagagtc 480
tggggtgcgctgtgttgktcattgtaaaaamcctttggagcatctgggaatgtgctgccc 540
cacatgtccaggctgtgtgtttgagggtgtgcagtatcaagaangggaggaattncagcc 600
agaa 604
<210> 53
<211> 854
<212> DNA
<213> Homo Sapiens
<220>
<221> SITE
<222> (805)
<223> n equals a,t,g, or c
<220>
<221> SITE
<222> (818)
<223> n equals a,t,g, or c
<220>
<221> SITE
<222> (829)
<223> n equals a,t,g, or c
<220>
<221> SITE
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
36
<222> (832)
<223> n equals a,t,g, or c
<400> 53
tcgagttttttttttttttttttttttttgcttttacatcttaagccctttattgactac 60
aatgcagaacattttattttaagacacagtgggttttgtttttgttgatgttttcaccaa 120
ttcaactgaagacgaaagtcaagacaatcaaatggtaactagtagcagcctatcagtaaa 180
tgagggcaagtatagagactgttctttggactgaggttaaatcaattagtcaataaaggc 240
ttttccactgtctaataattataacatattaacagtcgccaaatagtgttggatgggact 300
cctctagaaataactaaagcctttcattttatacatgaaatagccacaaaatgtagatgg 360
gttacatcaactcattggatttgcccatcttaaattactctgagattcagagaaatcaaa 420
atttcatcatatgttgtgcaacagtttcttcaaatactttctcatgctcaataaaattaa 480
catggcatccagacaccctttaatttcaacacatgacatataaatggtcacatccatcat 540
acacacatagatgattagaagacttgaggactacgcaaggatggcaagaaatcacttgat 600
tcttcggttatgccctagtgacaaatatatatttcttggcaaccagccaatcaattttgg 660
gaataatgttagcccatgacctaatacgttctaacagcctatcactcgctttttcatgca 720
ccatttagggccgggtcaccaatctcctgctgttcttttcccaggcatacattcctttct 780
ttgggaaacctggaaatgccccccnggataaatggggncccttctcccnggnggaaccac 840
cattttccccactc 854
<210>
54
<211>
814
<212>
DNA
<213> Sapiens
Homo
<220>
<221>
SITE
<222>
(341)
<223> als a,t,g,or c
n equ
<220>
<221>
SITE
<222>
(354)
<223> als a,t,g,or c
n equ
<220>
<221>
SITE
<222>
(804)
<223> als a,t,g,or c
n equ
<400>
54
aaggggaggaatttcagccagaaggaagcaaatgtaccaagtgttcctgcactggaggca 60
ggacacaatgtgtgagagaagtctgtcccattctctcctgtccccagcaccttagtcaca 120
tacccccaggacagtgctgccccaaatgtttgggtcagaggaaagtgtttgacctccctt 180
ttgggagctgcctctttcgaagtgatgtttatgacaatggatcctcatttctgtacgata 240
actgcacagcttgtacctgcagggactctactgtggtttgcaagaggaagtgctcccacc 300
ctggtggctgtgaccaaggccaggagggctgttgtgaarantgcctcctacgantgcccc 360
cagaagacatcaaagtatgcaaatttggcaacaagattttccaggatggagagatgtggt 420
cctctatcaattgtaccatctgtgcttgtgtgaaaggcaggacggagtgtcscaataagc 480
agtgcattcccatcagtagctgcccacagggcaaaattctcaacagaaaaggatgctgtc 540
ctatttgcactgaaaagcccggcgtttgcacggtgtttggagatccccactacaacactt 600
ttgacggtcggacatttaactttcaggggacgtgtcagtacgttttgacaaaagactgct 660
cctcccctgcctcgcccttccaggtgctggtgaagaacgacgcccgccggacacgctcct 720
tctcgtggaccaagtcggtggarctggtgctgggcgaracagggtcagcctgcagcagca 780
cctcaccgtgcgctggtacggctngcgcatcgct 814
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
37
<210> 55
<211> 322
<212> DNA
<213> Homo sapiens
<220>
<221> SITE
<222> (314)
<223> n equals a,t,g, or c
<400> 55
ggaggaatgaaagtggcggtctcgccggcagttggtcccgggccctggggctcgggagtc 60
gggggcggtgggacagtgcggctactcttgatcctctccggctgcttggtctacggcaca 120
gctgaaactgatgtaaatgtggtcatgcttcaggaatcccaagtttgtgaaaagcgtgcc 180
agccaacaattctgttacacaaatgtgcttatcccaaaatggcatgatatatggacacgg 240
atacagmtccgartamatagttccagattggttcgagtcacccaggtggarraatgarga 300
rgaaactgaaggancttagaac 322
<210> 56
<211> 3140
<212> DNA
<213> Homo Sapiens
<220>
<221> SITE
<222> (3)
<223> n equals a,t,g, or c
<220>
<221> SITE
<222> (5)
<223> n equals a,t,g, or c
<220>
<221> SITE
<222> (21)
<223> n equals a,t,g, or c
<220>
<221> SITE
<222> (1628)
<223> n equals a,t,g, or c
<220>
<221> SITE
<222> (1642)
<223> n equals a,t,g, or c
<220>
<221> SITE
<222> (1656)
<223> n equals a,t,g, or c
<220>
<221> SITE
<222> (2094)
<223> n equals a,t,g, or c
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
38
<400> 56
tgntncgagtcacccaggtgnagaatgaggagaaactgaaggagctagagcagatttgat 60
cccaaactctttctkgttttccttcttggacttatgctatttttttgtggagacttgctg 120
agcagaagtcaaattttctactactctactgggatgactgtgggaattgtggcctctctg 180
ctaatcatcatttttatactatctaagtttatgcctaagaaaagtcccatttacgtcatc 240
ctggtgggaggctggtctttttctctgtacctcattcaactagtttttaaaaatttacaa 300
gagatctggaggtgttactggcagtatcttttaagttatgtcctcacagttggattcatg 360
agttttgcagtatgttacaagtatgggcccttggagaatgaacgaagtatcaacctgctg 420
acctggaccttgcagctgatgggcctgtgtttcatgtattctggcatccagataccacat 480
attgcccttgccattatcatcattgctctttgtactaagaacctggaacaccctattcag 540
tggctgtacatcacctgcagaaaggtgtgtaagggagcagaaaagcctgttccccctcgt 600
ctcctgacagaagaagaatatcggatacaaggagaggtagaaacccgaaaggctttagag 660
gagctccgagaattttgtaacagtccagactgctctgcttggaagactgtttctcgaatc 720
cagtctccaaaaagatttgctgactttgtggaaggctcttcccacctcacgccaaatgaa 780
gtttctgtccatgagcaggagtatggattagggagcattattgcccaggatgaaatctat 840
gaggaagcatcctctgaggaggaggactcatattctcggtgtcctgctatcacacagaac 900
aactttctaacctaggtagtggtcagttatctttacgtggactggcttggtgccttggtc 960
catgttgcatgtgttgtgcaattgctttcaaccctttgaaacagagtgagatagataggg 1020
tagaaattctcctactgaaataagaggcctaaaaaggcctccctttggaaatgggaggtc 1080
tctatgggatccctgaggaaggagagtggataaagtagtgaatgctgggtagttcacttc 1140
ccattggttaagctaacagcccacttttatgtttccagagaaattggatggccacagcta 1200
gcatggcattctagctccttcttgaaagttgattcaatcatggcatttctgtcactggct 1260
ggctctccaaagtaagaactgttgttaagtgcaggaatgcttttagactataggctgcaa 1320
cttccagagagaaatccacaaatctgagcctccttcactccagcttttatttcrgtgact 1380
ttagaataattattgatttaactgttttgggaggaaaatagatttttattgttttgtttt 1440
ttaaatgaatgtcttttaaaaamcwtamcaaactcatgttccagaaccagcaagtgctcc 1500
agagtgacacaccccctaggcccctacatatttattaatatggattatccattaaagccc 1560
caggagctgttgttttaagctttgatttagttctcatacatatgatagaaagtcctattt 1620
gcctttangaacatgcctgtangctcttctgcaggntgagatgttctgggctttttatta 1680
tattcaactttcaattccatcttaaaaaacatttgtwttcttctcttcccattcttcctt 1740
accctgcctttgccctttcaggaagggtcagttcccttacctgtgaactatgtatgttca 1800
gagtagcattattcctgctagctaggagaagtcatcttgtttaggggatttggatgcttt 1860
ttatacgttctccattttcctgtcattgggtcatgttatctttgagttgctatgaaatca 1920
ggaaactgtctccttttcctttcccttcctttgtctacatgctctgtccattcctttcag 1980
ccttttctcaccacccatactcccccaaatctgggtaatttttaagccttgaaactatgt 2040
agtttcttgatacacaatttgtagttatgcagcagccacaatttgcattgccanggaaat 2100
argctccaggttatcttcatgcctctgggtgctcattcagctgtcaagtttccatgaact 2160
tacacttatttatgattgcgtttctgacctgagatgtatgctgcctgttattgcagtagc 2220
attagtttcagattcttttgccattgcaaagtaccccttataaaccagcaatgtcatctg 2280
tgaggaagcaaattctcaagtgtctgtcatttacttggttctttttctttgtggtcttca 2340
cccttataccctggaaaagtctgtaattaccttagccaggaagatagatggtcatggcaa 2400
gcgcacagcaccagacttactggctcaccaagatgatggaaaaaggcagatgatttttta 2460
aaaagccgtaatgactcctttagaccagccatttagcgtggtaattttgaaaggcctagc 2520
tccattgcagacttccaaagggtcagctctgagactgccctccaggtgggcagttgatta 2580
tttccaccagtgttttccagagccttaaactgtcctaagtgacaactacctcagttggca 2640
ggaaagagacatatagtagaaagtgaaaaatgagcagtatttgggcagatgctatgggtt 2700
acagttgaagggtaaaaggaactttacattgggaaacctttatacccttgtgaattatgt 2760
acatggtaaaatgttctctctctacaaagaactattaaaacttctgaaatatactatttt 2820
ttaccttatttatagaaattgagacctagcatatttaagcataagtttattttaaaaaat 2880
aattcaactcgtgcaagtggtctcaggattctctggagattttggtgcctcccctactta 2940
gggaggtgatagcttgcctawaagggtgacttttccygatcatgtctttatttcaatgag 3000
aaagcactgtgaaattgtgaaagawtctcctctttctctgtttaataaacccccatgaaa 3060
tataaaaaaaaaaaaaaagggcggccgctctagaggatccctcgaggggcccaagcttac 3120
gcgtgcatgcgacgtcatag 3140
<210> 57
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
39
<211> 1402
<212> DNA
<213> Homo Sapiens
<220>
<221> SITE
<222> (1335)
<223> n equals a,t,g, or c
<400> 57
ccgggccccccctcgagggtcatttttttttttttttttttttaacctttcaaaatattt60
ttatcattgtgaattggtaattgtattacagtcagtatcctttggggaaaaaagtctcaa120
aatttaacttcccttgttgaagaagctattttacagtgccatattattcattaagcatta180
attaaagaacagcaggataattagtaggatcatcaatattacaagaaacattagatcgct240
caagaaggaaataatttaactagaaacactttaaattttctcaagtagtgcagctgatag300
attagggagaaaaacaacaactccgcttaggggatatagactttaaatcattgtttagaa360
ataggtacttctgtatttattttgttaagttaaaataatagggctcaattaggctgagta420
actccctagaggcacagttgtaatagctggtctcagagaaggcaaaccctcctagttctc480
atcagaaggaaatgatctgtcattagtcctaggtaactgattaatatctcacagtggact540
ggaaatcgctgaatgttctctgcaagttttatactcaaggtgtgaattcataagcaacca600
tcctatttgaaagttagccattgcttagggttctctattagaatcttatcaagcacattc660
tcagttatgcctctcagcaacattttaggaacaacattggtgagtatatgagaatagccg720
tgacctccatatttcatcagccgggttttcgtatgtatgtcatgtgctaccagaattcga780
tcttcacagccctcttccaccaggagacgcacccttctaattcttttgttatcatcaggc840
atgtcaatatctgggccgagttggtaatgaagtagttcagtaccaaagagatcatattcc900
aagtagcagccaagttgagcaaactccaagagctctttcttatcaagaatagtcctatcc960
aggtgtgacatgactgttttggagatgtctgcgcctgcttcttgcaatattcggataatc1020
tgaaatggtgccctggagctccgtccaggatggataataacaggacaaccaagctgagcc1080
tgggcatgagctgtggcctggagaacctttctttcactctcagtcaaaggccaggagcaa1140
ccaatttctccaataatgccacacttgatactggttccatcagctccatggagaatttca1200
ttcataaggacatcggtaagctcgggtacaacgcgctgccttctccctgggaacttccgg1260
caggccaacttccctggcctttttagtctcccctgtgccctcccacttcgccctctggtt1320
ctcctcgtgccgaantcctgcagcccgggggatccactagttctagagcggccgccaccg1380
cggtggagctccagctttggas 1402
<210> 58
<211> 2306
<212> DNA
<213> Homo Sapiens
<400> 58
ggcacgagactgcatgcacctgggccttgtcagtctgattcttttctgtcaagctcttga 60
ggtggacatttccctccaagggcctgggattgtaccaggaagaagtgaggtttccctgag 120
tctccaggggcctagaggtggaggctgcttccccattgctacaggggccccttttattgt 180
cctcctgcccctgggtctctacctggtctttcacctctgttgcttctttgggctcttctg 240
cgctcacctccgtcttcgggagcctggctgggatcacctgatcatctaatgaaggaagtt 300
gaaggttaaacttgcctctgagacaagggatccttacggggctgaggtgtccaaacatta 360
tggagttgtgaggagacagcacgggtttcttccttgagggggggctccagaccacaggac 420
gcaggaccctctgtggggtgcccgtgttccgagggataagacacagcctcataggggcgc 480
cgtcccacctgactggaaaagaaggcccaagatgtcgctgacggttgaagaggagtggga 540
aacggcccacaattccccgggcaggcacaggtgcaggagctgcagggtgagcccggccag 600
ctgggaaggcctcacggacaagacgagcaggttgccgatggcatggccaggacctgcggc 660
agaaccaggaacaaaatacgcttagcgagttgcccattttgagtgagttgtgcacagacg 720
aaactaagggtcagaagcggagaggatactcctaagtcacccacttctctgtggctgggt 780
gcacactgggcatctgggagtttatgacatcactatggggctggtgacagagccagtgtg 840
tggaggagtgcttaggagcccagcgagggtgcctacaagaggagtcaaagggcaaagggt 900
gagacccttccaccggtccagctggactctagcctcagggatatcctgctcctgggggca 960
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
agtgtgtggccctggatgggcccccctgtggggctgttgggggtgcgaggctgatccgcc 1020
agagcccttccacctggcgcctggcccaggtgctggctagcacccagtggccctgttttg 1080
gccggccctgtcccccaggttacagggccagaacctggaagcagagcgcaggaccagcca 1140
gatcgcgccaggcttccccggggcctctccagtgcctctgtgccacctggagccaggccc 1200
gccttctccatggctgccgtggcctcaagggccaccagccttgctccgcaggttccaaag 1260
agaggacgcagtgccctgacctgactggatgcacctcttaccacatgcctccctggcagg 1320
cagggtctccactttttacaaatttgcctgagaccattcctcaggtcattcaggtggtca 1380
tggcccagccaggctttgaacccaggctgtgcgattccacagctggcgctctggcctgtg 1440
tgcctcatgatcatggatacagcatctattcttattttttcatgtagtcctggggtactt 1500
agcaccgtggcatatctgtaataagcacatgcacacctcgaaggaggtcttcacttcaac 1560
atacgagttgaccatggcatgctctgggctccagtcctctacaaagacgtagggcaggaa 1620
ctaccagttgtcaggacaggaccatcccacattgctcttctaatggagcctttcacccca 1680
gatgttctttctcgtctgatgggaaggatccaagtatgtaaagattatgttatagatcag 1740
ctttggtctgtcctaaaagaaatttgccagtggattattccatatggataaaagtcagtt 1800
tctctggtcttcctggaatgtgtctagaaagcaaatagattatttacaagttcatagtag 1860
atcaatgtattggattaaaatatgacaaacataatttggtcattgtgagcatgccagctt 1920
ggtcaactattcaccacacatgatgccctaaatataactctaggttttcttatgcccaag 1980
agagggacatactcttgggtgtctggactagggaaacatgtatgaaaaaccatttggcca 2040
ctctacatcttgttattggagaattgaaaccatctatattcaaagatattattaaaaggc 2100
aagaagttaaaaaataaaaaaataaattgcatgtctgttgttatggaattttataactgt 2160
acataccttttatttaatccattcacaggtcatattataggatatctttcatttaataag 2220
tatactaaatccataccttatactgttgagttttgatagtaccagctcgtgccgaattcg 2280
atatcaagcttatcgataccgtcgac 2306
<210> 59
<211> 391
<212> DNA
<213> Homo sapiens
<400>
59
gactgcatgcacctgggccttgtcagtctgattcttttctgtcaagctcttgaggtggac 60
atttccctccaagggcctgggattgtaccaggaagaagtgaggtttccctgagtctccag 120
gggcctagaggtggaggctgcttccccattgctacaggggccccttttattgtcctcctg 180
cccctgggtctctacctggtctttcacctctgttgcttctttgggctcttctgcgctcam 240
ctccgtcttcgggagcctggctgggatcacctgatcatctaatgaaggaagttgaaggtt 300
aaamttgcctctgagacaagggrtcccttacggggctgaggtgtccaaacattatggagt 360
tgtgaggagacagcacgggtttcttccttga 391
<210> 60
<211> 769
<212> DNA
<213> Homo sapiens
<400>
60
ctcgaggtcgacggtatcgataagcttgatatcgaattcggcacgagctggtactatcaa 60
aactcaacagtataaggtatggatttagtatacttattaaatgaaagatatcctataata 120
tgacctgtgaatggattaaataaaaggtatgtacagttataaaattccataayaacagac 180
atgcaatttatttttttattttttaacttcttgccttttaataatatctttgaatataga 240
tggtttcaattctccaataacaagatgtagagtggccaaatggtttttcatacatgtttc 300
cctagtccagacacccaagagtatgtccctctcttgggcataagaaaacctagagttata 360
tttagggcatcatgtgtggtgaatagttgaccaagctggcatgctcacaatgaccaaatt 420
atgtttgtcatattttaatccaatacattgatctactatgaacttgtaaataatctattt 480
gctttctagacacattccmggaagaccagagaaactgacttttatccatatggaataatc 540
cactggcaaatttcttttaggacagaccaaagctgatctataacataatctttacatact 600
trgatccttcccatcagacgagaaagaacatctggggtgaaaggctccattagaagagca 660
atgtgggatggtgctgtgctgacaactggtagttcctgccctacgtctttgtagaggact 720
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
41
ggagcccaga gcatgccatg gtcaactcgt atgttgaagt gaagacctc 769
<210> 61
<211> 2338
<212> DNA
<213> Homo sapiens
<220>
<221> SITE
<222> (4)
<223> n equals a,t,g, or c
<400>
61
ctcnctatggggcgaatgggkaccggccsccctttttttttttttttttaagattccagc 60
tttttttattttttccccttttacacaaaacaaagtagaagaaataatacaggattaaaa 120
ctgcaaaagtagttaatcagtagaatatgtatgcacacaaaaaatccagataggaagaca 180
ggcttatttacaatgaaagtaaggtaaaattaacgtagttttcttatgaaataataaaaa 240
aaaatcaaacatatgctacaatgggcaccactgttcacagcaatattaaagaggagaaaa 300
caacacttatttaatagggacagatataccacaaggtacaacatcagtgcaataaattca 360
caaaactatattacagctagttaatcagtttaagaattgttcccgtcagtcacatttttt 420
ggccctcagaagttcattcctaaagatttcaactactctaaatttctagctaccaagaag 480
ttaagaatgattataagaagctttccaaggagttatgaaatctttgtagaccagaggcca 540
actatcatcacctcaagtctgctctcaccaacagcccttgtatttttcagggagaaatct 600
ctaggaaaaaagtcagacaccagtgtagtcactatctcccatgtcaaacctaggggacta 660
aaatggtcagtattaccataaaatgataattttgaggtttaccttaaaaggcttattctg 720
gtctcaaaaattagataagattatcttctactgaaatgaatattgactaaacatagaaga 780
ctgctgtccttttgccagtcttagagtgaaagtcatagacatgagtcttaacctactgta 840
tactatagctaatgtcagctgaaaatctgaaattaaaagcatgctagaaatcctaaatgc 900
aatcttttggaagtctgctattaaaaagcctttaaggatttactaacttcgagtctaagt 960
gcaaggggacgaaagcttaagcctgtcagacattccttttcttggacaaaaagatcaaag 1020
tttcctacaaattgctaagctttgcacaagggagaagcctacatgtactagtgcatggaa 1080
tcagtttcatcttatttcatggggactcttctcccactggaaagaaacagaatgaggaat 1140
gaatcttaattggtctcttcatcagaagtggtaaacttggtctctatattcacgaagtca 1200
gacagttttttaagcagactgtggaagcagacagaaccagcttcctgtagccacagacca 1260
ctacatggtatctaagctaaagcaaagatgaacaattatccagattcacttgaactgtac 1320
taaagggcaaggttcaccactacaaaaaggaagttgtctaaaagcaagaattcaattaac 1380
gctgggtaagaaaagtcaaaacactaatgagttgtccatgaagccaactgctaagaacgc 1440
gctcaactatacgcgacatgaagacactatgcacgaagccttacttggcgagtctgaatt 1500
tctattaactaagggcagagtgagggagaacaaagagctacttccgtaacattttagtat 1560
ccagatagtacagcagaaacggttcccggggcaatgggtgctgcattaatcacactgatt 1620
aaagcagatgaatcattcgtttttcttttctttttgtttgagaagtttgtttatctccct 1680
cttggccattccaatgtacttcaaaatgattccatgttggtttagtccaggaagcaatag 1740
taaggaagtcactatcaggtaggtgagaagcaggttgtggacttgttgtcccacccaagc 1800
aaccgcagcaagggaaacgatcatggtcatgaagtacatcttaggtttttcttcctttag 1860
tgtgaagaggcgtttccaccaacccacagctctgcgtcgagtttttactagattgctgca 1920
aatttcatggaatctttgctgttgttcagtggtccatttattggagccaaaaattctagg 1980
cgctagaatgggaacaaggtagtcagccaagcacaaaaacataacaaaacaggaaacgcc 2040
ggacagaacagatggatctagatagtagataatcagaaacaccaaagaaaccacacccat 2100
gatggcaggtggaaaccaggctctttcccatcggaggactttatcagccatcagcatcac 2160
ttctccccatccttgcagctgttcttccagacttgcagtctctgcagccagcaggttggt 2220
gctgcgattatctccctccgccatcgtctcggggatgcagtctctacaagcgcaggccac 2280
ctccccaacgagtcctccaaccgaaacccgcaccggacgcgtgggcggacgcgtgggc 2338
<210> 62
<211> 560
<212> DNA
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
42
<213> Homo Sapiens
<220>
<221> SITE
<222> (517)
<223> n equals a,t,g, or c
<400> 62
tcgagtttttttttttttttttcattattgtttttattttgatgatgtgatgattactgc 60
atttcaaggccaaggctattttattgactgcaacctattaaaatgtaagagagtaataaa 120
aaaaagagaataatattaattaattcagactatatctctacatagagctggaaaaatatt 180
ataaaaaatatattttgcagttaaattgagtatctttcatatagataagctagatcacat 240
atatttaatgaagcaatataactagcattattcactagtagttctgttaccaaatacaat 300
cttaagtaatttaatgaattccagtgtcatcagtctggtgacattttaaaatttaaattc 360
caatacactttgattacagagtccaaaattatctttgctactaaaaaataacattttata 420
tcataagagcacgtctgtgataaacattgaaacgtgtattattgaaaaggagttattctt 480
agcattttctcctacgtgatctaccctttcttattcntctcgttctcttaacatattcct 540
tktaaawtaacgtagactat 560
<210> 63
<211> 685
<212> DNA
<213> Homo Sapiens
<220>
<221> SITE
<222> (303)
<223> n equals a,t,g, or c
<400> 63
cggcacgagcggcacgagcccacagcctgcaggaccgaggcccctgtgtgagggactggg 60
ggtgtgaggagttgccaggagaagcagaccctgccaggtgcccatctcctctcagcaccc 120
ctccccccaacacacttcctgtaatggcgtctccaatggggagcccattagtcctgctta 180
gggcagcacctaagtactgtggctgataaatataacatgagcccatcgattagaatcctc 240
ctagtcctacagcagctcggctccctaatggctcccctcccctcagcacactgaytaccc 300
acntctcaccccccamcccaggctgattttctggtttacttgaggctgtggggcaaacat 360
gagaggagcagaagtgaggtgatgggtgcaacacccagagcccagtcaaaggggccactg 420
cttggagacaggctggactggtytgggccctgaggcagcctggaagcccctcggtcagtt 480
aggggcagctaggggaggaccgcctgtgagttcccggggcccggcctgcggtargacatg 540
garcaaggaargcagarggccctactttytgtgrrgcscaagacakcactgcccttgggg 600
gccttgggggtctcctggtaatctcaaaaaaactgcaatgatccactctggcctgttacc 660
acgcctcttctcctgggattccttg 685
<210> 64
<211> 604
<212> DNA
<213> Homo Sapiens
<400> 64
ccgttagacg ccagttcgaa attaaccctc actaaaggga acaaaagctg gagctccacc 60
gcggtggcgg ccgctctaga actagtggat cccccgggct gcaggaattc ggcacgagca 120
gcgcgcactt accaccatgc agatccaggt cgcggggctg ctgcagtttg ccgtgcccct 180
cttctccact gcagaggaag acctgcttgc aatccagctc ctgctgaact cctcagagtc 240
cagccttcac cagctgaccg ccatggtgga ctgccgaggg ctgcacaagg attatctgga 300
cgctcttgct ggcatctgct acgacggcct ccagggcttg ctgtaccttg gcctcttctc 360
cttcctggcc gccctcgcct tctccaccat gatctgtgca gggccaaggg cctggaagca 420
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
43
cttcaccacc agaaacagag actacgatga cattgatgat gatgacccct ttaaccccca 480
agcctggcgc atggcggctc acagtccccc gaggggacag cttcacagct tctgcagcta 540
cagcagtggc ctgggaagtc agaccagcct gcagcccccg gcccagacca tctccaacgc 600
ccct 604
<210> 65
<211> 1520
<212> DNA
<213> Homoapiens
s
<220>
<221> SITE
<222> (716)
<223> n ls a,t,g,or c
equa
<220>
<221> SITE
<222> (748)
<223> n or c
equals
a,t,g,
<220>
<221> SITE
<222> (1398)
<223> n or c
equals
a,t,g,
<220>
<221> SITE
<222> (1410)
<223> n or c
equals
a,t,g,
<220>
<221> SITE
<222> (1424)
<223> n or c
equals
a,t,g,
<220>
<221> SITE
<222> (1432)
<223> n or c
equals
a,t,g,
<400> 65
tcgacccacgcgtccgctcagagaatgtccagagctaagtctgagcaaagggccagcttt 60
tgcttttggggctggggtacaggcagattcctgttggagagcatttattttacttttcaa 120
gtgtgggctttctggaaggtaatgggaaactgcaaggtgtcttgcaagcaggtgtttggc 180
ttatggtgatgtggttcccagggtggcttttgggcaggggcagggcggggtcctccagga 240
gctgtctgtcggtgctgagctcagtctgcttccctaccactcagcccactgcttggatgg 300
ttccctctgtcactctcatcttacactgtccaggttttagcactgaaagtcatatgtgtg 360
ggaagcctctcagtcccaggccaaccagaacagttggtcgccccgtgtctaacatacctg 420
tctaactgtctccagggcatggctcatcagcgtggctgtggcaccctctccctgccttct 480
cttgtctccttcactgtcaagtctgggaaagggctcctggtgggtgtccgatgaggcaca 540
gctgggtgacaaagatggctccagctctggttggggcgtgcacctcatggtgacacctcc 600
ctctgtgggacctccgagggtcaaggaaggaggcaccttccatcctaggtttaggattca 660
ttgcacttacacaggattccccgtggcagatcacctgtgttccagargctgtgtanttca 720
aggtgtgtgtgaggcagttgcatggtangggggcacctataactgggggtaagtctgtaa 780
tggtgcaatagatgatgttagaacagtgggggtccccctccctctcaggcctcaaaagca 840
caaccaccagarctggtgggtggcactgggccatggggagacctgaccagcaaggctccc 900
tctttcctctgtccaggccacccagcctgaratacagtgctggtcactctaaggaatgct 960
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
44
gtggcccccaagggacaccccttgggtggctcctcccwggcttctcactgckggctggca 1020
rgcargctggtgggcgggcaarcgtaatattacccctaagataaatgaagaagagtttca 1080
gcccttattggattatagtttttattgaactttataaattccaattagcagacccctggg 1140
caataagcgacacacagcccgtggctcgctttatagcgtgattatgaccctgtttatcct 1200
cccctgaacgagaagtattaatggaggaaacaaaggaaagggactcaaaaatacataaaa 1260
acagatattggtgacacagagataaacagacaggagtcagggagagaaaacaatctccgg 1320
gcaggcaagggggaacagagaatcagagattctaagaacccatgtgctcagaccaccgtg 1380
agcttggatgtccttctnggctcctcccanggcacaaacccctnagctcctnaagctgct 1440
ggggctgaagagacctccctcccaagcctactcaggatcttgctggggtggatgtgatta 1500
taatgaaatcaggtgccacc 1520
<210> 66
<211> 596
<212> DNA
<213> Homo Sapiens
<220>
<221> SITE
<222> (595)
<223> n equals a,t,g, or c
<400> 66
ggccgcccttttttttttttttttaatcttccactgaagcctccaagaaagatgacctgc 60
agaagggggaggaaggcgcaaacaaagccagacaaagtgaggagggcacgccatccttga 120
gcacacttctttctcctgggagatgcagcaagtcgggcaagggtttcagtccagaagagc 180
agtaccaccacccacctggctggctcaggaccccccagctcacccccacacctctctagg 240
gacagcttcttcccagctgctgttctgggtcatggctgttagaaatttaaagctgatgaa 300
cgcaaaccagagtatccaggaagaaatgaaaagcaggtggggttggagggcattcccagc 360
tgtggccaggcccttggaggctgcccatctgtctgtgggctctcaggwyctccaggtgga 420
agctcctgggagacagatcttacttggttcaattcaaactcaaggattcttaatcttatg 480
ctttcagtcccctggaggarggggcagaatgcatgatcagttttcagagcttggacaacc 540
ccctgtacctataggcaaaattacttgtgcgtgcatgtgtacataatgcatgtgna 596
<210> 67
<211> 2093
<212> DNA
<213> Homo Sapiens
<220>
<221> SITE
<222> (2080)
<223> n equals a,t,g, or c
<400> 67
ggctaggtggcgcgcgggtccggcgggcggttggcttgagcgggaccggagctgaggcag 60
gaagagccggcgccatggtggagaaggaggaggctggcggcggcattagcgaggaggagg 120
cggcacagtatgaccggcagatccgcctgtggggactggaggcccagaaacggctgcggg 180
cctctcgggtgcttcttgtcggcttgaaaggacttggggctgaaattgccaagaatctca 240
tcttggcaggagtgaaaggactgaccatgctggatcacgaacaggtaactccagaagatc 300
ccggagctcagttcttgattcgtactgggtctgttggccgaaatagggctgaagcctctt 360
tggagcgagctcagaatctcaaccccatggtggatgtgaaggtggacactgaggatatag 420
agaagaaaccagagtcatttttcactcaattcgatgctgtgtgtctgacttgctgctcca 480
gggatgtcatagttaaagttgaccagatctgtcacaaaaatagcatcaagttctttacag 540
gagatgtttttggctaccatggatacacatttgccaatctaggagagcatgagtttgtag 600
aggagaaaactaaagttgccaaagttagccaaggagtagaagatgggcccgacaccaaga 660
gagcaaaacttgattcttctgagacaacgatggtcaaaaagaaggtggtcttctgccctg 720
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
ttaaagaagccctggaggtggactggagcagtgagaaagcaaaggctgctctgaagcgca 780
cgacctccgactactttctccttcaagtgctcttaaagttccgtacagataaaggaagag 840
atcccagttctgatacatatgaggaagattctgagttgttgctccagatacgaaatgatg 900
tgcttgactcactgggtattagtcctgacctgcttcctgaggactttgtcaggtactgct 960
tctccgagatggccccagtgtgtgcggtggttggagggattttggcacaggaaattgtga 1020
aggccctgtctcagcgggaccctcctcacaacaacttcttcttcttcgatggcatgaagg 1080
ggaatgggattgtggagtgccttggccccaagtgaactcaagatttggcagccccagaga 1140
tgccaactgcagcatgcccacctgtattccctgtccccttccttcatgaaggcatctcca 1200
ggcaaggaaaactgaagtcattggcccgatacaaaacatttcctgcaacgaaggaggtgg 1260
tgccgacgtgctgcttcccatcaccagcagctgctcgacaaggggcgcagggtggctgtc 1320
tttgttccagcactgttcaggctgcctgtcatcccgggcctgccagctcccctgagtgat 1380
gagcacttccaagcacccctctgccctttctctgtccttatgctgtcccggcctcgccag 1440
ccctctggggcattgtgggagatgcctgccaggaatgagcaagctctgttgctcgggagc 1500
ctcttgtcaccttcttggacttattccccacctgataccttatagagaaaagtgtgaatt 1560
caggtggagagtaggcccaggccccatgaggcaccagtggaagcacagctccaagttcag 1620
acaggtgcccttagagaggaaaaccatgacaggcaaatgcatttcctctggagtttgaga 1680
ccctgacaaacaacaggtggcatctggtgtgctgttcttgagttttcgtttaggattagt 1740
tgagttccagctgggttttgggagaaaggagatgctaccaagtcttggatgttagggcga 1800
gaccctgcaagttgagtattagagagcttgtctttcaaggcaggttcctggggcttcagg 1860
gctaggagggaggagcctgcccttttaacagaaccccagtcacatgcggctcaagtcact 1920
cagaggctgttgcatttcagggctatgttggtcctttgtttacctcctaaaccacagctg 1980
tttgtgtttcacatatgttgtgaattttccttggttctttttaaaggaatgataataaag 2040
ttacttgctttaggaaaaaaaaaaaaaaaaatcgccgganggggggggccccg 2093
<210> 68
<211> 261
<212> PRT
<213> Homo Sapiens
<400> 68
Met Ala Val Thr Ala Cys Gln Gly Leu Gly Phe Val Val Ser Leu Ile
1 5 10 15
Gly Ile Ala Gly Ile Ile Ala Ala Thr Cys Met Asp Gln Trp Ser Thr
20 25 30
Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala Val Phe Asn Tyr Gln Gly
35 40 45
Leu Trp Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg
55 60
Gly Tyr Phe Thr~Leu Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg
65 70 75 80
Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val
85 90 95
Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser
100 105 110
Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser
115 120 125
Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val
130 135 140
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
46
Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly
145 150 155 160
Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe
165 170 175
Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met
180 185 190
Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala
195 200 205
Val Ser Tyr His.Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly
210 215 220
Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Arg
225 230 235 240
Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser
245 250 255
Lys His Asp Tyr Val
260
<210> 69
<211> 27
<212> PRT
<213> Homo sapiens
<400> 69
Trp Ser Pro Leu Val Trp Leu Trp Pro Met Val Trp Thr Phe Thr Lys
1 5 10 15
Leu Glu Ser Thr His Pro Ser Ser Leu Thr Phe
20 25
<210> 70
<211> 149
<212> PRT
<213> Homo Sapiens
<400> 70
Met Val Leu Leu Trp Ala Ser Val Leu Phe Pro Ala Pro Glu Asp Trp
1 5 10 15
Ala Glu Leu Gln Gly Ala Val Tyr Arg Leu Leu Val Val Leu Leu Cys
20 25 30
Cys Leu Ala Thr Arg Lys Leu Pro His Phe Leu His Pro Gln Arg Asn
35 40 45
Leu Leu Gln Gly Ser Gly Leu Asp Leu Gly Ala Ile Tyr Gln Arg Val
50 55 60
Glu Gly Phe Ala Ser Gln Pro Glu Ala Ala Leu Arg Ile His Ala Thr
65 70 75 80
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His Leu Gly Arg Ser Pro Pro Pro Arg Ile Gly Ser Gly Leu Lys Ala
85 90 95
Leu Leu Gln Leu Pro Ala Ser Asp Pro Thr Tyr Trp Ala Thr Ala Tyr
100 105 110
Phe Asp Val Leu Leu Asp Lys Phe Gln Val Phe Asn Ile Gln Asp Lys
115 120 125
Asp Arg Ile Ser Ala Met Gln Ser Ile Phe Gln Lys Thr Arg Thr Leu
130 135 140
Gly Gly Glu Glu Ser
145
<210> 71
<211> 547
<212> PRT
<213> Homo sapiens
<400> 71
Met Ala Met Gly Leu Phe Arg Val Cys Leu Val Val Val Thr Ala Ile
1 5 10 15
Ile Asn His Pro Leu Leu Phe Pro Arg Glu Asn Ala Thr Val Pro Glu
20 25 30
Asn Glu Glu Glu Ile Ile Arg Lys Met Gln Ala His Gln Glu Lys Leu
35 40 45
Gln Leu Glu Gln Leu Arg Leu Glu Glu Glu Val Ala Arg Leu Ala Ala
50 55 60
Glu Lys Glu Ala Leu Glu Gln Val Ala Glu Glu Gly Arg Gln Gln Asn
65 70 75 80
Glu Thr Arg Val Ala Trp Asp Leu Trp Ser Thr Leu Cys Met Ile Leu
85 90 95
Phe Leu Met Ile Glu Val Trp Arg Gln Asp His Gln Glu Gly Pro Ser
100 105 110
Pro Glu Cys Leu Gly Gly Glu Glu Asp Glu Leu Pro Gly Leu Gly Gly
115 120 125
Ala Pro Leu Gln Gly Leu Thr Leu Pro Asn Lys Ala Thr Leu Gly His
130 135 140
Phe Tyr Glu Arg Cys Ile Arg Gly Ala Thr Ala Asp Ala Ala Arg Thr
145 150 155 160
Arg Glu Phe Leu Glu Gly Phe Val Asp Asp Leu Leu Glu Ala Leu Arg
165 170 175
Ser Leu Cys Asn Arg Asp Thr Asp Met Glu Val Glu Asp Phe Ile Gly
180 185 190
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Val Asp Ser Met Tyr Glu Asn Trp Gln Val Asp Arg Pro Leu Leu Cys
195 200 205
His Leu Phe Val Pro Phe Thr Pro Pro Glu Pro Tyr Arg Phe His Pro
210 215 220
Glu Leu Trp Cys Ser Gly Arg Ser Val Pro Leu Asp Arg Gln Gly Tyr
225 230 235 240
Gly Gln Ile Lys Val Val Arg Ala Asp Gly Asp Thr Leu Ser Cys Ile
245 250 255
Cys Gly Lys Thr Lys Leu Gly Glu Asp Met Leu Cys Leu Leu His Gly
260 265 270
Arg Asn Ser Met Ala Pro Pro Cys Gly Asp Met Glu Asn Leu Leu Cys
275 280 285
Ala Thr Asp Ser Leu Tyr Leu Asp Thr Met Gln Val Met Lys Trp Phe
290 295 300
Gln Thr Ala Leu Thr Arg Ala Trp Lys Gly Ile Ala His Lys Tyr Glu
305 310 315 320
Phe Asp Leu Ala Phe Gly Gln Leu Asp Ser Pro Gly Ser Leu Lys Ile
325 330 335
Lys Phe Arg Ser Gly Lys Phe Met Pro Phe Asn Leu Ile Pro Val Ile
340 345 350
Gln Cys Asp Asp Ser Asp Leu Tyr Phe Val Ser His Leu Pro Arg Glu
355 360 365
Pro Ser Glu Gly Thr Pro Ala Ser Ser Thr Asp Trp Leu Leu Ser Phe
370 375 380
Ala Val Tyr Glu Arg His Phe Leu Arg Thr Thr Leu Lys Ala Leu Pro
385 390 395 400
Glu Gly Ala Cys His Leu Ser Cys Leu Gln Ile Ala Ser Phe Leu Leu
405 410 415
Ser Lys Gln Ser Arg Leu Thr Gly Pro Ser Gly Leu Ser Ser Tyr His
420 425 430
Leu Lys Thr Ala Leu Leu His Leu Leu Leu Leu Arg Gln Ala Ala Asp
435 440 445
Trp Lys Ala Gly Gln Leu Asp Ala Arg Leu His Glu Leu Leu Cys Phe
450 455 460
Leu Glu Lys Ser Leu Leu Gln Lys Lys Leu His His Phe Phe Ile Gly
465 470 475 480
Asn Arg Lys Val Pro Glu Ala Met Gly Leu Pro Glu Ala Val Leu Arg
485 490 495
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Ala Glu Pro Leu Asn Leu Phe Arg Pro Phe Val Leu Gln Arg Ser Leu
500 505 510
Tyr Arg Lys Thr Leu Asp Ser Phe Tyr Glu Met Leu Lys Asn Ala Pro
515 520 525
Ala Leu Ile Ser Glu Tyr Ser Leu His Val Pro Ser Asp Gln Pro Thr
530 535 540
Pro Lys Ser
545
<210> 72
<211> 245
<212> PRT
<213> Homo sapiens
<400> 72
Met Leu Gln Ala Arg Asn Gln Ser Pro Ser Ser Gln Arg Pro Leu Asp
1 5 10 15
Val Leu Arg Arg Asn Gln Asp Pro Gln Ser Pro Ala Ser Ile Ser Val
20 25 30
Ile Ile Phe Ile Thr Pro Lys Glu Glu Pro Ala Leu Gln Glu Gly Leu
35 40 45
His Leu Gln Glu Asp Gly Leu Pro Ala Thr Ala Glu Asp Ala Ala Thr
50 55 60
Cys Leu Thr Val Leu Ser Ser Gln Pro Ala Ser Cys Arg Ala Ser Cys
65 70 75 80
Cys Leu Arg Ala Asp Gly Pro Gly Met Leu Ala His Thr Cys Glu His
85 90 95
Ser Thr Gly Lys Trp Glu His Ser Thr Arg Lys Trp Glu His Ser Thr
100 105 110
Gly Lys Trp Glu His Ser Thr Gly Lys Trp Gly Leu Thr Ala Leu Gln
115 120 125
Asn Gly Ser Thr Val Leu Gly Asn Gly Ser Thr Val Leu Gly Ser Gly
130 135 140
Ser Thr Val Leu Arg Ser Gly Ser Thr Val Leu Arg Asn Gly Ser Thr
145 150 155 160
Leu Leu Arg Asn Gly Ser Thr Val Leu Gly Asn Gly His Thr Val Leu
165 170 175
Gly Asn Gly His Thr Val Leu Arg Asn Gly Ser Thr Val Leu Gly Asn
180 185 190
Gly Ser Thr Val Leu Gly Asn Gly Ser Pro Gln Tyr Trp Glu Arg Gly
195 200 205
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Val His Ser Thr Arg Lys Trp Glu His Ser Thr Gly Lys Trp Glu His
210 215 220
Ser Thr Gly Lys Trp Glu His Ser Thr Gly Lys Pro Gln Thr Trp Ile
225 230 235 240
Leu Ser Phe Ser Ala
245
<210> 73
<211> 434
<212> PRT
<213> Homo sapiens
<400> 73
Met Gly Met Gly Arg Gly Ala Gly Arg Ser Ala Leu Gly Phe Trp Pro
1 5 10 15
Thr Leu Ala Phe Leu Leu Cys Ser Phe Pro Ala Ala Thr Ser Pro Cys
20 25 30
Lys Ile Leu Lys Cys Asn Ser Glu Phe Trp Ser Ala Thr Ser Gly Ser
35 40 45
His Ala Pro Ala Ser Asp Asp Thr Pro Glu Phe Cys Ala Ala Leu Arg
50 55 60
Ser Tyr Ala Leu Cys Thr Arg Arg Thr Ala Arg Thr Cys Arg Gly Asp
65 70 75 80
Leu Ala Tyr His Ser Ala Val His Gly Ile Glu Asp Leu Met Ser Gln
85 90 95
His Asn Cys Ser Lys Asp Gly Pro Thr Ser Gln Pro Arg Leu Arg Thr
100 105 110
Leu Pro Pro Ala Gly Asp Ser Gln Glu Arg Ser Asp Ser Pro Glu Ile
115 120 125
Cys His Tyr Glu Lys Ser Phe His Lys His Ser Ala Thr Pro Asn Tyr
130 135 140
Thr His Cys Gly Leu Phe Gly Asp Pro His Leu Arg Thr Phe Thr Asp
145 150 155 160
Arg Phe Gln Thr Cys Lys Val Gln Gly Ala Trp Pro Leu Ile Asp Asn
165 170 175
Asn Tyr Leu Asn Val Gln Val Thr Asn Thr Pro Val Leu Pro Gly Ser
180 185 190
Ala Ala Thr Ala Thr Ser Lys Leu Thr Ile Ile Phe Lys Asn Phe Gln
195 200 205
Glu Cys Val Asp Gln Lys Val Tyr Gln Ala Glu Met Asp Glu Leu Pro
210 215 220
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Ala Ala Phe Val Asp Gly Ser Lys Asn Gly Gly Asp Lys His Gly Ala
225 230 235 240
Asn Ser Leu Lys Ile Thr Glu Lys Val Ser Gly Gln His Val Glu Ile
245 250 255
Gln Ala Lys Tyr Ile Gly Thr Thr Ile Val Val Arg Gln Val Gly Arg
260 265 270
Tyr Leu Thr Phe Ala Val Arg Met Pro Glu Glu Val Val Asn Ala Val
275 ' 280 285
Glu Asp Trp Asp Ser Gln Gly Leu Tyr Leu Cys Leu Arg Gly Cys Pro
290 295 300
Leu Asn Gln Gln Ile Asp Phe Gln Ala Phe His Thr Asn Ala Glu Gly
305 310 315 320
Thr Gly Ala Arg Arg Leu Ala Ala Ala Ser Pro Ala Pro Thr Ala Pro
325 330 335
Glu Thr Phe Pro Tyr Glu Thr Ala Val Ala Lys Cys Lys Glu Lys Leu
340 345 350
Pro Val Glu Asp Leu Tyr Tyr Gln Ala Cys Val Phe Asp Leu Leu Thr
355 360 365
Thr Gly Asp Val Asn Phe Thr Leu Ala Ala Tyr Tyr Ala Leu Glu Asp
370 375 380
Val Lys Met Leu His Ser Asn Lys Asp Lys Leu His Leu Tyr Glu Arg
385 390 395 400
Thr Arg Asp Leu Pro Gly Arg Ala Ala Ala Gly Leu Pro Leu Ala Pro
405 410 415
Arg Pro Leu Leu Gly Ala Leu Val Pro Leu Leu Ala Leu Leu Pro Val
420 425 430
Phe Cys
<210> 74
<211> 94
<212> PRT
<213> Homo Sapiens
<400> 74
Met Leu Leu Gly Phe Leu Val Leu Ile Pro Trp Gly Ser Leu Ile Leu
1 5 10 15
Gly Ser Ser Asp Leu Asp Pro Ser Ser Leu Pro Leu Gly Thr Arg Gly
20 25 30
His Gly Trp Arg Trp Pro Pro Leu Ser Pro Val Gln Ile Leu Tyr Pro
35 40 45
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Leu Ala Gly Asp Pro His Ala Ala Val Ser Cys Ser Cys Cys Gly Glu
50 55 60
Thr Glu Leu Arg Ala Leu Leu Thr Gly Ser Leu Pro Met Glu Ala Phe
65 70 75 80
Ser Gly Leu His Ser Ile Glu Tyr Ser Ser Arg Thr Ala Cys
85 90
<210> 75
<211> 135
<212> PRT
<213> Homo Sapiens
<400> 75
Met Leu His Leu Lys Ala Leu Gly Arg Gly Leu Leu Gln Ala Cys Leu
1 5 10 15
Leu Ala Thr Gly Gly Ser Met Ala Cys Asp Ser Ile Thr Pro Ile Phe
20 25 30
Thr Gln His Ser Pro Arg Val Pro Leu Ser Pro Ser Phe Pro Leu Leu
35 40 45
Gln Glu Thr Gly Ala Phe Ala Pro Ser Pro Arg Leu Glu Gly Ser Gly
50 55 60
Met Ile Leu Ala His Cys Cys Leu Glu Leu Leu Cys Ser Ser Asp Pro
65 70 75 80
Pro Ala Ser Ala Ile Gln Asn Cys Trp Asp Tyr Arg Cys Glu Pro Arg
85 90 95
His Leu Ala Ser Ile Ser Ser Phe Tyr Lys Asp Ile Gly Arg Ile Gly
100 105 110
Leu Gly Pro Val Leu Leu Gln Tyr Asn Leu Ile Leu Val Asn Tyr Ile
115 120 125
Cys His Asn Ser Val Ser Lys
130 135
<210> 76
<211> 280
<212> PRT
<213> Homo sapiens
<220>
<221> SITE
<222> (94)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (95)
<223> Xaa equals any of the naturally occurring L-amino acids
CA 02396719 2002-07-24
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53
<400> 76
Met His Ser Gln Cys Gln Gly Phe Phe Ser Ser Leu Thr Met Leu Asn
1 5 10 15
Pro Leu Arg Gln Leu Phe Lys Leu Met Ala Ser Leu Phe Leu Ser Val
20 25 30
Phe Thr Leu Gly Leu Pro Phe Ala Leu Phe Gln Tyr Tyr Ala Tyr Thr
35 40 45
Gln Phe Cys Leu Pro Gly Ser Ala Arg Pro Ile Pro Glu Pro Leu Val
50 55 60
Gln Leu Ala Val Asp Lys Gly Tyr Arg Ile Ala Glu Gly Asn Glu Pro
65 70 75 80
Pro Trp Cys Phe Trp Asp Val Pro Leu Ile Tyr Ser Tyr Xaa Xaa Asp
85 90 95
Val Tyr Trp Asn Val Gly Phe Leu Lys Tyr Tyr Glu Leu Lys Gln Val
100 105 110
Pro Asn Phe Leu Leu Ala Ala Pro Val Ala Ile Leu Val Ala Trp Ala
115 120 125
Thr Trp Thr Tyr Val Thr Thr His Pro Trp Leu Cys Leu Thr Leu Gly
130 135 140
Leu Gln Arg Ser Lys Asn Asn Lys Thr Leu Glu Lys Pro Asp Leu Gly
145 150 155 160
Phe Leu Ser Pro Gln Val Phe Val Tyr Val Val His Ala Ala Val Leu
165 170 175
Leu Leu Phe Gly Gly Leu Cys Met His Val Gln Val Leu Thr Arg Phe
180 185 190
Leu Gly Ser Ser Thr Pro Ile Met Tyr Trp Phe Pro Ala His Leu Leu
195 200 205
Gln Asp Gln Glu Pro Leu Leu Arg Ser Leu Lys Thr Val Pro Trp Lys
210 215 220
Pro Leu Ala Glu Asp Ser Pro Pro Gly Gln Lys Val Pro Arg Asn Pro
225 230 235 240
Ile Met Gly Leu Leu Tyr His Trp Lys Thr Cys Ser Pro Val Thr Arg
245 250 255
Tyr Ile Leu Gly Tyr Phe Leu Thr Tyr Trp Leu Leu Gly Leu Leu Leu
260 265 270
His Cys Asn Phe Leu Pro Trp Thr
275 280
<210> 77
CA 02396719 2002-07-24
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S4
<211> 105
<212> PRT
<213> Homo Sapiens
<400> 77
Met Phe Ala Leu Ala Trp Lys Val Ile Phe Ser Val Met Leu Gln Asn
1 5 10 15
Pro Ile Arg Tyr Pro Ser Val Leu Gly Ile Lys Ser Ser Leu Leu Ser
20 25 30
Ser Leu Val Leu Val Met Val Trp Gly Asn Glu Lys Ser Gly Pro Cys
35 40 45
Pro Thr Pro Lys Ser Arg Lys Gly Arg Arg Ser Cys Pro Ala Gln Val
50 55 60
Gly Arg Gly Glu Glu Gly Ser Tyr Trp Asp Pro Glu Phe Arg Leu Ser
65 70 75 80
Arg Lys Ser Asn Gln Gly Leu Arg Arg Asp Tyr Leu Ser Leu Tyr His
85 90 95
Phe Asn Leu His Phe Arg Asp Thr Phe
100 105
<210> 78
<211> 92
<212> PRT
<213> Homo Sapiens
<400> 78
Met Thr Ser Leu Leu Glu Gly Arg Met Val Leu Cys Val Ser Cys Leu
1 5 10 15
Leu Leu Pro Leu Leu Leu Leu Leu Lys His Phe Asn Gly Leu Met Thr
20 25 30
Pro Tyr Leu Ala His Asn Val Tyr Cys Pro Ile Glu Tyr Ile Ser Phe
35 40 45
Phe Pro Phe His Glu Lys Asn Ile Glu Tyr Ile Ser Ile Trp Phe Ile
50 55 60
Phe Asp Ser Phe Lys Phe Ile Tyr Ser Arg Leu Leu Cys Ile Ser Gln
65 70 75 80
Ile Tyr Val Leu Tyr Arg Ala Tyr Thr Leu Pro His
85 90
<210> 79
<211> 445
<212> PRT
<213> Homo Sapiens
<220>
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
<221> SITE
<222> (147)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (288)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (293)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (332)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (443)
<223> Xaa equals any of the naturally occurring L-amino acids
<400> 79
Met Leu Trp Phe Ser Gly Val Gly Ala Leu Ala Glu Arg Tyr Cys Arg
1 5 10 15
Arg Ser Pro Gly Ile Thr Cys Cys Val Leu Leu Leu Leu Asn Cys Ser
20 25 30
Gly Val Pro Met Ser Leu Ala Ser Ser Phe Leu Thr Gly Ser Val Ala
35 40 45
Lys Cys Glu Asn Glu Gly Glu Val Leu Gln Ile Pro Phe Ile Thr Asp
50 55 60
Asn Pro Cys Ile Met Cys Val Cys Leu Asn Lys Glu Val Thr Cys Lys
70 75 80
Arg Glu Lys Cys Pro Val Leu Ser Arg Asp Cys Ala Leu Ala Ile Lys
85 90 95
Gln Arg Gly Ala Cys Cys Glu Gln Cys Lys Gly Cys Thr Tyr Glu Gly
100 105 110
Asn Thr Tyr Asn Ser Ser Phe Lys Trp Gln Ser Pro Ala Glu Pro Cys
115 120 125
Val Leu Arg Gln Cys Gln Glu Gly Val Val Thr Glu Ser Gly Val Arg
130 135 140
Cys Val Xaa His Cys Lys Asn Pro Leu Glu His Leu Gly Met Cys Cys
145 150 155 160
Pro Thr Cys Pro Gly Cys Val Phe Glu Gly Val Gln Tyr Gln Glu Gly
165 170 175
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Glu Glu Phe Gln Pro Glu Gly Ser Lys Cys Thr Lys Cys Ser Cys Thr
180 185 190
Gly Gly Arg Thr Gln Cys Val Arg Glu Val Cys Pro Ile Leu Ser Cys
195 200 205
Pro Gln His Leu Ser His Ile Pro Pro Gly Gln Cys Cys Pro Lys Cys
210 215 220
Leu Gly Gln Arg Lys Val Phe Asp Leu Pro Phe Gly Ser Cys Leu Phe
225 230 235 240
Arg Ser Asp Val Tyr Asp Asn Gly Ser Ser Phe Leu Tyr Asp Asn Cys
245 250 255
Thr Ala Cys Thr Cys Arg Asp Ser Thr Val Val Cys Lys Arg Lys Cys
260 265 270
Ser His Pro Gly Gly Cys Asp Gln Gly Gln Glu Gly Cys Cys Glu Xaa
275 280 285
Cys Leu Leu Arg Xaa Pro Pro Glu Asp Ile Lys Val Cys Lys Phe Gly
290 295 300
Asn Lys Ile Phe Gln Asp Gly Glu Met Trp Ser Ser Ile Asn Cys Thr
305 310 315 320
Ile Cys Ala Cys Val Lys Gly Arg Thr Glu Cys Xaa Asn Lys Gln Cys
325 330 335
Ile Pro Ile Ser Ser Cys Pro Gln Gly Lys Ile Leu Asn Arg Lys Gly
340 345 350
Cys Cys Pro Ile Cys Thr Glu Lys Pro Gly Val Cys Thr Val Phe Gly
355 360 365
Asp Pro His Tyr Asn Thr Phe Asp Gly Arg Thr Phe Asn Phe Gln Gly
370 375 380
Thr Cys Gln Tyr Val Leu Thr Lys Asp Cys Ser Ser Pro Ala Ser Pro
385 390 395 400
Phe Gln Val Leu Val Lys Asn Asp Ala Arg Arg Thr Arg Ser Phe Ser
405 410 415
Trp Thr Lys Ser Val Glu Leu Val Leu Gly Glu Thr Gly Ser Ala Cys
420 425 . 430
Ser Ser Thr Ser Pro Cys Ala Gly Thr Ala Xaa Ala Ser
435 440 445
<210> 80
<211> 273
<212> PRT
<213> Homo sapiens
<400> 80
CA 02396719 2002-07-24
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Met Leu Phe Phe Cys Gly Asp Leu Leu Ser Arg Ser Gln Ile Phe Tyr
1 5 10 15
Tyr Ser Thr Gly Met Thr Val Gly Ile Val Ala Ser Leu Leu Ile Ile
20 25 30
Ile Phe Ile Leu Ser Lys Phe Met Pro Lys Lys Ser Pro Ile Tyr Val
35 40 45
Ile Leu Val Gly Gly Trp Ser Phe Ser Leu Tyr Leu Ile Gln Leu Val
50 55 60
Phe Lys Asn Leu Gln Glu Ile Trp Arg Cys Tyr Trp Gln Tyr Leu Leu
65 70 75 80
Ser Tyr Val Leu Thr Val Gly Phe Met Ser Phe Ala Val Cys Tyr Lys
85 90 95
Tyr Gly Pro Leu Glu Asn Glu Arg Ser Ile Asn Leu Leu Thr Trp Thr
100 105 110
Leu Gln Leu Met Gly Leu Cys Phe Met Tyr Ser Gly Ile Gln Ile Pro
115 120 125
His Ile Ala Leu Ala Ile Ile Ile Ile Ala Leu Cys Thr Lys Asn Leu
130 135 140
Glu His Pro Ile Gln Trp Leu Tyr Ile Thr Cys Arg Lys Val Cys Lys
145 150 155 160
Gly Ala Glu Lys Pro Val Pro Pro Arg Leu Leu Thr Glu Glu Glu Tyr
165 170 175
Arg Ile Gln Gly Glu Val Glu Thr Arg Lys Ala Leu Glu Glu Leu Arg
180 185 190
Glu Phe Cys Asn Ser Pro Asp Cys Ser Ala Trp Lys Thr Val Ser Arg
195 200 205
Ile Gln Ser Pro Lys Arg Phe Ala Asp Phe Val Glu Gly Ser Ser His
210 215 220
Leu Thr Pro Asn Glu Val Ser Val His Glu Gln Glu Tyr Gly Leu Gly
225 230 235 240
Ser Ile Ile Ala Gln Asp Glu Ile Tyr Glu Glu Ala Ser Ser Glu Glu
245 250 255
Glu Asp Ser Tyr Ser Arg Cys Pro Ala Ile Thr Gln Asn Asn Phe Leu
260 265 270
Thr
<210> 81
<211> 503
<212> PRT
CA 02396719 2002-07-24
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58
<213> Homo sapiens
<400> 81
Met Glu Gln Arg His Val Leu Leu Lys Gln Lys Glu Leu Gly Gly Glu
1 5 10 15
Glu Pro Glu Pro Ser Leu Arg Glu Gly Pro Gly Gly Leu Val Met Glu
20 25 30
Gly His Leu Phe Lys Arg Ala Ser Asn Ala Phe Lys Thr Trp Ser Arg
35 40 45
Arg Trp Phe Thr Ile Gln Ser Asn Gln Leu Val Tyr Gln Lys Lys Tyr
50 55 60
Lys Asp Pro Val Thr Val Val Val Asp Asp Leu Arg Leu Cys Thr Val
65 70 75 80
Lys Leu Cys Pro Asp Ser Glu Arg Arg Phe Cys Phe Glu Val Val Ser
85 90 95
Thr Ser Lys Ser Cys Leu Leu Gln Ala Asp Ser Glu Arg Leu Leu Gln
100 105 110
Leu Trp Val Ser Ala Val Gln Ser Ser Ile Ala Ser Ala Phe Ser Gln
115 120 125
Ala Arg Leu Asp Asp Ser Pro Arg Gly Pro Gly Gln Gly Ser Gly His
130 135 140
Leu Ala Ile Gly Ser Ala Ala Thr Leu Gly Ser Gly Gly Met Ala Arg
145 150 155 160
Gly Arg Glu Pro Gly Gly Val Gly His Val Val Ala Gln Val Gln Ser
165 170 175
Val Asp Gly Asn Ala Gln Cys Cys Asp Cys Arg Glu Pro Ala Pro Glu
180 185 190
Trp Ala Ser Ile Asn Leu Gly Val Thr Leu Cys Ile Gln Cys Ser Gly
195 200 205
Ile His Arg Ser Leu Gly Val His Phe Ser Lys Val Arg Ser Leu Thr
210 215 220
Leu Asp Ser Trp Glu Pro Glu Leu Val Lys Leu Met Cys Glu Leu Gly
225 230 235 240
Asn Val Ile Ile Asn Gln Ile Tyr Glu Ala Arg Val Glu Ala Met Ala
245 250 255
Val Lys Lys Pro Gly Pro Ser Cys Ser Arg Gln Glu Lys Glu Ala Trp
260 265 270
Ile His Ala Lys Tyr Val Glu Lys Lys Phe Leu Thr Lys Leu Pro Glu
275 280 285
Ile Arg Gly Arg Arg Gly Gly Arg Gly Arg Pro Arg Gly Gln Pro Pro
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290 295 300
Val Pro Pro Lys Pro Ser Ile Arg Pro Arg Pro Gly Ser Leu Arg Ser
305 310 315 320
Lys Pro Glu Pro Pro Ser Glu Asp Leu Gly Ser Leu His Pro Gly Ala
325 330 335
Leu Leu Phe Arg Ala Ser Gly His Pro Pro Ser Leu Pro Thr Met Ala
340 345 350
Asp Ala Leu Ala His Gly Ala Asp Val Asn Trp Val Asn Gly Gly Gln
355 360 365
Asp Asn Ala Thr Pro Leu Ile Gln Ala Thr Ala Ala Asn Ser Leu Leu
370 375 380
Ala Cys Glu Phe Leu Leu Gln Asn Gly Ala Asn Val Asn Gln Ala Asp
385 390 395 400
Ser Ala Gly Arg Gly Pro Leu His His Ala Thr Ile Leu Gly His Thr
405 410 415
Gly Leu Ala Cys Leu Phe Leu Lys Arg Gly Ala Asp Leu Gly Ala Arg
420 425 430
Asp Ser Glu Gly Arg Asp Pro Leu Thr Ile Ala Met Glu Thr Ala Asn
435 440 445
Ala Asp Ile Val Thr Leu Leu Arg Leu Ala Lys Met Arg Glu Ala Glu
450 455 460
Ala Ala Gln Gly Gln Ala Gly Asp Glu Thr Tyr Leu Asp Ile Phe Arg
465 470 475 480
Asp Phe Ser Leu Met Ala Ser Asp Asp Pro Glu Lys Leu Ser Arg Arg
485 490 495
Ser His Asp Leu His Thr Leu
500
<210> 82
<211> 230
<212> PRT
<213> Homo sapiens
<220>
<221> SITE
<222> (63)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (66)
<223> Xaa equals any of the naturally occurring L-amino acids
<400> 82
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
Met Gly Asn Ser Leu Ser Val Phe Cys Ser Trp Phe Cys Arg Arg Ser
1 5 10 15
Trp Pro Cys His Arg Gln Pro Ala Arg Leu Val Arg Glu Ala Phe Pro
20 25 30
Ala Gly Arg Ala His Pro Ala Ala Pro Ala Pro Val Pro Ala Arg Gly
35 40 45
Ile Val Gly Arg Phe Pro Leu Leu Phe Asn Arg Gln Arg His Xaa Gly
50 55 60
Pro Xaa Phe Pro Val Arg Trp Asp Gly Ala Pro Met Arg Leu Cys Leu
70 75 80
Ile Pro Arg Asn Thr Gly Thr Pro Gln Arg Val Leu Arg Pro Val Val
85 90 95
Trp Ser Pro Pro Ser Arg Lys Lys Pro Val Leu Ser Pro His Asn Ser
100 105 110
Ile Met Phe Gly His Leu Ser Pro Val Arg Ile Pro Cys Leu Arg Gly
115 120 125
Lys Phe Asn Leu Gln Leu Pro Ser Leu Asp Asp Gln Val Ile Pro Ala
130 135 140
Arg Leu Pro Lys Thr Glu Val Ser Ala Glu Glu Pro Lys Glu Ala Thr
145 150 155 160
Glu Val Lys Asp Gln Val Glu Thr Gln Gly Gln Glu Asp Asn Lys Arg
165 170 175
Gly Pro Cys Ser Asn Gly Glu Ala Ala Ser Thr Ser Arg Pro Leu Glu
180 185 190
Thr Gln Gly Asn Leu Thr Ser Ser Trp Tyr Asn Pro Arg Pro Leu Glu
195 200 205
Gly Asn Val His Leu Lys Ser Leu Thr Glu Lys Asn Gln Thr Asp Lys
210 215 220
Ala Gln Val His Ala Val
225 230
<210> 83
<211> 155
<212> PRT
<213> Homo sapiens
<400> 83
Met Gly Val Val Ser Leu Val Phe Leu Ile Ile Tyr Tyr Leu Asp Pro
1 5 10 15
Ser Val Leu Ser Gly Val Ser Cys Phe Val Met Phe Leu Cys Leu Ala
20 25 30
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Asp Tyr Leu Val Pro Ile Leu Ala Pro Arg Ile Phe Gly Ser Asn Lys
35 40 45
Trp Thr Thr Glu Gln Gln Gln Arg Phe His Glu Ile Cys Ser Asn Leu
50 55 60
Val Lys Thr Arg Arg Arg Ala Val Gly Trp Trp Lys Arg Leu Phe Thr
65 70 75 80
Leu Lys Glu Glu Lys Pro Lys Met Tyr Phe Met Thr Met Ile Val Ser
85 90 95
Leu Ala Ala Val Ala Trp Val Gly Gln Gln Val His Asn Leu Leu Leu
100 105 110
Thr Tyr Leu Ile Val Thr Ser Leu Leu Leu Leu Pro Gly Leu Asn Gln
115 120 125
His Gly Ile Ile Leu Lys Tyr Ile Gly Met Ala Lys Arg Glu Ile Asn
130 135 140
Lys Leu Leu Lys Gln Lys Glu Lys Lys Asn Glu
145 150 155
<210> 84
<211> 155
<212> PRT
<213> Homo sapiens
<220>
<221> SITE
<222> (92)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (105)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (125)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (130)
<223> Xaa equals any of the naturally occurring L-amino acids
<400> 84
Met Gln His Gln Leu His Leu Leu Ile Cys Trp Gly Lys Gly Ser Lys
1 5 10 15
Ser Asn Thr Ser Cys Leu Gly Pro Val Leu Ser Cys Ser Asn Met Trp
20 25 30
Ser Leu Ala Leu Leu Val Val Ala Gly Ser Met Gly Val Ala Tyr Ser
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35 40 45
Ser Val Val Met Tyr Val Leu Leu Trp Val Pro Leu Pro Leu Pro Ser
50 55 60
His Phe Leu Pro Ser Gly Ala Pro Glu Ala Gln Pro Thr Thr Trp Ala
65 70 75 80
Gln Ser Pro His Ser Val Cys Lys Cys Gly Thr Xaa Leu Gly Pro Ala
85 90 95
Lys Pro Gln Gly Pro Ser Leu Pro Xaa Pro Pro Cys Leu Ile Met Leu
100 105 110
Leu Ser Cys Arg Arg Gln Leu Gly Leu Ala Pro Ser Xaa Trp Leu Pro
115 120 125
Gly Xaa Gly Ser His Gly Gly Glu Leu Arg Gly Cys Ser Gln Gly Trp
130 135 140
Ala Pro Gly Ile Ala His Leu Asn Ile Cys Thr
145 150 155
<210> 85
<211> 140
<212> PRT
<213> Homo Sapiens
<400> 85
Ile Pro Leu Cys Ser Ile Phe Gly Ala Leu Ile Ala Val Cys Leu Ile
1 5 10 15
Met Gly Leu Phe Asp Gly Cys Phe Ile Ser Ile Met Ala Pro Ile Ala
20 25 30
Phe Glu Leu Val Gly Ala Gln Asp Val Ser Gln Ala Ile Gly Phe Leu
35 40 45
Leu Gly Phe Met Ser Ile Pro Met Thr Val Gly Pro Pro Ile Ala Gly
50 55 60
Leu Leu Arg Asp Lys Leu Gly Ser Tyr Asp Val Ala Phe Tyr Leu Ala
65 70 75 80
Gly Val Pro Pro Leu Ile Gly Gly Ala Val Leu Cys Phe Ile Pro Trp
85 90 95
Ile His Ser Lys Lys Gln Arg Glu Ile Ser Lys Thr Thr Gly Lys Glu
100 105 110
Lys Met Glu Lys Met Leu Glu Asn Gln Asn Ser Leu Leu Ser Ser Ser
115 120 125
Ser Gly Met Phe Lys Lys Glu Ser Asp Ser Ile Ile
130 135 140
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<210> 86
<211> 25
<212> PRT
<213> Homo Sapiens
<400> 86
Met Ser Pro Ser Ile Arg Ile Leu Leu Val Leu Gln Gln Leu Gly Ser
1 5 10 15
Leu Met Ala Pro Leu Pro Ser Ala His
20 25
<210> 87
<211> 712
<212> PRT
<213> Homo Sapiens
<400> 87
Met Gly Gln Gly Leu Lys Ala Trp Pro Arg Tyr Arg Val Val Gly Ser
1 5 10 15
Ala Asp Ala Gly Gln Tyr Asn Leu Glu Ile Thr Asp Ala Glu Leu Ser
20 25 30
Asp Asp Ala Ser Tyr Glu Cys Gln Ala Thr Glu Ala Ala Leu Arg Ser
35 40 45
Arg Arg Ala Lys Leu Thr Val Leu Ile Pro Pro Glu Asp Thr Arg Ile
50 55 60
Asp Gly Gly Pro Val Ile Leu Leu Gln Ala Gly Thr Pro His Asn Leu
65 70 75 80
Thr Cys Arg Ala Phe Asn Ala Lys Pro Ala Ala Thr Ile Ile Trp Phe
85 90 95
Arg Asp Gly Thr Gln Gln Glu Gly Ala Val Ala Ser Thr Glu Leu Leu
100 105 110
Lys Asp Gly Lys Arg Glu Thr Thr Val Ser Gln Leu Leu Ile Asn Pro
115 120 125
Thr Asp Leu Asp Ile Gly Arg Val Phe Thr Cys Arg Ser Met Asn Glu
130 135 140
Ala Ile Pro Ser Gly Lys Glu Thr Ser Ile Glu Leu Asp Val His His
145 150 155 160
Pro Pro Thr Val Thr Leu Ser Ile Glu Pro Gln Thr Val Gln Glu Gly
165 170 175
Glu Arg Val Val Phe Thr Cys Gln Ala Thr Ala Asn Pro Glu Ile Leu
180 185 190
Gly Tyr Arg Trp Ala Lys Gly Gly Phe Leu Ile Glu Asp Ala His Glu
195 200 205
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Ser Arg Tyr Glu Thr Asn Val Asp Tyr Ser Phe Phe Thr Glu Pro Val
210 215 220
Ser Cys Glu Val His Asn Lys Val Gly Ser Thr Asn Val Ser Thr Leu
225 230 235 240
Val Asn Val His Phe Ala Pro Arg Ile Val Val Asp Pro Lys Pro Thr
245 250 255
Thr Thr Asp Ile Gly Ser Asp Val Thr Leu Thr Cys Val Trp Val Gly
260 265 270
Asn Pro Pro Leu Thr Leu Thr Trp Thr Lys Lys Asp Ser Asn Met Gly
275 280 285
Pro Arg Pro Pro Gly Ser Pro Pro Glu Ala Ala Leu Ser Ala Gln Val
290 295 300
Leu Ser Asn Ser Asn Gln Leu Leu Leu Lys Ser Val Thr Gln Ala Asp
305 310 315 320
Ala Gly Thr Tyr Thr Cys Arg Ala Ile Val Pro Arg Ile Gly Val Ala
325 330 335
Glu Arg Glu Val Pro Leu Tyr Val Asn Gly Pro Pro Ile Ile Ser Ser
340 345 350
Glu Ala Val Gln Tyr Ala Val Arg Gly Asp Gly Gly Lys Val Glu Cys
355 360 365
Phe Ile Gly Ser Thr Pro Pro Pro Asp Arg Ile Ala Trp Ala Trp Lys
370 375 380
Glu Asn Phe Leu Glu Val Gly Thr Leu Glu Arg Tyr Thr Val Glu Arg
385 390 395 400
Thr Asn Ser Gly Ser Gly Val Leu Ser Thr Leu Thr Ile Asn Asn Val
405 410 415
Met Glu Ala Asp Phe Gln Thr His Tyr Asn Cys Thr Ala Trp Asn Ser
420 425 430
Phe Gly Pro Gly Thr Ala Ile Ile Gln Leu Glu Glu Arg Glu Val Leu
435 440 445
Pro Val Gly Ile Ile Ala Gly Ala Thr Ile Gly Ala Ser Ile Leu Leu
450 455 460
Ile Phe Phe Phe Ile Ala Leu Val Phe Phe Leu Tyr Arg Arg Arg Lys
465 470 475 480
Gly Ser Arg Lys Asp Val Thr Leu Arg Lys Leu Asp Ile Lys Val Glu
485 490 495
Thr Val Asn Arg Glu Pro Leu Thr Met His Ser Asp Arg Glu Asp Asp
500 505 510
Thr Ala Ser Val Ser Thr Ala Thr Arg Val Met Lys Ala Ile Tyr Ser
CA 02396719 2002-07-24
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515 520 525
Ser Phe Lys Asp Asp Val Asp Leu Lys Gln Asp Leu Arg Cys Asp Thr
530 535 540
Ile Asp Thr Arg Glu Glu Tyr Glu Met Lys Asp Pro Thr Asn Gly Tyr
545 550 555 560
Tyr Asn Val Arg Ala His Glu Asp Arg Pro Ser Ser Arg Ala Val Leu
565 570 575
Tyr Ala Asp Tyr Arg Ala Pro Gly Pro Ala Arg Phe Asp Gly Arg Pro
580 585 590
Ser Ser Arg Leu Ser His Ser Ser Gly Tyr Ala Gln Leu Asn Thr Tyr
595 600 605
Ser Arg Gly Pro Ala Ser Asp Tyr Gly Pro Glu Pro Thr Pro Pro Gly
610 615 620
Pro Ala Ala Pro Ala Gly Thr Asp Thr Thr Ser Gln Leu Ser Tyr Glu
625 630 635 640
Asn Tyr Glu Lys Phe Asn Ser His Pro Phe Pro Gly Ala Ala Gly Tyr
645 650 655
Pro Thr Tyr Arg Leu Gly Tyr Pro Gln Ala Pro Pro Ser Gly Leu Glu
660 665 670
Arg Thr Pro Tyr Glu Ala Tyr Asp Pro Ile Gly Lys Tyr Ala Thr Ala
675 680 685
Thr Arg Phe Ser Tyr Thr Ser Gln His Ser Asp Tyr Gly Gln Arg Phe
690 695 700
Gln Gln Arg Met Gln Thr His Val
705 710
<210> 88
<211> 213
<212> PRT
<213> Homo sapiens
<400> 88
Met Gln Ile Gln Val Ala Gly Leu Leu Gln Phe Ala Val Pro Leu Phe
1 5 10 15
Ser Thr Ala Glu Glu Asp Leu Leu Ala Ile Gln Leu Leu Leu Asn Ser
20 25 30
Ser Glu Ser Ser Leu His Gln Leu Thr Ala Met Val Asp Cys Arg Gly
35 40 45
Leu His Lys Asp Tyr Leu Asp Ala Leu Ala Gly Ile Cys Tyr Asp Gly
50 55 60
Leu Gln Gly Leu Leu Tyr Leu Gly Leu Phe Ser Phe Leu Ala Ala Leu
CA 02396719 2002-07-24
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65 70 75 80
Ala Phe Ser Thr Met Ile Cys Ala Gly Pro Arg Ala Trp Lys His Phe
85 90 95
Thr Thr Arg Asn Arg Asp Tyr Asp Asp Ile Asp Asp Asp Asp Pro Phe
100 105 110
Asn Pro Gln Ala Trp Arg Met Ala Ala His Ser Pro Pro Arg Gly Gln
115 120 125
Leu His Ser Phe Cys Ser Tyr Ser Ser Gly Leu Gly Ser Gln Thr Ser
130 135 140
Leu Gln Pro Pro Ala Gln Thr Ile Ser Asn Ala Pro Val Ser Glu Tyr
145 150 155 160
Met Asn Gln Ala Met Leu Phe Gly Arg Asn Pro Arg Tyr Glu Asn Val
165 170 175
Pro Leu Ile Gly Arg Ala Ser Pro Pro Pro Thr Tyr Ser Pro Ser Met
180 185 190
Arg Ala Thr Tyr Leu Ser Val Ala Asp Glu His Leu Arg His Tyr Gly
195 200 205
Asn Gln Phe Pro Ala
210
<210> 89
<211> 346
<212> PRT
<213> Homo sapiens
<400> 89
Met Val Glu Lys Glu Glu Ala Gly Gly Gly Ile Ser Glu Glu Glu Ala
1 5 10 15
Ala Gln Tyr Asp Arg Gln Ile Arg Leu Trp Gly Leu Glu Ala Gln Lys
20 25 30
Arg Leu Arg Ala Ser Arg Val Leu Leu Val Gly Leu Lys Gly Leu Gly
35 40 45
Ala Glu Ile Ala Lys Asn Leu Ile Leu Ala Gly Val Lys Gly Leu Thr
50 55 60
Met Leu Asp His Glu Gln Val Thr Pro Glu Asp Pro Gly Ala Gln Phe
65 70 75 80
Leu Ile Arg Thr Gly Ser Val Gly Arg Asn Arg Ala Glu Ala Ser Leu
85 90 95
Glu Arg Ala Gln Asn Leu Asn Pro Met Val Asp Val Lys Val Asp Thr
100 105 110
Glu Asp Ile Glu Lys Lys Pro Glu Ser Phe Phe Thr Gln Phe Asp Ala
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115 120 125
Val Cys Leu Thr Cys Cys Ser Arg Asp Val Ile Val Lys Val Asp Gln
130 135 140
Ile Cys His Lys Asn Ser Ile Lys Phe Phe Thr Gly Asp Val Phe Gly
145 150 155 160
Tyr His Gly Tyr Thr Phe Ala Asn Leu Gly Glu His Glu Phe Val Glu
165 170 1'75
Glu Lys Thr Lys Val Ala Lys Val Ser Gln Gly Val Glu Asp Gly Pro
180 185 190
Asp Thr Lys Arg Ala Lys Leu Asp Ser Ser Glu Thr Thr Met Val Lys
195 200 205
Lys Lys Val Val Phe Cys Pro Val Lys Glu Ala Leu Glu Val Asp Trp
210 215 220
Ser Ser Glu Lys Ala Lys Ala Ala Leu Lys Arg Thr Thr Ser Asp Tyr
225 230 235 240
Phe Leu Leu Gln Val Leu Leu Lys Phe Arg Thr Asp Lys Gly Arg Asp
245 250 255
Pro Ser Ser Asp Thr Tyr Glu Glu Asp Ser Glu Leu Leu Leu Gln Ile
260 265 270
Arg Asn Asp Val Leu Asp Ser Leu Gly Ile Ser Pro Asp Leu Leu Pro
275 280 285
Glu Asp Phe Val Arg Tyr Cys Phe Ser Glu Met Ala Pro Val Cys Ala
290 295 300
Val Val Gly Gly Ile Leu Ala Gln Glu Ile Val Lys Ala Leu Ser Gln
305 310 315 320
Arg Asp Pro Pro His Asn Asn Phe Phe Phe Phe Asp Gly Met Lys Gly
325 330 335
Asn Gly Ile Val Glu Cys Leu Gly Pro Lys
340 345
<210> 90
<211> 261
<212> PRT
<213> Homo sapiens
<220>
<221> SITE
<222> (125)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (127)
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<223> Xaa equals any of the naturally occurring L-amino acids
<400> 90
Met Ala Val Thr Ala Cys Gln Gly Leu Gly Phe Val Val Ser Leu Ile
1 5 10 15
Gly Ile Ala Gly Ile Ile Ala Ala Thr Cys Met Asp Gln Trp Ser Thr
20 25 30
Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala Val Phe Asn Tyr Gln Gly
35 40 45
Leu Trp Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg
50 55 60
Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg
65 70 75 80
Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val
85 90 95
Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser
100 105 110
Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Xaa Ile Xaa Ser
115 120 125
Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val
130 135 140
Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly
145 150 155 160
Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe
165 170 175
Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met
180 185 190
Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala
195 200 205
Val Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly
210 215 220
Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Arg
225 230 235 240
Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser
245 250 255
Lys His Asp Tyr Val
260
<210> 91
<211> 360
<212> PRT
CA 02396719 2002-07-24
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<213> Homo Sapiens
<400> 91
Met Ala Ala Glu Trp Ala Ser Arg Phe Trp Leu Trp Ala Thr Leu Leu
1 5 10 15
Ile Pro Ala Ala Ala Val Tyr Glu Asp Gln Val Gly Lys Phe Asp Trp
20 25 30
Arg Gln Gln Tyr Val Gly Lys Val Lys Phe Ala Ser Leu Glu Phe Ser
35 40 45
Pro Gly Ser Lys Lys Leu Val Val Ala Thr Glu Lys Asn Val Ile Ala
50 55 60
Ala Leu Asn Ser Arg Thr Gly Glu Ile Leu Trp Arg His Val Asp Lys
65 70 75 80
Gly Thr Ala Glu Gly Ala Val Asp Ala Met Leu Leu His Gly Gln Asp
85 90 95
Val Ile Thr Val Ser Asn Gly Gly Arg Ile Met Arg Ser Trp Glu Thr
100 105 110
Asn Ile Gly Gly Leu Asn Trp Glu Ile Thr Leu Asp Ser Gly Ser Phe
115 120 125
Gln Ala Leu Gly Leu Val Gly Leu Gln Glu Ser Val Arg Tyr Ile Ala
130 135 140
Val Leu Lys Lys Thr Thr Leu Ala Leu His His Leu Ser Ser Gly His
145 150 155 160
Leu Lys Trp Val Glu His Leu Pro Glu Ser Asp Ser Ile His Tyr Gln
165 170 175
Met Val Tyr Ser Tyr Gly Ser Gly Val Val Trp Ala Leu Gly Val Val
180 185 190
Pro Phe Ser His Val Asn Ile Val Lys Phe Asn Val Glu Asp Gly Glu
195 200 205
Ile Val Gln Gln Val Arg Val Ser Thr Pro Trp Leu Gln His Leu Ser
210 215 220
Gly Ala Cys Gly Val Val Asp Glu Ala Val Leu Val Cys Pro Asp Pro
225 230 235 240
Ser Ser Arg Ser Leu Gln Thr Leu Ala Leu Glu Thr Glu Trp Glu Leu
245 250 255
Arg Gln Ile Pro Leu Gln Ser Leu Asp Leu Glu Phe Gly Ser Gly Phe
260 265 270
Gln Pro Arg Val Leu Pro Thr Gln Pro Asn Pro Val Asp Ala Ser Arg
275 280 285
Ala Gln Phe Phe Leu His Leu Ser Pro Ser His Tyr Ala Leu Leu Gln
CA 02396719 2002-07-24
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290 295 300
Tyr His Tyr Gly Thr Leu Ser Leu Leu Lys Asn Phe Pro Gln Thr Ala
305 310 315 320
Leu Val Ser Phe Ala Thr Thr Gly Glu Lys Thr Val Ala Ala Val Met
325 330 335
Ala Cys Arg Asn Glu Val Gln Lys Thr Ser Ser Ser Glu Asp Gly Ser
340 345 350
Met Gly Glu Leu Phe Gly Glu Val
355 360
<210> 92
<211> 485
<212> PRT
<213> Homo Sapiens
<400> 92
Ala Arg Lys Pro Arg Ser Gln Ile Lys Asn Glu Ile Asn Ile Asp Thr
1 5 10 15
Leu Ala Arg Asp Glu Phe Asn Leu Gln Lys Met Met Val Met Val Thr
20 25 30
Ala Ser Gly Lys Leu Phe Gly Ile Glu Ser Ser Ser Gly Thr Ile Leu
35 40 45
Trp Lys Gln Tyr Leu Pro Asn Val Lys Pro Asp Ser Ser Phe Lys Leu
50 55 60
Met Val Gln Arg Thr Thr Ala His Phe Pro His Pro Pro Gln Cys Thr
65 70 75 80
Leu Leu Val Lys Asp Lys Glu Ser Gly Met Ser Ser Leu Tyr Val Phe
85 90 95
Asn Pro Ile Phe Gly Lys Trp Ser Gln Val Ala Pro Pro Val Leu Lys
100 105 110
Arg Pro Ile Leu Gln Ser Leu Leu Leu Pro Val Met Asp Gln Asp Tyr
115 120 125
Ala Lys Val Leu Leu Leu Ile Asp Asp Glu Tyr Lys Val Thr Ala Phe
130 135 140
Pro Ala Thr Arg Asn Val Leu Arg Gln Leu His Glu Leu Ala Pro Ser
145 150 155 160
Ile Phe Phe Tyr Leu Val Asp Ala Glu Gln Gly Arg Leu Cys Gly Tyr
165 170 175
Arg Leu Arg Lys Asp Leu Thr Thr Glu Leu Ser Trp Glu Leu Thr Ile
180 185 190
Pro Pro Glu Val Gln Arg Ile Val Lys Val Lys Gly Lys Arg Ser Ser
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195 200 205
Glu His Val His Ser Gln Gly Arg Val Met Gly Asp Arg Ser Val Leu
210 215 220
Tyr Lys Ser Leu Asn Pro Asn Leu Leu Ala Val Val Thr Glu Ser Thr
225 230 235 240
Asp Ala His His Glu Arg Thr Phe Ile Gly Ile Phe Leu Ile Asp Gly
245 250 255
Val Thr Gly Arg Ile Ile His Ser Ser Val Gln Lys Lys Ala Lys Gly
260 265 270
Pro Val His Ile Val His Ser Glu Asn Trp Val Val Tyr Gln Tyr Trp
275 280 285
Asn Thr Lys Ala Arg Arg Asn Glu Phe Thr Val Leu Glu Leu Tyr Glu
290 295 300
Gly Thr Glu Gln Tyr Asn Ala Thr Ala Phe Ser Ser Leu Asp Arg Pro
305 310 315 320
Gln Leu Pro Gln Val Leu Gln Gln Ser Tyr Ile Phe Pro Ser Ser Ile
325 330 335
Ser Ala Met Glu Ala Thr Ile Thr Glu Arg Gly Ile Thr Ser Arg His
340 345 350
Leu Leu Ile Gly Leu Pro Ser Gly Ala Ile Leu Ser Leu Pro Lys Ala
355 360 365
Leu Leu Asp Pro Arg Arg Pro Glu Ile Pro Thr Glu Gln Ser Arg Glu
370 375 380
Glu Asn Leu Ile Pro Tyr Ser Pro Asp Val Gln Ile His Ala Glu Arg
385 390 395 400
Phe Ile Asn Tyr Asn Gln Thr Val Ser Arg Met Arg Gly Ile Tyr Thr
405 410 415
Ala Pro Ser Gly Leu Glu Ser Thr Cys Leu Val Val Ala Tyr Gly Leu
420 425 430
Asp Ile Tyr Gln Thr Arg Val Tyr Pro Ser Lys Gln Phe Asp Val Leu
435 440 445
Lys Asp Asp Tyr Asp Tyr Val Leu Ile Ser Ser Val Leu Phe Gly Leu
450 455 460
Val Phe Ala Thr Met Ile Thr Lys Arg Leu Ala Gln Val Lys Leu Leu
465 470 475 480
Asn Arg Ala Trp Arg
485
<210> 93
CA 02396719 2002-07-24
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<211> 149
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (140)
<223> Xaa equals any of the naturally occurring L-amino acids
<400> 93
Met Val Leu Leu Trp Ala Ser Val Leu Phe Pro Ala Pro Glu Asp Trp
1 5 10 15
Ala Glu Leu Gln Gly Ala Val Tyr Arg Leu Leu Val Val Leu Leu Cys
20 25 30
Cys Leu Ala Thr Arg Lys Leu Pro His Phe Leu His Pro Gln Arg Asn
35 40 45
Leu Leu Gln Gly Ser Gly Leu Asp Leu Gly Ala Ile Tyr Gln Arg Val
50 55 60
Glu Gly Phe Ala Ser Gln Pro Glu Ala Ala Leu Arg Ile His Ala Thr
65 70 75 80
His Leu Gly Arg Ser Pro Pro Pro Arg Ile Gly Ser Gly Leu Lys Ala
85 90 95
Leu Leu Gln Leu Pro Ala Ser Asp Pro Thr Tyr Trp Ala Thr Ala Tyr
100 105 110
Phe Asp Val Leu Leu Asp Lys Phe Gln Val Phe Asn Ile Gln Asp Lys
115 120 125
Asp Arg Ile Ser Ala Met Gln Ser Ile Phe Gln Xaa Thr Arg Thr Leu
130 135 140
Gly Gly Glu Glu Ser
145
<210> 94
<211> 209
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (136)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (161)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
CA 02396719 2002-07-24
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73
<222> (169)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (197)
<223> Xaa equals any of the naturally occurring L-amino acids
<400> 94
Met Ala Met Gly Leu Phe Arg Val Cys Leu Val Val Val Thr Ala Ile
1 5 10 15
Ile Asn His Pro Leu Leu Phe Pro Arg Glu Asn Ala Thr Val Pro Glu
20 25 30
Asn Glu Glu Glu Ile Ile Arg Lys Met Gln Ala His Gln Glu Lys Leu
35 40 45
Gln Leu Glu Gln Leu Arg Leu Glu Glu Glu Val Ala Arg Leu Ala Ala
50 55 60
Glu Lys Glu Ala Leu Glu Gln Val Ala Glu Glu Gly Arg Gln Gln Asn
65 70 75 80
Glu Thr Arg Val Ala Trp Asp Leu Trp Ser Thr Leu Cys Met Ile Leu
85 90 95
Phe Leu Met Ile Glu Val Trp Arg Gln Asp His Gln Glu Gly Pro Ser
100 105 110
Pro Glu Cys Leu Gly Gly Glu Glu Asp Glu Leu Pro Gly Trp Gly Ala
115 120 125
Pro Pro Cys Arg Ala Ser Pro Xaa Pro Thr Arg His Ala Cys His Phe
130 135 140
Tyr Glu Arg Cys Ile Arg Gly Ala Thr Ala Asp Ala Ala Arg Thr Arg
145 150 155 160
Xaa Phe Leu Glu Gly Phe Val Asp Xaa Leu Leu Glu Ala Leu Arg Ser
165 170 175
Leu Cys Asn Arg Asp Thr Asp Met Glu Val Glu Asp Phe Ile Gly Val
180 185 190
Asp Ser Met Tyr Xaa Asn Trp Gln Val Asp Arg Pro Leu Leu Cys His
195 200 205
Leu
<210> 95
<211> 107
<212> PRT
<213> Homo sapiens
<400> 95
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Met Leu Val Leu Leu Leu Asp Phe Leu Gly Leu Val His Leu Gly Gln
1 5 10 15
Leu Leu Ile Phe His Ile Tyr Leu Lys Ala Lys Lys Met Thr Thr Phe
20 25 30
Glu Tyr Leu Ile Asn Asn Arg Lys Glu Glu Ser Ser Lys His Gln Ala
35 40 45
Val Arg Lys Asp Pro Tyr Val Gln Met Asp Lys Gly Val Leu Gln Gln
50 55 60
Gly Ala Gly Ala Leu Gly Ser Ser Ala Gln Gly Val Lys Ala Lys Ser
65 70 75 80
Ser Leu Leu Ile His Lys His Leu Cys His Phe Cys Thr Ser Val Asn
85 90 95
Gln Asp Gly Asp Ser Thr Ala Arg Val His Leu
100 105
<210> 96
<211> 6
<212> PRT
<213> Homo Sapiens
<400> 96
Val Phe Gln Ile Tyr Leu
1 5
<210> 97
<211> 6
<212> PRT
<213> Homo Sapiens
<400> 97
Val Phe Gln Ile Tyr Leu
1 5
<210> 98
<211> 357
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (140)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (325)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
CA 02396719 2002-07-24
WO 01/54708 PCT/USO1/01434
<221> SITE
<222> (329)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (335)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (338)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (339)
<223> Xaa equals any of the naturally occurring L-amino acids
<400> 98
Met Gly Met Gly Arg Gly Ala Gly Arg Ser Ala Leu Gly Phe Trp Pro
1 5 10 15
Thr Leu Ala Phe Leu Leu Cys Ser Phe Pro Ala Ala Thr Ser Pro Cys
20 25 30
Lys Ile Leu Lys Cys Asn Ser Glu Phe Trp Ser Ala Thr Ser Gly Ser
35 40 45
His Ala Pro Ala Ser Asp Asp Thr Pro Glu Phe Cys Ala Ala Leu Arg
50 55 60
Ser Tyr Ala Leu Cys Thr Arg Arg Thr Ala Arg Thr Cys Arg Gly Asp
65 70 75 80
Leu Ala Tyr His Ser Ala Val His Gly Ile Glu Asp Leu Met Ser Gln
90 95
His Asn Cys Ser Lys Asp Gly Pro Thr Ser Gln Pro Arg Leu Arg Thr
100 105 110
Leu Pro Pro Ala Gly Asp Ser Gln Glu Arg Ser Asp Ser Pro Glu Ile
115 120 125
Cys His Tyr Glu Lys Ser Phe His Lys His Ser Xaa Thr Pro Asn Tyr
130 135 140
Thr His Cys Gly Leu Phe Gly Asp Pro His Leu Arg Thr Phe Thr Asp
145 150 155 160
Arg Phe Gln Thr Cys Lys Val Gln Gly Ala Trp Pro Leu Ile Asp Asn
165 170 175
Asn Tyr Leu Asn Val Gln Val Thr Asn Thr Pro Val Leu Pro Gly Ser
180 185 190
Ala Ala Thr Ala Thr Ser Lys Leu Thr Ile Ile Phe Lys Asn Phe Gln
195 200 205
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Glu Cys Val Asp Gln Lys Val Tyr Gln Ala Glu Met Asp Glu Leu Pro
210 215 220
Ala Ala Phe Val Asp Gly Ser Lys Asn Gly Gly Asp Lys His Gly Ala
225 230 235 240
Asn Ser Leu Lys Ile Thr Glu Lys Val Ser Gly Gln His Val Glu Ile
245 250 255
Gln Ala Lys Tyr Ile Gly Thr Thr Ile Val Val Arg Gln Val Gly Arg
260 265 270
Tyr Leu Thr Phe Ala Val Arg Met Pro Glu Glu Val Val Asn Ala Val
275 280 285
Glu Asp Trp Asp Ser Gln Gly Leu Tyr Leu Cys Leu Arg Gly Cys Pro
290 295 300
Leu Asn Gln Gln Ile Asp Phe Gln Ala Phe His Thr Asn Ala Glu Gly
305 310 315 320
Thr Gly Ala Arg Xaa Leu Ala Ala Xaa Ser Leu Asp Pro Gln Xaa Pro
325 330 335
Arg Xaa Xaa His Thr Arg Gln Ala Val Ala Lys Cys Lys Glu Lys Leu
340 345 350
Pro Val Glu Asp Leu
355
<210> 99
<211> 179
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (91)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (140)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (169)
<223> Xaa equals any of the naturally occurring L-amino acids
<400> 99
Met Gly Met Gly Arg Gly Ala Gly Arg Ser Ala Leu Gly Phe Trp Pro
1 5 10 15
Thr Leu Ala Phe Leu Leu Cys Ser Phe Pro Ala Ala Thr Ser Pro Cys
20 25 30
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Lys Ile Leu Lys Cys Asn Ser Glu Phe Trp Ser Ala Thr Ser Gly Ser
35 40 45
His Ala Pro Ala Ser Asp Asp Thr Pro Glu Phe Cys Ala Ala Leu Arg
50 55 60
Ser Tyr Ala Leu Cys Thr Arg Arg Thr Ala Arg Thr Cys Arg Gly Asp
65 70 75 80
Leu Ala Tyr His Ser Ala Val His Gly Ile Xaa Asp Leu Met Ser Gln
85 90 95
His Asn Cys Ser Lys Asp Gly Pro Thr Ser Gln Pro Arg Leu Arg Thr
100 105 110
Leu Pro Pro Ala Glu Thr Ala Arg Ser Ala Arg Thr Ala Pro Arg Ser
115 120 125
Ala Ile Thr Arg Arg Ala Phe Thr Ser Thr Arg Xaa Pro Pro Thr Thr
130 135 140
Arg Thr Val Ala Ser Ser Gly Thr His Thr Phe Arg Thr Phe Thr Asp
145 150 155 160
Arg Phe Gln Thr Cys Lys Val Gln Xaa Arg Leu Ala Ala His Arg Gln
165 170 175
Leu Ile Thr
<210> 100
<211> 213
<212> PRT
<213> Homo sapiens
<400> 100
Leu Leu Phe Leu Ser Leu Leu Gln Met Gln Glu Leu Leu Gly Arg Gly
1 5 10 15
Ala Trp Ala Pro Gly Cys Gly Arg Arg Pro Ser Gly Trp Gly Gln Leu
20 25 30
Ala Cys Pro Asp Pro Leu Leu Pro Pro His Asn Pro Lys Ser Pro Gln
35 40 45
Pro Gly Pro Ser Thr Ser Gly Val Trp Gly Glu Glu Gln Gly Leu Arg
50 55 60
Thr Leu Ser Ser Glu His Pro Trp Gln Gly Leu Gln Pro Leu Ile Ser
65 70 75 80
Ser Leu Lys Pro Cys Gly His Thr Ala Arg Arg Asp Leu Pro Leu Ala
85 90 95
Pro Ala Ser Phe Gln Pro Arg Val Leu Ile Gln Gly Pro Arg Thr Val
100 105 110
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Pro Pro Val Leu Leu Cys Pro Gln His Lys Ala Arg Leu His Ser Gln
115 120 125
Lys Cys Ser Gln Ala Leu Glu Gly Asp Pro Ala Ser Ser Pro Thr Ala
130 135 140
Pro His Pro Thr His Pro Ser Ala Ala Pro Leu Leu Phe Pro Arg Asp
145 150 155 160
Leu Ser Tyr Thr Gly Gln Glu Ala Ala Glu Arg Val Ser Pro Pro Pro
165 170 175
Ser Lys Arg Ser Cys Ser Leu Cys Gln Asn Arg Val Trp Ala Gly Gly
180 185 190
Arg Ala Leu Gly Ala Arg Pro Leu Pro Leu Pro Ala Gly Phe Ser Trp
195 200 205
Ser Leu Cys Trp Lys
210
<210> 101
<211> 94
<212> PRT
<213> Homo sapiens
<400> 101
Met Leu Leu Gly Phe Leu Val Leu Ile Pro Trp Gly Ser Leu Ile Leu
1 5 10 15
Gly Ser Ser Asp Leu Asp Pro Ser Ser Leu Pro Leu Gly Thr Arg Gly
20 25 30
His Gly Trp Arg Trp Pro Pro Leu Ser Pro Val Gln Ile Leu Tyr Pro
35 40 45
Leu Ala Gly Asp Pro His Ala Ala Val Ser Cys Ser Cys Cys Gly Glu
50 55 60
Thr Glu Leu Arg Ala Leu Leu Thr Gly Ser Leu Pro Met Glu Ala Phe
65 70 75 80
Ser Gly Leu His Ser Ile Glu Tyr Ser Ser Arg Thr Ala Cys
85 90
<210> 102
<211> 135
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (125)
<223> Xaa equals any of the naturally occurring L-amino acids
CA 02396719 2002-07-24
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79
<400> 102
Met Leu His Leu Lys Ala Leu Gly Arg Gly Leu Leu Gln Ala Cys Leu
1 5 10 15
Leu Ala Thr Gly Gly Ser Met Ala Cys Asp Ser Ile Thr Pro Ile Phe
20 25 30
Thr Gln His Ser Pro Arg Val Pro Leu Ser Pro Ser Phe Pro Leu Leu
35 40 45
Glri Glu Thr Gly Ala Phe Ala Pro Ser Pro Arg Leu Glu Gly Ser Gly
50 55 60
Met Ile Leu Ala His Cys Cys Leu Glu Leu Leu Cys Ser Ser Asp Pro
65 70 75 80
Pro Ala Ser Ala Ile Gln Asn Cys Trp Asp Tyr Arg Cys Glu Pro Arg
85 90 95
His Leu Ala Ser Ile Ser Ser Phe Tyr Lys Asp Ile Gly Arg Ile Gly
100 105 110
Leu Gly Pro Val Leu Leu Gln Tyr Asn Leu Ile Leu Xaa Asn Tyr Ile
115 120 125
Cys His Asn Ser Val Ser Lys
130 135
<210> 103
<211> 92
<212> PRT
<213> Homo sapiens
<220>
<221> SITE
<222> (68)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (86)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (90)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (92)
<223> Xaa equals any of the naturally occurring L-amino acids
<400> 103
Met Leu His Leu Lys Ala Leu Gly Arg Gly Leu Leu Gln Ala Cys Leu
1 5 10 15
CA 02396719 2002-07-24
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Leu Ala Thr Gly Gly Ser Met Ala Cys Asp Ser Ile Thr Pro Ile Phe
20 25 30
Thr Gln His Ser Pro Arg Val Pro Leu Ser Pro Ser Phe Pro Leu Leu
35 40 45
Gln Glu Thr Gly Ala Phe Ala Pro Ser Pro Arg Leu Glu Gly Ser Gly
50 55 60
Met Ile Leu Xaa His Cys Cys Leu Glu Leu Leu Cys Ser Ser Asp Pro
65 70 75 80
Pro Ala Ser Ala Ile Xaa Asn Cys Trp Xaa Tyr Xaa
90
<210> 104
<211> 75
<212> PRT
<213> Homo Sapiens
<400> 104
Met Leu Asn Pro Leu Arg Gln Leu Phe Lys Leu Met Ala Ser Leu Phe
1 5 10 15
Leu Ser Val Phe Thr Leu Gly Leu Pro Phe Ala Leu Phe Gln Tyr Tyr
20 25 30
Ala Tyr Thr Gln Phe Cys Leu Pro Gly Ser Ala Arg Pro Ile Pro Glu
35 40 45
Pro Leu Val Gln Leu Ala Val Asp Lys Gly Tyr Arg Ile Ala Glu Gly
50 55 60
Asn Glu Pro Leu Gly Ala Ser Gly Met Phe His
65 70 75
<210> 105
<211> 28
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (3)
<223> Xaa equals any of the naturally occurring L-amino acids
<400> 105
Glu Cys Xaa Arg Lys Pro Thr Pro Arg Ala Glu Phe Leu Gln Pro Gly
1 5 10 15
Gly Ser Thr Ser Ser Arg Ala Ala Ala Thr Ala Val
20 25
<210> 106
<211> 105
CA 02396719 2002-07-24
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81
<212> PRT
<213> Homo sapiens
<220>
<221> SITE
<222> (69)
<223> Xaa equals any of the naturally occurring L-amino acids
<400> 106
Met Phe Ala Leu Ala Trp Lys Val Ile Phe Ser Val Met Leu Gln Asn
1 5 10 15
Pro Ile Arg Tyr Pro Ser Val Leu Gly Ile Lys Ser Ser Leu Leu Ser
20 25 30
Ser Leu Val Leu Val Met Val Trp Gly Asn Glu Lys Ser Gly Pro Cys
35 40 45
Pro Thr Pro Lys Ser Arg Lys Gly Arg Arg Ser Cys Pro Ala Gln Val
50 55 60
Gly Arg Gly Glu Xaa Gly Ser Tyr Trp Asp Pro Glu Phe Arg Leu Ser
65 70 75 80
Arg Lys Ser Asn Gln Gly Leu Arg Arg Asp Tyr Leu Ser Leu Tyr His
85 90 95
Phe Asn Leu His Phe Arg Asp Thr Phe
100 105
<210> 107
<211> 92
<212> PRT
<213> Homo sapiens
<400> 107
Met Thr Ser Leu Leu Glu Gly Arg Met Val Leu Cys Val Ser Cys Leu
1 5 10 15
Leu Leu Pro Leu Leu Leu Leu Leu Lys His Phe Asn Gly Leu Met Thr
20 25 30
Pro Tyr Leu Ala His Asn Val Tyr Cys Pro Ile Glu Tyr Ile Ser Phe
35 40 45
Phe Pro Phe His Glu Lys Asn Ile Glu Tyr Ile Ser Ile Trp Phe Ile
50 55 60
Phe Asp Ser Phe Lys Phe Ile Tyr Ser Arg Leu Leu Cys Ile Ser Gln
65 70 75 80
Ile Tyr Val Leu Tyr Arg Ala Tyr Thr Leu Pro His
85 90
<210> 108
<211> 92
CA 02396719 2002-07-24
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82
<212> PRT
<213> Homo.sapiens
<400> 108
Met Thr Ser Leu Leu Glu Gly Arg Met Val Leu Cys Val Ser Cys Leu
1 5 10 15
Leu Leu Pro Leu Leu Leu Leu Leu Lys His Phe Asn Gly Leu Met Thr
20 25 30
Pro Tyr Leu Ala His Asn Val Tyr Cys Pro Ile Glu Tyr Ile Ser Phe
35 40 45
Phe Pro Phe His Glu Lys Asn Ile Glu Tyr Ile Ser Ile Trp Phe Ile
50 55 60
Phe Asp Ser Phe Lys Phe Ile Tyr Ser Arg Leu Leu Cys Ile Ser Gln
65 70 75 80
Ile Tyr Val Leu Tyr Arg Ala Tyr Thr Leu Pro His
85 90
<210> 109
<211> 182
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (104)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (147)
<223> Xaa equals any of the riaturally occurring L-amino acids
<220>
<221> SITE
<222> (151)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (176)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (179)
<223> Xaa equals any of the naturally occurring L-amino acids
<400> 109
Met Leu Trp Phe Ser Gly Val Gly Ala Leu Ala Glu Arg Tyr Cys Arg
1 5 10 15
Arg Ser Pro Gly Ile Thr Cys Cys Val Leu Leu Leu Leu Asn Cys Ser
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20 25 30
Gly Val Pro Met Ser Leu Ala Ser Ser Phe Leu Thr Gly Ser Val Ala
35 40 45
Lys Cys Glu Asn Glu Gly Glu Val Leu~Gln Ile Pro Phe Ile Thr Asp
50 55 60
Asn Pro Cys Ile Met'Cys Val Cys Leu Asn Lys Glu Val Thr Cys Lys
65 70 75 80
Arg Glu Lys Cys Pro Val Leu Ser Arg Asp Cys Ala Leu Ala Ile Lys
85 90 95
Gln Arg Gly Ala Cys Cys Glu Xaa Cys Lys Gly Cys Thr Tyr Glu Gly
100 105 110
Asn Thr Tyr Asn Ser Ser Phe Lys Trp Gln Ser Pro Ala Glu Pro Cys
115 120 125
Val Leu Arg Gln Cys Gln Glu Gly Val Val Thr Glu Ser Gly Val Arg
130 135 140
Cys Val Xaa His Cys Lys Xaa Pro Leu Glu His Leu Gly Met Cys Cys
145 150 155 160
Pro Thr Cys Pro Gly Cys Val Phe Glu Gly Val Gln Tyr Gln Glu Xaa
165 170 175
Glu Glu Xaa Gln Pro Glu
180
<210> 110
<211> 47
<212> PRT
<213> Homo Sapiens
<400> 110
Ser Ser Ser Leu Leu Ile Ile Tyr Val Cys Met Met Asp Val Thr Ile
1 5 10 15
Tyr Met Ser Cys Val Glu Ile Lys Gly Cys Leu Asp Ala Met Leu Ile
20 25 30
Leu Leu Ser Met Arg Lys Tyr Leu Lys Lys Leu Leu His Asn Ile
35 40 45
<210> 111
<211> 96
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (81)
<223> Xaa equals any of the naturally occurring L-amino acids
CA 02396719 2002-07-24
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84
<220>
<221> SITE
<222> (83)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (84)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (96)
<223> Xaa equals any of the naturally occurring L-amino acids
<400> 111
Met Lys Val Ala Val Ser Pro Ala Val Gly Pro Gly Pro Trp Gly Ser
1 5 10 15
Gly Val Gly Gly Gly Gly Thr Val Arg Leu Leu Leu Ile Leu Ser Gly
20 25 30
Cys Leu Val Tyr Gly Thr Ala Glu Thr Asp Val Asn Val Val Met Leu
35 40 45
Gln Glu Ser Gln Val Cys Glu Lys Arg Ala Ser Gln Gln Phe Cys Tyr
50 55 60
Thr Asn Val Leu Ile Pro Lys Trp His Asp Ile Trp Thr Arg Ile Gln
65 70 75 80
Xaa Arg Xaa Xaa Ser Ser Arg Leu Val Arg Val Thr Gln Val Glu Xaa
85 90 95
<210> 112
<211> 273
<212> PRT
<213> Homo Sapiens
<400> 112
Met Leu Phe Phe Cys Gly Asp Leu Leu Ser Arg Ser Gln Ile Phe Tyr
1 5 10 15
Tyr Ser Thr Gly Met Thr Val Gly Ile Val Ala Ser Leu Leu Ile Ile
20 25 30
Ile Phe Ile Leu Ser Lys Phe Met Pro Lys Lys Ser Pro Ile Tyr Val
35 40 45
Ile Leu Val Gly Gly Trp Ser Phe Ser Leu Tyr Leu Ile Gln Leu Val
50 55 60
Phe Lys Asn Leu Gln Glu Ile Trp Arg Cys Tyr Trp Gln Tyr Leu Leu
CA 02396719 2002-07-24
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65 70 75 80
Ser Tyr Val Leu Thr Val Gly Phe Met Ser Phe Ala Val Cys Tyr Lys
85 90 95
Tyr Gly Pro Leu Glu Asn Glu Arg Ser Ile Asn Leu Leu Thr Trp Thr
100 105 110
Leu Gln Leu Met Gly Leu Cys Phe Met Tyr Ser Gly Ile Gln Ile Pro
115 120 125
His Ile Ala Leu Ala Ile Ile Ile Ile Ala Leu Cys Thr Lys Asn Leu
130 135 140
Glu His Pro Ile Gln Trp Leu Tyr Ile Thr Cys Arg Lys Val Cys Lys
145 150 155 160
Gly Ala Glu Lys Pro Val Pro Pro Arg Leu Leu Thr Glu Glu Glu Tyr
165 170 175
Arg Ile Gln Gly Glu Val Glu Thr Arg Lys Ala Leu Glu Glu Leu Arg
180 185 190
Glu Phe Cys Asn Ser Pro Asp Cys Ser Ala Trp Lys Thr Val Ser Arg
195 200 205
Ile Gln Ser Pro Lys Arg Phe Ala Asp Phe Val Glu Gly Ser Ser His
210 215 220
Leu Thr Pro Asn Glu Val Ser Val His Glu Gln Glu Tyr Gly Leu Gly
225 230 235 240
Ser Ile Ile Ala Gln Asp Glu Ile Tyr Glu Glu Ala Ser Ser Glu Glu
245 250 255
Glu Asp Ser Tyr Ser Arg Cys Pro Ala Ile Thr Gln Asn Asn Phe Leu
260 265 270
Thr
<210> 113
<211> 265
<212> PRT
<213> Homo sapiens
<220>
<221> SITE
<222> (22)
<223> Xaa equals any of the naturally occurring L-amino acids
<400> 113
Pro Lys Leu Glu Leu His Arg Gly Gly Gly Arg Ser Arg Thr Ser Gly
1 5 10 15
Ser Pro Gly Leu Gln Xaa Phe Gly Thr Arg Arg Thr Arg Gly Arg Ser
20 25 30
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Gly Arg Ala Gln Gly Arg Leu Lys Arg Pro Gly Lys Leu Ala Cys Arg
35 40 45
Lys Phe Pro Gly Arg Arg Gln Arg Val Val Pro Glu Leu Thr Asp Val
50 55 60
Leu Met Asn Glu Ile Leu His Gly Ala Asp Gly Thr Ser Ile Lys Cys
65 70 75 80
Gly Ile Ile Gly Glu Ile Gly Cys Ser Trp Pro Leu Thr Glu Ser Glu
85 90 95
Arg Lys Val Leu Gln Ala Thr Ala His Ala Gln Ala Gln Leu Gly Cys
100 105 110
Pro Val Ile Ile His Pro Gly Arg Ser Ser Arg Ala Pro Phe Gln Ile
115 120 125
Ile Arg Ile Leu Gln Glu Ala Gly Ala Asp Ile Ser Lys Thr Val Met
130 135 140
Ser His Leu Asp Arg Thr Ile Leu Asp Lys Lys Glu Leu Leu Glu Phe
145 150 155 160
Ala Gln Leu Gly Cys Tyr Leu Glu Tyr Asp Leu Phe Gly Thr Glu Leu
165 170 175
Leu His Tyr Gln Leu Gly Pro Asp Ile Asp Met Pro Asp Asp Asn Lys
180 185 190
Arg Ile Arg Arg Val Arg Leu Leu Val Glu Glu Gly Cys Glu Asp Arg
195 200 205
Ile Leu Val Ala His Asp Ile His Thr Lys Thr Arg Leu Met Lys Tyr
210 215 220
Gly Gly His Gly Tyr Ser His Ile Leu Thr Asn Val Val Pro Lys Met
225 230 235 240
Leu Leu Arg Gly Ile Thr Glu Asn Val Leu Asp Lys Ile Leu Ile Glu
245 250 255
Asn Pro Lys Gln Trp Leu Thr Phe Lys
260 265
<210> 114
<211> 91
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (78)
<223> Xaa equals any of the naturally occurring L-amino acids
<400> 114
CA 02396719 2002-07-24
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87
Met His Leu Gly Leu Val Ser Leu Ile Leu Phe Cys Gln Ala Leu Glu
1 5 10 15
Val Asp Ile Ser Leu Gln Gly Pro Gly Ile Val Pro Gly Arg Ser Glu
20 25 30
Val Ser Leu Ser Leu Gln Gly Pro Arg Gly Gly Gly Cys Phe Pro Ile
35 40 45
Ala Thr Gly Ala Pro Phe Ile Val Leu Leu Pro Leu Gly Leu Tyr Leu
50 55 60
Val Phe His Leu Cys Cys Phe Phe Gly Leu Phe Cys Ala Xaa Leu Arg
65 70 75 80
Leu Arg Glu Pro Gly Trp Asp His Leu Ile Ile
85 90
<210> 115
<211> 81 .
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (56)
<223> Xaa equals any of the naturally occurring L-amino acids
<400> 115
Gly Leu His Phe Asn Ile Arg Val Asp His Gly Met Leu Trp Ala Pro
1 5 10 15
Val Leu Tyr Lys Asp Val Gly Gln Glu Leu Pro Val Val Ser Thr Ala
20 25 30
Pro Ser His Ile Ala Leu Leu Met Glu Pro Phe Thr Pro Asp Val Leu
35 40 45
Ser Arg Leu Met Gly Arg Ile Xaa Val Cys Lys Asp Tyr Val Ile Asp
50 55 60
Gln Leu Trp Ser Val Leu Lys Glu Ile Cys Gln Trp Ile Ile Pro Tyr
65 70 75 80
Gly
<210> 116
<211> 234
<212> PRT
<213> Homo Sapiens
<400> 116
Pro Thr Arg Pro Pro Thr Arg Pro Val Arg Val Ser Val Gly Gly Leu
1 5 10 15
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Val Gly Glu Val Ala Cys Ala Cys Arg Asp Cys Ile Pro Glu Thr Met
20 25 30
Ala Glu Gly Asp Asn Arg Ser Thr Asn Leu Leu Ala Ala Glu Thr Ala
35 40 45
Ser Leu Glu Glu Gln Leu Gln Gly Trp Gly Glu Val Met Leu Met Ala
50 55 60
Asp Lys Val Leu Arg Trp Glu Arg Ala Trp Phe Pro Pro Ala Ile Met
65 70 75 80
Gly Val Val Ser Leu Val Phe Leu Ile Ile Tyr Tyr Leu Asp Pro Ser
85 90 95
Val Leu Ser Gly Val Ser Cys Phe Val Met Phe Leu Cys Leu Ala Asp
100 105 110
Tyr Leu Val Pro Ile Leu Ala Pro Arg Ile Phe Gly Ser Asn Lys Trp
115 120 125
Thr Thr Glu Gln Gln Gln Arg Phe His Glu Ile Cys Ser Asn Leu Val
130 135 ' 140
Lys Thr Arg Arg Arg Ala Val Gly Trp Trp Lys Arg Leu Phe Thr Leu
145 150 155 160
Lys Glu Glu Lys Pro Lys Met Tyr Phe Met Thr Met Ile Val Ser Leu
165 170 ' 175
Ala Ala Val Ala Trp Val Gly Gln Gln Val His Asn Leu Leu Leu Thr
180 185 190
Tyr Leu Ile Val Thr Ser Leu Leu Leu Leu Pro Gly Leu Asn Gln His
195 200 205
Gly Ile Ile Leu Lys Tyr Ile Gly Met Ala Lys Arg Glu Ile Asn Lys
210 215 220
Leu Leu Lys Gln Lys Glu Lys Lys Asn Glu
225 230
<210> 117
<211> 42
<212> PRT
<213> Homo Sapiens
<400> 117 '
Tyr Thr Phe Gln Cys Leu Ser Gln Thr Cys Ser Tyr Asp Ile Lys Cys
1 5 10 15
Tyr Phe Leu Val Ala Lys Ile Ile Leu Asp Ser Val Ile Lys Val Tyr
20 25 30
Trp Asn Leu Asn Phe Lys Met Ser Pro Asp
35 40
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<210> 118
<211> 25
<212> PRT
<213> Homo sapiens
<400> 118
Met Ser Pro Ser Ile Arg Ile Leu Leu Val Leu Gln Gln Leu Gly Ser
1 5 10 15
Leu Met Ala Pro Leu Pro Ser Ala His
20 25
<210> 119
<211> 156
<212> PRT
<213> Homo sapiens
<400> 119
Met Gln Ile Gln Val Ala Gly Leu Leu Gln Phe Ala Val Pro Leu Phe
1 5 10 15
Ser Thr Ala Glu Glu Asp Leu Leu Ala Ile Gln Leu Leu Leu Asn Ser
20 25 30
Ser Glu Ser Ser Leu His Gln Leu Thr Ala Met Val Asp Cys Arg Gly
35 40 45
Leu His Lys Asp Tyr Leu Asp Ala Leu Ala Gly Ile Cys Tyr Asp Gly
50 55 60
Leu Gln Gly Leu Leu Tyr Leu Gly Leu Phe Ser Phe Leu Ala Ala Leu
65 70 75 80
Ala Phe Ser Thr Met Ile Cys Ala Gly Pro Arg Ala Trp Lys His Phe
85 90 95
Thr Thr Arg Asn Arg Asp Tyr Asp Asp Ile Asp Asp Asp Asp Pro Phe
100 105 110
Asn Pro Gln Ala Trp Arg Met Ala Ala His Ser Pro Pro Arg Gly Gln
115 120 125
Leu His Ser Phe Cys Ser Tyr Ser Ser Gly Leu Gly Ser Gln Thr Ser
130 135 140
Leu Gln Pro Pro Ala Gln Thr Ile Ser Asn Ala Pro
145 150 155
<210> 120
<211> 42
<212> PRT
<213> Homo sapiens
<400> 120
Met Val Pro Ser Val Thr Leu Ile Leu His Cys Pro Gly Phe Ser Thr
CA 02396719 2002-07-24
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1 5 10 15
Glu Ser His Met Cys Gly Lys Pro Leu Ser Pro Arg Pro Thr Arg Thr
20 25 30
Val Gly Arg Pro Val Ser Asn Ile Pro Val
35 40
<210> 121
<211> 89
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (17)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (47)
<223> Xaa equals any of the naturally occurring L-amino acids
<400> 121
Val Gln Gly Val Val Gln Ala Leu Lys Thr Asp His Ala Phe Cys Pro
1 5 10 15
Xaa Leu Gln Gly Thr Glu Ser Ile Arg Leu Arg Ile Leu Glu Phe Glu
20 25 30
Leu Asn Gln Val Arg Ser Val Ser Gln Glu Leu Pro Pro Gly Xaa Pro
35 40 45
Glu Ser Pro Gln Thr Asp Gly Gln Pro Pro Arg Ala Trp Pro Gln Leu
50 55 60
Gly Met Pro Ser Asn Pro Thr Cys Phe Ser Phe Leu Pro Gly Tyr Ser
65 , 70 75 80
Gly Leu Arg Ser Ser Ala Leu Asn Phe
<210> 122
<211> 370
<212> PRT
<213> Homo Sapiens
<400> 122
Leu Gly Gly Ala Arg Val Arg Arg Ala Val Gly Leu Ser Gly Thr Gly
1 5 10 15
Ala Glu Ala Gly Arg Ala Gly Ala Met Val Glu Lys Glu Glu Ala Gly
20 25 30
Gly Gly Ile Ser Glu Glu Glu Ala Ala Gln Tyr Asp Arg Gln Ile Arg
35 40 45
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Leu Trp Gly Leu Glu Ala Gln Lys Arg Leu Arg Ala Ser Arg Val Leu
50 55 60
Leu Val Gly Leu Lys Gly Leu Gly Ala Glu Ile Ala Lys Asn Leu Ile
65 70 75 80
Leu Ala Gly Val Lys Gly Leu Thr Met Leu Asp His Glu Gln Val Thr
85 90 95
Pro Glu Asp Pro Gly Ala Gln Phe Leu Ile Arg Thr Gly Ser Val Gly
100 105 110
Arg Asn Arg Ala Glu Ala Ser Leu Glu Arg Ala Gln Asn Leu Asn Pro
115 120 125
Met Val Asp Val Lys Val Asp Thr Glu Asp Ile Glu Lys Lys Pro Glu
130 135 140
Ser Phe Phe Thr Gln Phe Asp Ala Val Cys Leu Thr Cys Cys Ser Arg
145 150 155 160
Asp Val Ile Val Lys Val Asp Gln Ile Cys His Lys Asn Ser Ile Lys
165 170 175
Phe Phe Thr Gly Asp Val Phe Gly Tyr His Gly Tyr Thr Phe Ala Asn
180 185 190
Leu Gly Glu His Glu Phe Val Glu Glu Lys Thr Lys Val Ala Lys Val
195 200 205
Ser Gln Gly Val Glu Asp Gly Pro Asp Thr Lys Arg Ala Lys Leu Asp
210 215 220
Ser Ser Glu Thr Thr Met Val Lys Lys Lys Val Val Phe Cys Pro Val
225 230 235 240
Lys Glu Ala Leu Glu Val Asp Trp Ser Ser Glu Lys Ala Lys Ala Ala
245 250 255
Leu Lys Arg Thr Thr Ser Asp Tyr Phe Leu Leu Gln Val Leu Leu Lys
260 265 270
Phe Arg Thr Asp Lys Gly Arg Asp Pro Ser Ser Asp Thr Tyr Glu Glu
275 280 285
Asp Ser Glu Leu Leu Leu Gln Ile Arg Asn Asp Val Leu Asp Ser Leu
290 295 300
Gly Ile Ser Pro Asp Leu Leu Pro Glu Asp Phe Val Arg Tyr Cys Phe
305 310 315 320
Ser Glu Met Ala Pro Val Cys Ala Val Val Gly Gly Ile Leu Ala Gln
325 330 335
Glu Ile Val Lys Ala Leu Ser Gln Arg Asp Pro Pro His Asn Asn Phe
340 345 350
CA 02396719 2002-07-24
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Phe Phe Phe Asp Gly Met Lys Gly Asn Gly Ile Val Glu Cys Leu Gly
355 360 365
Pro Lys
370
<210> 123
<211> 276
<212> PRT
<213> Homo Sapiens
<400> 123
Gly Thr Ser Leu Ser Cys Gly Ser Val Ser Thr Leu Cys Ala Thr Met
1 5 10 15
Ala Val Thr Ala Cys Gln Gly Leu Gly Phe Val Val Ser Leu Ile Gly
20 25 30
Ile Ala Gly Ile Ile Ala Ala Thr Cys Met Asp Gln Trp Ser Thr Gln
35 40 45
Asp Leu Tyr Asn Asn Pro Val Thr Ala Val Phe Asn Tyr Gln Gly Leu
50 55 60
Trp Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg Gly
65 70 75 80
Tyr Phe Thr Leu Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg Ala
85 90 95
Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val Ser
100 105 110
Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser Ala
115 120 125
Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser Gly
130 135 140
Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val Thr
145 150 155 160
Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly Met
165 170 175
Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe Val
180 . 185 190
Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met Cys
195 200 205
Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala Val
210 215 220
Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly Phe
225 230 235 240
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Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Arg Tyr
245 250 255
Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser Lys
260 265 270
His Asp Tyr Val
275
<210> 124
<211> 75
<212> PRT
<213> Homo sapiens
<400> 124
Met Arg Gly Ile Tyr Thr Ala Pro Ser Gly Leu Glu Ser Thr Cys Leu
1 ~ 5 10 15
Val Val Ala Tyr Gly Leu Asp Ile Tyr Gln Thr Arg Val Tyr Pro Ser
20 25 30
Lys Gln Phe Asp Val Leu Lys Asp Asp Tyr Asp Tyr Val Leu Ile Ser
35 40 45
Ser Val Leu Phe Gly Leu Val Phe Ala Thr Met Ile Thr Lys Arg Leu
50 55 60
Ala Gln Val Lys Leu Leu Asn Arg Ala Trp Arg
65 70 75
<210> 125
<211> 171
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (162)
<223> Xaa equals any of the naturally occurring L-amino acids
<400> 125
Gly Val Asn His Glu Ser Trp Arg Asp Ser Gly Gln Thr Asp Gly Leu
1 5 10 15
Thr Phe Gly His Leu Lys Met Val Leu Leu Trp Ala Ser Val Leu Phe
20 25 30
Pro Ala Pro Glu Asp Trp Ala Glu Leu Gln Gly Ala Val Tyr Arg Leu
35 40 45
Leu Val Val Leu Leu Cys Cys Leu Ala Thr Arg Lys Leu Pro His Phe
50 55 60
Leu His Pro Gln Arg Asn Leu Leu Gln Gly Ser Gly Leu Asp Leu Gly
65 70 75 80
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94
Ala Ile Tyr Gln Arg Val Glu Gly Phe Ala Ser Gln Pro Glu Ala Ala
85 90 95
Leu Arg Ile His Ala Thr His Leu Gly Arg Ser Pro Pro Pro Arg Ile
100 105 110
Gly Ser Gly Leu Lys Ala Leu Leu Gln Leu Pro Ala Ser Asp Pro Thr
115 120 125
Tyr Trp Ala Thr Ala Tyr Phe Asp Val Leu Leu Asp Lys Phe Gln Val
130 135 140
Phe Asn Ile Gln Asp Lys Asp Arg Ile Ser Ala Met Gln Ser Ile Phe
145 150 155 160
Gln Xaa Thr Arg Thr Leu Gly Gly Glu Glu Ser
165 170
<210> 126
<211> 205
<212> PRT
<213> Homo sapiens
<400> 126
Met Gln Glu Leu Leu Gly Arg Gly Ala Trp Ala Pro Gly Cys Gly Arg
1 5 10 15
Arg Pro Ser Gly~Trp Gly Gln Leu Ala Cys Pro Asp Pro Leu Leu Pro
20 25 30
Pro His Asn Pro Lys Ser Pro Gln Pro Gly Pro Ser Thr Ser Gly Val
35 40 45
Trp Gly Glu Glu Gln Gly Leu Arg Thr Leu Ser Ser Glu His Pro Trp
50 55 60
Gln Gly Leu Gln Pro Leu Ile Ser Ser Leu Lys Pro Cys Gly His Thr
65 70 75 80
Ala Arg Arg Asp Leu Pro Leu Ala Pro Ala Ser Phe Gln Pro Arg Val
85 90 95
Leu Ile Gln Gly Pro Arg Thr Val Pro Pro Val Leu Leu Cys Pro Gln
100 105 110
His Lys Ala Arg Leu His Ser Gln Lys Cys Ser Gln Ala Leu Glu Gly
115 120 125
Asp Pro Ala Ser Ser Pro Thr Ala Pro His Pro Thr His Pro Ser Ala
130 135 140
Ala Pro Leu Leu Phe Pro Arg Asp Leu Ser Tyr Thr Gly Gln Glu Ala
145 150 155 160
Ala Glu Arg Val Ser Pro Pro Pro Ser Lys Arg Ser Cys Ser Leu Cys
165 170 175
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Gln Asn Arg Val Trp Ala Gly Gly Arg Ala Leu Gly Ala Arg Pro Leu
180 185 190
Pro Leu Pro Ala Gly Phe Ser Trp Ser Leu Cys Trp Lys
195 200 205
<210> 127
<211> 142
<212> PRT
<213> Homo sapiens
<400> 127
Met Phe His Thr Phe Thr Leu Gln Gly Arg Gly Glu Gly Ser Cys Ser
1 5 10 15
Pro Ser His Thr Ala Leu Leu Ser Asn Ile Thr Ala Pro Val Thr Thr
20 25 30
Thr Leu Ser Arg Ala Leu Glu Gly Glu Gly Ser Met Pro Glu Pro Leu
35 40 45
Ala Ala Gly Gly Pro Trp Arg Ser Ala Gly Pro Trp Trp Thr Arg Gln
50 55 60
Glu Ile Cys Thr Pro Trp Ala Val Pro Ala His Thr Trp Gly Ser Arg
65 70 75 80
Glu Asp Gly Ala Arg Ala Ser Leu His Ile Tyr Ala Ser Ser Arg Thr
85 90 95
Gln Gly Ala Gly Pro Gly Ala Gly Pro Gly Arg Pro Gly Gly Ala Gly
100 105 110
Gly Pro Gly Ala Ala Pro Gln Pro Pro Cys Leu Ala Gly Pro Glu Ser
115 120 125
Ser His Thr Gly Ala Val Cys Leu Cys Trp Ser Gly Ala Ser
130 135 140
<210> 128
<211> 152
<212> PRT
<213> Homo sapiens
<400> 128
Met Gly Arg Glu Ala Thr Gly Gln Lys Ala Pro Gly Gly His Asp Leu
1 5 10 15
Leu Ala Lys Arg Phe Gln Gln Ser Ala Lys Val Pro Ser Ser Ala Pro
20 25 30
Gly Leu Pro Cys Leu Gly Pro Ser Pro Lys Asp Ala Ser Leu Cys Gly
35 40 45
Trp Gly Val Pro Ala Gly Leu Gly Gly Ser Ser Val Leu Ser Cys Lys
50 . 55 60
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Ser Val Cys Val Arg Val Ala Gly Thr Gly Gly Pro Thr Asp Gln Arg
65 70 75 80
Ala Ala Val Gly Arg Ala Phe Gln Cys Ile Ala Arg Gly Lys Glu Leu
85 90 95
Thr Leu Thr Val Pro Ser Val Ser Leu Val Gly Val Gly Gly Phe Ser
1'00 105 110
Leu Arg Gly Cys Phe Thr His Ser His Cys Arg Gly Gly Ala Arg Gly
115 120 125
Ala Ala Leu Pro Leu Thr Gln Leu Tyr Cys Gln Thr Ser Arg His Gln
130 135 140
Ser Pro Gln Pro Cys His Val His
145 150
<210> 129
<211> 471
<212> PRT
<213> Homo sapiens
<400> 129
Pro Arg Val Arg Ser Glu Ala Ala Leu Ser Ser Gln Arg Asp Leu Phe
1 5 10 15
Ser Ser Phe Phe Leu Asn Ser Ser Ser Arg Glu Arg Leu Val Val Thr
20 25 30
Gly Arg Ala Gly Trp Met Gly Met Gly Arg Gly Ala Gly Arg Ser Ala
35 40 45
Leu Gly Phe Trp Pro Thr Leu Ala Phe Leu Leu Cys Ser Phe Pro Ala
50 55 60
Ala Thr Ser Pro Cys Lys Ile Leu Lys Cys Asn Ser Glu Phe Trp Ser
65 70 75 80
Ala Thr Ser Gly Ser His Ala Pro Ala Ser Asp Asp Thr Pro Glu Phe
85 90 95
Cys Ala Ala Leu Arg Ser Tyr Ala Leu Cys Thr Arg Arg Thr Ala Arg
100 105 110
Thr Cys Arg Gly Asp Leu Ala Tyr His Ser Ala Val His Gly Ile Glu
115 120 125
Asp Leu Met Ser Gln His Asn Cys Ser Lys Asp Gly Pro Thr Ser Gln
130 135 140
Pro Arg Leu Arg Thr Leu Pro Pro Ala Gly Asp Ser Gln Glu Arg Ser
145 150 155 160
Asp Ser Pro Glu Ile Cys His Tyr Glu Lys Ser Phe His Lys His Ser
165 170 175
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Ala Thr Pro Asn Tyr Thr His Cys Gly Leu Phe Gly Asp Pro His Leu
180 185 190
Arg Thr Phe Thr Asp Arg Phe Gln Thr Cys Lys Val Gln Gly Ala Trp
195 200 205
Pro Leu Ile Asp Asn Asn Tyr Leu Asn Val Gln Val Thr Asn Thr Pro
210 215 220
Val Leu Pro Gly Ser Ala Ala Thr Ala Thr Ser Lys Leu Thr Ile Ile
225 230 235 240
Phe Lys Asn Phe Gln Glu Cys Val Asp Gln Lys Val Tyr Gln Ala Glu
245 250 255
Met Asp Glu Leu Pro Ala Ala Phe Val Asp Gly Ser Lys Asn Gly Gly
260 265 270
Asp Lys His Gly Ala Asn Ser Leu Lys Ile Thr Glu Lys Val Ser Gly
275 280 285
Gln His Val Glu Ile Gln Ala Lys Tyr Ile Gly Thr Thr Ile Val Val
290 295 300.
Arg Gln Val Gly Arg Tyr Leu Thr Phe Ala Val Arg Met Pro Glu Glu
305 310 315 320
Val Val Asn Ala Val Glu Asp Trp Asp Ser Gln Gly Leu Tyr Leu Cys
325 330 335
Leu Arg Gly Cys Pro Leu Asn Gln Gln Ile Asp Phe Gln Ala Phe His
340 345 350
Thr Asn Ala Glu Gly Thr Gly Ala Arg Arg Leu Ala Ala Ala Ser Pro
355 360 365
Ala Pro Thr Ala Pro Glu Thr Phe Pro Tyr Glu Thr Ala Val Ala Lys
370 375 380
Cys Lys Glu Lys Leu Pro Val Glu Asp Leu Tyr Tyr Gln Ala Cys Val
385 390 395 400
Phe Asp Leu Leu Thr Thr Gly Asp Val Asn Phe Thr Leu Ala Ala Tyr
405 410 415
Tyr Ala Leu Glu Asp Val Lys Met Leu His Ser Asn Lys Asp Lys Leu
420 425 430
His Leu Tyr Glu Arg Thr Arg Asp Leu Pro Gly Arg Ala Ala Ala Gly
435 440 445
Leu Pro Leu Ala Pro Arg Pro Leu Leu Gly Ala Leu Val Pro Leu Leu
450 455 460
Ala Leu Leu Pro Val Phe Cys
465 470
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98
<210> 130
<211> 164
<212> PRT
<213> Homo sapiens
<400> 130
Met Arg His His Thr Trp Leu Ile Leu Tyr Ile Tyr Phe Leu Tyr Phe
1 5 10 15
Gln Glu Gly Val Leu Pro Cys Cys Pro Gly Trp Ser Ala Val Ala Arg
20 . 25 30
Ser Trp Leu Thr Ala Thr Ser Ala Phe Arg Val Gln Ala Val Leu Cys
35 40 45
Leu Ser Leu Pro Ser Ser Trp Asp Tyr Arg Cys Pro Pro Pro His Pro
50 55 60
Ala Asn Phe Cys Val Cys Val Cys Val Phe Leu Val Glu Met Gly Phe
65 70 75 80
His His Leu Gly Gln Ala Gly Leu Glu Leu Leu Thr Leu Asp Pro Pro
85 90 95
Val Ser Ala Ser Gln Ser Asp Gly Ile Thr Gly Val Ser His Cys Ala
100 105 110
Gln Pro Asn Phe Val Tyr Leu Val Glu Met Gly Phe Leu His Val Gly
115 120 125
Gln Ala Gly Leu Lys Leu Pro Asn Leu Gly Asp Pro Pro Ala Ser Ala
130 135 140
Ser Gln Ser Ala Gly Ile Thr Asp His Glu Val Arg Ser Ser Arg Leu
145 150 155 160
Ala Trp Pro Thr
<210> 131
<211> 311
<212> PRT
<213> Homo sapiens
<220>
<221> SITE
<222> (125)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (126)
<223> Xaa equals any of the naturally occurring L-amino acids
<400> 131
Arg Thr Arg Gly Gly Arg Val Trp Thr Ser Val Leu Leu Phe Ala Phe
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99
1 5 10 15
Ala Thr Gly Val Arg Ser Asn Gly Leu Val Ser Val Gly Phe Leu Met
20 ~ 25 30
His Ser Gln Cys Gln Gly Phe Phe Ser Ser Leu Thr Met Leu Asn Pro
35 40 45
Leu Arg Gln Leu Phe Lys Leu Met Ala Ser Leu Phe Leu Ser Val Phe
50 55 60
Thr Leu Gly Leu Pro Phe Ala Leu Phe Gln Tyr Tyr Ala Tyr Thr Gln
65 70 75 80
Phe Cys Leu Pro Gly Ser Ala Arg Pro Ile Pro Glu Pro Leu Val Gln
85 90 95
Leu Ala Val Asp Lys Gly Tyr Arg Ile Ala Glu Gly Asn Glu Pro Pro
100 105 110
Trp Cys Phe Trp Asp Val Pro Leu Ile Tyr Ser Tyr Xaa Xaa Asp Val
115 120 125
Tyr Trp Asn Val Gly Phe Leu Lys Tyr Tyr Glu Leu Lys Gln Val Pro
130 135 140
Asn Phe Leu Leu Ala Ala Pro Val Ala Ile Leu Val Ala Trp Ala Thr
145 150 155 160
Trp Thr Tyr Val Thr Thr His Pro Trp Leu Cys Leu Thr Leu Gly Leu
165 170 175
Gln Arg Ser Lys Asn Asn Lys Thr Leu Glu Lys Pro Asp Leu Gly Phe
180 185 190
Leu Ser Pro Gln Val Phe Val Tyr Val Val His Ala Ala Val Leu Leu
195 200 205
Leu Phe Gly Gly Leu Cys Met His Val Gln Val Leu Thr Arg Phe Leu
210 215 220
Gly Ser Ser Thr Pro Ile Met Tyr Trp Phe Pro Ala His Leu Leu Gln
225 230 235 240
Asp Gln Glu Pro Leu Leu Arg Ser Leu Lys Thr Val Pro Trp Lys Pro
245 250 255
Leu Ala Glu Asp Ser Pro Pro Gly Gln Lys Val Pro Arg Asn Pro Ile
260 265 . 270
Met Gly Leu Leu Tyr His Trp Lys Thr Cys Ser Pro Val Thr Arg Tyr
275 280 285
Ile Leu Gly Tyr Phe Leu Thr Tyr Trp Leu Leu Gly Leu Leu Leu His
290 295 300
Cys Asn Phe Leu Pro Trp Thr
305 310
CA 02396719 2002-07-24
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100
<210> 132
<211> 111
<212> PRT
<213> Homo sapiens
<220>
<221> SITE
<222> (75)
<223> Xaa equals any of the naturally occurring L-amino acids
<400> 132
Arg His Thr His Gln Gly Met Phe Ala Leu Ala Trp Lys Val Ile Phe
1 5 10 15
Ser Val Met Leu Gln Asn Pro Ile Arg Tyr Pro Ser Val Leu Gly Ile
20 25 30
Lys Ser Ser Leu Leu Ser Ser Leu Val Leu Val Met Val Trp Gly Asn
35 40 45
Glu Lys Ser Gly Pro Cys Pro Thr Pro Lys Ser Arg Lys Gly Arg Arg
50 55 60
Ser Cys Pro Ala Gln Val Gly Arg Gly Glu Xaa Gly Ser Tyr Trp Asp
65 70 75 80
Pro Glu Phe Arg Leu Ser Arg Lys Ser Asn Gln Gly Leu Arg Arg Asp
85 90 95
Tyr Leu Ser Leu Tyr His Phe Asn Leu His Phe Arg Asp Thr Phe
100 105 110
<210> 133
<211> 119
<212> PRT
<213> Homo sapiens
<400> 133
Met Gln Glu Phe His Leu Leu His Pro Val Pro Leu Ile Gln Gln Glu
1 5 10 15
Thr Phe Ala Pro Gln Val Leu Ala Val Gln Gly Ser Pro Asn Ser Ala
20 25 30
Ser Val Arg Leu Glu Cys Val Ser Lys Met Gln Val Glu Val Ile Gln
35 40 45
Ala Gln Val Ile Pro Pro Gln Pro Leu Val Gly Phe Pro Ala Gln Ser
50 55 60
Glu Phe Trp Val Pro Ile Gly Pro Leu Phe Ser Pro Pro His Leu Cys
65 70 75 80
Trp Thr Ala Pro Ser Ser Leu Pro Gly Leu Trp Cys Gly Ala Gly Pro
85 90 95
CA 02396719 2002-07-24
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101
Thr Leu Phe Ile Ser Pro Asn His His Gln His Lys Thr Thr Glu Gln
100 105 110
Gly Gly Leu Tyr Thr Lys Asp
115
<210> 134
<211> 103
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (10)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (21)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (23)
<223> Xaa equals any of the naturally occurring L-amino acids
<400> 134
Met Thr Ser His Ala Arg Val Arg Lys Xaa Gly Ser Ser Arg Ala Ala
1 5 10 15
Ala Leu Phe Phe Xaa Phe Xaa Phe Phe Phe Phe Phe Leu Lys Arg Pro
20 25 30
Glu Ser Cys Ser Val Pro Gln Pro Gly Val Gln Trp Cys Asn Leu Gly
35 40 45
Ser Leu Gln Leu Pro Pro Pro Arg Phe Lys Arg Phe Ser Cys Leu Ser
50 55 60
Leu Pro Gly Ser Trp Asp Tyr Arg Cys Thr Pro Pro Arg Leu Ala Asn
65 70 75 80
Phe Leu Ile Phe Ser Arg Asp Arg Val Ser Pro Cys Trp Pro Gly Trp
85 90 95
Ser Arg Thr Pro Asp Leu Lys
100
<210> 135
<211> 127
<212> PRT
<213> Homo Sapiens
<400> 135
Glu Tyr Ala Ser Ser Asn Thr Thr His Tyr Arg Glu Ser Trp Tyr Ala
CA 02396719 2002-07-24
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102
1 5 10 15
Cys Arg Tyr Arg Ser Gly Ile Pro Gly Leu Leu Ile Phe Pro Gly Pro
20 25 30
Gly Thr Gly Met Thr Ser Leu Leu Glu Gly Arg Met Val Leu Cys Val
35 40 45
Ser Cys Leu Leu Leu Pro Leu Leu Leu Leu Leu Lys His Phe Asn Gly
50 55 60
Leu Met Thr Pro Tyr Leu Ala His Asn Val Tyr Cys Pro Ile Glu Tyr
65 70 75 80
Ile Ser Phe Phe Pro Phe His Glu Lys Asn Ile Glu Tyr Ile Ser Ile
85 90 95
Trp Phe Ile Phe Asp Ser Phe Lys Phe Ile Tyr Ser Arg Leu Leu Cys
100 105 110
Ile Ser Gln Ile Tyr Val Leu Tyr Arg Ala Tyr Thr Leu Pro His
115 120 125
<210> 136
<211> 464
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (166)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (307)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (312)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (351)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (462)
<223> Xaa equals any of the naturally occurring L-amino acids
<400> 136
Asn Ser Ala Arg Gly Gly Ala Gly Pro Ala Val Ala Arg Asp Ser Leu
1 5 10 15
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Gln Val Thr Met Leu Trp Phe Ser Gly Val Gly Ala Leu Ala Glu Arg
20 25 30
Tyr Cys Arg Arg Ser Pro Gly Ile Thr Cys Cys Val Leu Leu Leu Leu
35 40 45
Asn Cys Ser Gly Val Pro Met Ser Leu Ala Ser Ser Phe Leu Thr Gly
50 55 60
Ser Val Ala Lys Cys Glu Asn Glu Gly Glu Val Leu Gln Ile Pro Phe
65 70 75 80
Ile Thr Asp Asn Pro Cys Ile Met Cys Val Cys Leu Asn Lys Glu Val
85 90 95
Thr Cys Lys Arg Glu Lys Cys Pro Val Leu Ser Arg Asp Cys Ala Leu
100 105 110
Ala Ile Lys Gln Arg Gly Ala Cys Cys Glu Gln Cys Lys Gly Cys Thr
115 120 125
Tyr Glu Gly Asn Thr Tyr Asn Ser Ser Phe Lys Trp Gln Ser Pro Ala
130 135 140
Glu Pro Cys Val Leu Arg Gln Cys Gln Glu Gly Val Val Thr Glu Ser
145 150 155 160
Gly Val Arg Cys Val Xaa His Cys Lys Asn Pro Leu Glu His Leu Gly
165 170 175
Met Cys Cys Pro Thr Cys Pro Gly Cys Val Phe Glu Gly Val Gln Tyr
180 185 190
Gln Glu Gly Glu Glu Phe Gln Pro Glu Gly Ser Lys Cys Thr Lys Cys
195 200 205
Ser Cys Thr Gly Gly Arg Thr Gln Cys Val Arg Glu Val Cys Pro Ile
210 215 220
Leu Ser Cys Pro Gln His Leu Ser His Ile Pro Pro Gly Gln Cys Cys
225 230 235 240
Pro Lys Cys Leu Gly Gln Arg Lys Val Phe Asp Leu Pro Phe Gly Ser
245 250 255
Cys Leu Phe Arg Ser Asp Val Tyr Asp Asn Gly Ser Ser Phe Leu Tyr
260 265 270
Asp Asn Cys Thr Ala Cys Thr Cys Arg Asp Ser Thr Val Val Cys Lys
275 280 285
Arg Lys Cys Ser His Pro Gly Gly Cys Asp Gln Gly Gln Glu Gly Cys
290 295 300
Cys Glu Xaa Cys Leu Leu Arg Xaa Pro Pro Glu Asp Ile Lys Val Cys
305 310 315 320
Lys Phe Gly Asn Lys Ile Phe Gln Asp Gly Glu Met Trp Ser Ser Ile
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325 330 335
Asn Cys Thr Ile Cys Ala Cys Val Lys Gly Arg Thr Glu Cys Xaa Asn
340 345 350
Lys Gln Cys Ile Pro Ile Ser Ser Cys Pro Gln Gly Lys Ile Leu Asn
355 360 365
Arg Lys Gly Cys Cys Pro Ile Cys Thr Glu Lys Pro Gly Val Cys Thr
370 375 380
Val Phe Gly Asp Pro His Tyr Asn Thr Phe Asp Gly Arg Thr Phe Asn
385 390 395 400
Phe Gln Gly Thr Cys Gln Tyr Val Leu Thr Lys Asp Cys Ser Ser Pro
405 410 415
Ala Ser Pro Phe Gln Val Leu Val Lys Asn Asp Ala Arg Arg Thr Arg
420 425 430
Ser Phe Ser Trp Thr Lys Ser Val Glu Leu Val Leu Gly Glu Thr Gly
435 440 445
Ser Ala Cys Ser Ser Thr Ser Pro Cys Ala Gly Thr Ala Xaa Ala Ser
450 455 460
<210> 137
<211> 273
<212> PRT
<213> Homo sapiens
<400> 137
Met Leu Phe Phe Cys Gly Asp Leu Leu Ser Arg Ser Gln Ile Phe Tyr
1 5 10 15
Tyr Ser Thr Gly Met Thr Val Gly Ile Val Ala Ser Leu Leu Ile Ile
20 25 30
Ile Phe Ile Leu Ser Lys Phe Met Pro Lys Lys Ser Pro Ile Tyr Val
35 40 45
Ile Leu Val Gly Gly Trp Ser Phe Ser Leu Tyr Leu Ile Gln Leu Val
50 55 60
Phe Lys Asn Leu Gln Glu Ile Trp Arg Cys Tyr Trp Gln Tyr Leu Leu
65 70 75 80
Ser Tyr Val Leu Thr Val Gly Phe Met Ser Phe Ala Val Cys Tyr Lys
85 90 95
Tyr Gly Pro Leu Glu Asn Glu Arg Ser Ile Asn Leu Leu Thr Trp Thr
100 105 110
Leu Gln Leu Met Gly Leu Cys Phe Met Tyr Ser Gly Ile Gln Ile Pro
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115 120 125
His Ile Ala Leu Ala Ile Ile Ile Ile Ala Leu Cys Thr Lys Asn Leu
130 135 140
Glu His Pro Ile Gln Trp Leu Tyr Ile Thr Cys Arg Lys Val Cys Lys
145 150 155 160
Gly Ala Glu Lys Pro Val Pro Pro Arg Leu Leu Thr Glu Glu Glu Tyr
165 170 175
Arg Ile Gln Gly Glu Val Glu Thr Arg Lys Ala Leu Glu Glu Leu Arg
180 185 190
Glu Phe Cys Asn Ser Pro Asp Cys Ser Ala Trp Lys Thr Val Ser Arg
195 200 205
Ile Gln Ser Pro Lys Arg Phe Ala Asp Phe Val Glu Gly Ser Ser His
210 215 220
Leu Thr Pro Asn Glu Val Ser Val His Glu Gln Glu Tyr Gly Leu Gly
225 230 235 240
Ser Ile Ile Ala Gln Asp Glu Ile Tyr Glu Glu Ala Ser Ser Glu Glu
245 250 255
Glu Asp Ser Tyr Ser Arg Cys Pro Ala Ile Thr Gln Asn Asn Phe Leu
260 265 270
Thr
<210> 138
<211> 286
<212> PRT
<213> Homo sapiens
<400> 138
Phe Asp Pro Lys Leu Phe Leu Val Phe Leu Leu Gly Leu Met Leu Phe
1 5 10 15
Phe Cys Gly Asp Leu Leu Ser Arg Ser Gln Ile Phe Tyr Tyr Ser Thr
20 25 30
Gly Met Thr Val Gly Ile Val Ala Ser Leu Leu Ile Ile Ile Phe Ile
35 40 45
Leu Ser Lys Phe Met Pro Lys Lys Ser Pro Ile Tyr Val Ile Leu Val
50 55 60
Gly Gly Trp Ser Phe Ser Leu Tyr Leu Ile Gln Leu Val Phe Lys Asn
65 70 75 80
Leu Gln Glu Ile Trp Arg Cys Tyr Trp Gln Tyr Leu Leu Ser Tyr Val
85 90 95
Leu Thr Val Gly Phe Met Ser Phe Ala Val Cys Tyr Lys Tyr Gly Pro
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100 105 110
Leu Glu Asn Glu Arg Ser Ile Asn Leu Leu Thr Trp Thr Leu Gln Leu
115 ~ 120 125
Met Gly Leu Cys Phe Met Tyr Ser Gly Ile Gln Ile Pro His Ile Ala
130 135 ~ 140
Leu Ala Ile Ile Ile Ile Ala Leu Cys Thr Lys Asri Leu Glu His Pro
145 150 155 160
Ile Gln Trp Leu Tyr.Ile Thr Cys Arg Lys Val Cys Lys Gly Ala Glu
165 170 175
Lys Pro Val Pro Pro Arg Leu Leu Thr Glu Glu Glu Tyr Arg Ile Gln
180 185 190
Gly Glu Val Glu Thr Arg Lys Ala Leu Glu Glu Leu Arg Glu Phe Cys
195 200 205
Asn Ser Pro Asp Cys Ser Ala Trp Lys Thr Val Ser Arg Ile Gln Ser
210 215 220
Pro Lys Arg Phe Ala Asp Phe Val Glu Gly Ser Ser His Leu Thr Pro
225 230 235 240
Asn Glu Val Ser Val His Glu Gln Glu Tyr Gly Leu Gly Ser Ile Ile
245 250 255
Ala Gln Asp Glu Ile Tyr Glu Glu Ala Ser Ser Glu Glu Glu Asp Ser
260 265 270
Tyr Ser Arg Cys Pro Ala Ile Thr Gln Asn Asn Phe Leu Thr
275 280 285
<210> 139
<211> 560
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (11)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (20)
<223> Xaa equals any of the naturally occurring L-amino acids
<400> 139
Leu Arg Thr Arg Gly Ser Leu Thr Ser Trp Xaa Leu Cys Cys Val Trp
1 5 10 15
Trp Arg Pro Xaa Leu Pro Ile Ser Ala Gly His Glu Glu Leu Ser Arg
20 25 30
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Leu Ser Gln Tyr Arg Lys Glu Leu Gly Ala Gln Leu His Gln Leu Val
35 40 45
Leu Asn Ser Ala Arg Glu Lys Arg Asp Met Glu Gln Arg His Val Leu
50 55 60
Leu Lys Gln Lys Glu Leu Gly Gly Glu Glu Pro Glu Pro Ser Leu Arg
65 70 75 80
Glu Gly Pro Gly Gly Leu Val Met Glu Gly His Leu Phe Lys Arg Ala
85 90 95
Ser Asn Ala Phe Lys Thr Trp Ser Arg Arg Trp Phe Thr Ile Gln Ser
100 105 110
Asn Gln Leu Val Tyr Gln Lys Lys Tyr Lys Asp Pro Val Thr Val Val
115 120 125
Val Asp Asp Leu Arg Leu Cys Thr Val Lys Leu Cys Pro Asp Ser Glu
130 135 140
Arg Arg Phe Cys Phe Glu Val Val Ser Thr Ser Lys Ser Cys Leu Leu
145 150 155 160
Gln Ala Asp Ser Glu Arg Leu Leu Gln Leu Trp Val Ser Ala Val Gln
165 170 175
Ser Ser Ile Ala Ser Ala Phe Ser Gln Ala Arg Leu Asp Asp Ser Pro
180 185 190
Arg Gly Pro Gly Gln Gly Ser Gly His Leu Ala Ile Gly Ser Ala Ala
195 200 205
Thr Leu Gly Ser Gly Gly Met Ala Arg Gly Arg Glu Pro Gly Gly Val
210 215 220
Gly His Val Val Ala Gln Val Gln Ser Val Asp Gly Asn Ala Gln Cys
225 230 235 240
Cys Asp Cys Arg Glu Pro Ala Pro Glu Trp Ala Ser Ile Asn Leu Gly
245 250 255
Val Thr Leu Cys Ile Gln Cys Ser Gly Ile His Arg Ser Leu Gly Val
260 265 270
His Phe Ser Lys Val Arg Ser Leu Thr Leu Asp Ser Trp Glu Pro Glu
275 280 285
Leu Val Lys Leu Met Cys Glu Leu Gly Asn Val Ile Ile Asn Gln Ile
290 295 300
Tyr Glu Ala Arg Val Glu Ala Met Ala Val Lys Lys Pro Gly Pro Ser
305 310 315 320
Cys Ser Arg Gln Glu Lys Glu Ala Trp Ile His Ala Lys Tyr Val Glu
325 330 335
Lys Lys Phe Leu Thr Lys Leu Pro Glu Ile Arg Gly Arg Arg Gly Gly
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340 345 350
Arg Gly Arg Pro Arg Gly Gln Pro Pro Val Pro Pro Lys Pro Ser Ile
355 360 365
Arg Pro Arg Pro Gly Ser Leu Arg Ser Lys Pro Glu Pro Pro Ser Glu
370 375 380
Asp Leu Gly Ser Leu His Pro Gly Ala Leu Leu Phe Arg Ala Ser Gly
385 390 395 400
His Pro Pro Ser Leu Pro Thr Met Ala Asp Ala Leu Ala His Gly Ala
405 410 415
Asp Val Asn Trp Val Asn Gly Gly Gln Asp Asn Ala Thr Pro Leu Ile
420 425 430
Gln Ala Thr Ala Ala Asn Ser Leu Leu Ala Cys Glu Phe Leu Leu Gln
435 440 445
Asn Gly Ala Asn Val Asn Gln Ala Asp Ser Ala Gly Arg Gly Pro Leu
450 455 460
His His Ala Thr Ile Leu Gly His Thr Gly Leu Ala Cys Leu Phe Leu
465 470 475 480
Lys Arg Gly Ala Asp Leu Gly Ala Arg Asp Ser Glu Gly Arg Asp Pro
485 490 495
Leu Thr Ile Ala Met Glu Thr Ala Asn Ala Asp Ile Val Thr Leu Leu
500 505 510
Arg Leu Ala Lys Met Arg Glu Ala Glu Ala Ala Gln Gly Gln Ala Gly
515 520 525
Asp Glu Thr Tyr Leu Asp Ile Phe Arg Asp Phe Ser Leu Met Ala Ser
530 535 540
Asp Asp Pro Glu Lys Leu Ser Arg Arg Ser His Asp Leu His Thr Leu
545 550 555 560
<210> 140
<211> 90
<212> PRT
<213> Homo sapiens
<220>
<221> SITE
<222> (85)
<223> Xaa equals any of the naturally occurring L-amino acids
<400> 140
Gly Leu Arg Gly Phe Arg Glu Ala Arg Arg Asp Phe~Trp Arg Gly Ala
1 5 10 15
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Glu Ser Leu Glu Ala Ala Leu Thr His Asn Ala Glu Val Pro Arg Arg
20 25 30
Arg Ala Gln Glu Ala Glu Glu Ala Gly Ala Ala Leu Arg Thr Ala Arg
35 40 45
Ala Gly Tyr Arg Gly Arg Ala Leu Asp Tyr Ala Leu Gln Ile Asn Val
50 55 60
Ile Glu Asp Lys Arg Lys Phe Asp Ile Met Glu Phe Val Leu Arg Leu
65 70 75 80
Val Glu Ala Gln Xaa Thr His Phe Ser Arg
85 90
<210> 141
<211> 234
<212> PRT
<213> Homo Sapiens
<400> 141
Pro Thr Arg Pro Pro Thr Arg Pro Val Arg Val Ser Val Gly Gly Leu
1 5 10 15
Val Gly Glu Val Ala Cys Ala Cys Arg Asp Cys Ile Pro Glu Thr Met
20 25 30
Ala Glu Gly Asp Asn Arg Ser Thr Asn Leu Leu Ala Ala Glu Thr Ala
35 40 45
Ser Leu Glu Glu Gln Leu Gln Gly Trp Gly Glu Val Met Leu Met Ala
50 55 60
Asp Lys Val Leu Arg Trp Glu Arg Ala Trp Phe Pro Pro Ala Ile Met
65 70 75 80
Gly Val Val Ser Leu Val Phe Leu Ile Ile Tyr Tyr Leu Asp Pro Ser
85 90 95
Val Leu Ser Gly Val Ser Cys Phe Val Met Phe Leu Cys Leu Ala Asp
100 105 110
Tyr Leu Val Pro Ile Leu Ala Pro Arg Ile Phe Gly Ser Asn Lys Trp
115 120 125
Thr Thr Glu Gln Gln Gln Arg PY~,e His Glu Ile Cys Ser Asn Leu Val
130 135 140
Lys Thr Arg Arg Arg Ala Val Gly Trp Trp Lys Arg Leu Phe Thr Leu
145 150 155 160
Lys Glu Glu Lys Pro Lys Met Tyr Phe Met Thr Met Ile Val Ser Leu
165 170 175
Ala Ala Val Ala Trp Val Gly Gln Gln Val His Asn Leu Leu Leu Thr
180 185 190
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Tyr Leu Ile Val Thr Ser Leu Leu Leu Leu Pro Gly Leu Asn Gln His
195 200 205
Gly Ile Ile Leu Lys Tyr Ile Gly Met Ala Lys Arg Glu Ile Asn Lys
210 215 220
Leu Leu Lys Gln Lys Glu Lys Lys Asn Glu
225 230
<210> 142
<211> 75
<212> PRT
<213> Homo Sapiens
<400> 142
Met Gly Ser Cys Tyr Ile Tyr Gln Pro Gln Tyr Leu Gly Ala Ala Leu
1 5 10 15
Ser Arg Thr Asn Gly Leu Pro Ile Gly Asp Ala Ile Thr Gly Ser Val
20 25 30
Leu Gly Gly Gly Val Leu Arg Gly Asp Gly His Leu Ala Gly Ser Ala
35 40 45
Ser Pro Gly Asn Ser Ser His Pro Gln Ser Leu Thr Gln Gly Pro Arg
50 55 60
Ser Cys Arg Leu Trp Ala Arg Ala Ala Arg Ala
65 70 75
<210> 143
<211> 95
<212> PRT
<213> Homo Sapiens
<400> 143
Met Pro Ala Leu Ala Leu Ser Pro Arg Pro Leu Leu Pro Gly Ile Ser
1 5 10 15
Cys Pro Ser Pro Arg Cys Thr Cys Pro Ser Pro Ala Asn Pro Leu Cys
20 25 30
Leu His Phe Thr Arg Leu Gln Gly Val His Ile Ser Ser Ser Asp Gln
35 40 45
Gly Ser Glu Gly Arg Gly Cys Val Ser Cys Ile Ser Leu Gly Pro Leu
50 55 60
Arg Gly Arg Asp Cys Phe Pro Arg Val Gly Ala Leu Glu Val Met Arg
65 70 75 80
Leu Phe Pro Pro Asn His Gln Ile Trp Thr Glu Asn Cys Ile Gly
85 90 95
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<210> 144
<211> 234
<212> PRT
<213> Homo Sapiens
<400> 144
Met Ala Thr Thr Thr Cys Val Pro Met Lys Thr Ala Arg Leu Pro Gly
1 5 10 15
Gln Cys Ser Met Leu Thr Thr Val Pro Leu Ala Leu Pro Ala Ser Thr
20 25 30
Ala Ala Pro His Pro Val Ser Pro Thr Pro Ala Ala Met Pro Ser Ser
35 40 45
Thr Pro Ile Ala Gly Ala Leu Pro Leu Thr Met Ala Leu Ser Pro His
50 55 60
Pro Leu Ala Leu Leu Pro Gln Leu Ala Leu Thr Gln Pro Ala Ser Cys
65 70 75 80
Pro Thr Arg Thr Met Arg Ser Ser Thr Pro Ile Pro Ser Leu Gly Gln
85 90 95
Leu Gly Thr Pro Pro Thr Asp Trp Ala Thr Pro Arg Pro His Pro Leu
100 105 110
Ala Trp Ser Gly Pro His Met Arg Arg Met Thr Pro Leu Ala Ser Thr
115 120 125
Pro Gln Pro Leu Asp Ser Pro Thr Pro Pro Ser Thr Arg Thr Thr Ala
130 135 140
Ser Asp Ser Ser Ser Ala Cys Arg Leu Thr Cys Arg Gly Gln Ser Leu
145 ' 150 155 160
Ala Gly Ala Ser Leu Arg Gly Arg Gly Glu Gly Phe His Ser Cys Ser
165 170 175
Leu Ile Phe Arg Gly Ile Ala His Cys Ser Leu Leu Gly Pro Ala Phe
180 185 190
Phe Leu Pro Pro Trp Gln Val Gly Ser Arg Ser Pro Arg Asn Thr Pro
195 200 205
Ser Arg Gly Trp Cys Ser Val His Ala Pro Ala Ser Trp Ala Cys Pro
210 215 220
Ser Leu Phe Phe Gly Arg Met Cys Leu Phe
225 230
<210> 145
<211> 131
<212> PRT
<213> Homo Sapiens
<400> 145
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Met Asp Arg Val Thr Val Gly Gly Trp Cys Thr Ser Ser Ser Met Glu
1 5 10 15
Val Ser Leu Pro Leu Gly Met Ala Ser Phe Met Leu Arg Gln Val Lys
20 25 30
Thr Arg Pro Met Ser Arg Ser Val Gly Leu Ile Ser Ser Trp Leu Thr
35 40 45
Val Val Ser Leu Phe Pro Ser Phe Ser Asn Ser Val Leu Ala Thr Ala
50 55 60
Pro Ser Cys Cys Val Pro Ser Arg Asn Gln Met Met Val Ala Ala Gly
65 70 75 80
Phe Ala Leu Lys Ala Arg His Val Arg Leu Trp Gly Val Pro Ala Cys
85 90 95
Ser Arg Ile Thr Gly Pro Pro Ser Ile Leu Val Ser Ser Gly Gly Met
100 105 110
Ser Thr Val Ser Leu Ala Arg Arg Glu Arg Arg Ala Ala Ser Val Ala
115 120 125
Trp His Ser
130
<210>
146
<211>
221
<212>
PRT
<213>
Homo
Sapiens
<400>
146
His Glu Arg AlaLeuThr ThrMetGln IleGln ValAlaGly Leu
Gln
1 5 10 15
Leu Gln Ala ValProLeu PheSerThr AlaGlu GluAspLeu Leu
Phe
20 25 30
Ala Ile Leu LeuLeuAsn SerSerGlu SerSer LeuHisGln Leu
Gln
35 40 45
Thr Ala Val AspCysArg GlyLeuHis LysAsp TyrLeuAsp Ala
Met
50 55 60
Leu Ala Ile CysTyrAsp GlyLeuGln GlyLeu LeuTyrLeu Gly
Gly
65 70 75 80
Leu Phe Phe LeuAlaAla LeuAlaPhe SerThr MetIleCys Ala
Ser
85 90 95
Gly Pro Ala TrpLysHis PheThrThr ArgAsn ArgAspTyr Asp
Arg
100 105 110
Asp Ile Asp AspAspPro PheAsnPro GlnAla TrpArgMet Ala
Asp
115 120 125
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Ala His Ser Pro Pro Arg Gly Gln Leu His Ser Phe Cys Ser Tyr Ser
130 135 140
Ser Gly Leu Gly Ser Gln Thr Ser Leu Gln Pro Pro Ala Gln Thr Ile
145 150 155 160
Ser Asn Ala Pro Val Ser Glu Tyr Met Asn Gln Ala Met Leu Phe Gly
165 170 175
Arg Asn Pro Arg Tyr Glu Asn Val Pro Leu Ile Gly Arg Ala Ser Pro
180 185 190
Pro Pro Thr Tyr Ser Pro Ser Met Arg Ala Thr Tyr Leu Ser Val Ala
195 200 205
Asp Glu His Leu Arg His Tyr Gly Asn Gln Phe Pro Ala
210 215 220
DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
CECI EST LE TOME 2 DE 2
~~ TTENANT LES PAGES 284 A 436
NOTE : Pour les tomes additionels, veuillez contacter 1e Bureau canadien des
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CONTAINING PAGES 284 TO 436
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