U.S. patent application number 13/607384 was filed with the patent office on 2013-04-25 for polypeptides and antibodies.
This patent application is currently assigned to Amgen Inc.. The applicant listed for this patent is Xiao Feng, Alison Fitch, Ian Foltz, Stephen Foster, Brian Gliniak, Randal R. Ketchem, Sharon Wong-Madden, Xiao-Dong Yang. Invention is credited to Xiao Feng, Alison Fitch, Ian Foltz, Stephen Foster, Brian Gliniak, Randal R. Ketchem, Sharon Wong-Madden, Xiao-Dong Yang.
Application Number | 20130101589 13/607384 |
Document ID | / |
Family ID | 37546951 |
Filed Date | 2013-04-25 |
United States Patent
Application |
20130101589 |
Kind Code |
A1 |
Gliniak; Brian ; et
al. |
April 25, 2013 |
POLYPEPTIDES AND ANTIBODIES
Abstract
Polypeptides are provided. Antibodies or antigen binding domains
are provided which bind such polypeptides. Also provided are
methods of obtaining an antibody that binds tumor necrosis factor
(TNF)-related apoptosis-inducing ligand ("TRAIL") Receptor-2 (TR-2)
comprising administering at least one of such polypeptides to an
animal and obtaining an antibody that binds TR-2 from the animal.
Antibodies reactive with TR-2 are provided. Also provided are cells
producing antibodies reactive with TR-2, pharmaceutical
compositions comprising antibodies reactive with TR-2, methods
using antibodies reactive with TR-2, and kits comprising antibodies
reactive with TR-2. Also provided are methods of decreasing or
preventing binding of an antibody to TR-2 by administering such a
polypeptide.
Inventors: |
Gliniak; Brian; (Seattle,
WA) ; Yang; Xiao-Dong; (Palo Alto, CA) ;
Wong-Madden; Sharon; (Bellevue, WA) ; Foltz; Ian;
(Burnaby, CA) ; Feng; Xiao; (The Woodlands,
TX) ; Fitch; Alison; (Vancouver, CA) ; Foster;
Stephen; (Newbury Park, CA) ; Ketchem; Randal R.;
(Snohomish, WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Gliniak; Brian
Yang; Xiao-Dong
Wong-Madden; Sharon
Foltz; Ian
Feng; Xiao
Fitch; Alison
Foster; Stephen
Ketchem; Randal R. |
Seattle
Palo Alto
Bellevue
Burnaby
The Woodlands
Vancouver
Newbury Park
Snohomish |
WA
CA
WA
TX
CA
WA |
US
US
US
CA
US
CA
US
US |
|
|
Assignee: |
Amgen Inc.
|
Family ID: |
37546951 |
Appl. No.: |
13/607384 |
Filed: |
September 7, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12417438 |
Apr 2, 2009 |
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13607384 |
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11512051 |
Aug 28, 2006 |
7521048 |
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12417438 |
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60713433 |
Aug 31, 2005 |
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60713478 |
Aug 31, 2005 |
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Current U.S.
Class: |
424/139.1 |
Current CPC
Class: |
A61K 38/00 20130101;
A61P 37/00 20180101; A61P 35/00 20180101; C07K 2317/92 20130101;
C07K 2317/73 20130101; A61K 39/39558 20130101; C07K 2317/21
20130101; C07K 2317/24 20130101; C07K 16/2878 20130101 |
Class at
Publication: |
424/139.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395 |
Claims
1-113. (canceled)
114. A method of treating cancer in a patient that has cancer
associated with tissue expressing TRAIL receptor-2 (TR-2)
comprising administering to the patient a therapeutically effective
amount of an antibody comprising a heavy chain and a light chain,
wherein the heavy chain comprises CDR1 of SEQ ID NO: 30, CDR2 of
SEQ ID NO: 30, and CDR3 of SEQ ID NO: 30, and wherein the light
chain comprises CDR1 of SEQ ID NO: 64, CDR2 of SEQ ID NO: 64, and
CDR3 of SEQ ID NO: 64, and wherein the antibody binds TR-2 and
induces cancer cell apoptosis.
115. The method of claim 114, wherein the heavy chain comprises the
amino acid sequence of SEQ ID NO: 30, and wherein the light chain
comprises the amino acid sequence of SEQ ID NO: 64.
116. The method of claim 114, wherein the antibody is a fully human
antibody.
117. The method of claim 115, wherein the antibody is a fully human
antibody.
118. The method of claim 114, wherein the light chain comprises the
amino acid sequence of SEQ ID NO: 64.
119. The method of claim 118, wherein the antibody is a fully human
antibody.
120. The method of claim 114, wherein the heavy chain comprises the
amino acid sequence of SEQ ID NO: 30 in which one of the cysteine
residues of SEQ ID NO: 30 is replaced by one non-cysteine
residue.
121. The method of claim 120, wherein the antibody is a fully human
antibody.
122. The method of claim 120, wherein the light chain comprises the
amino acid sequence of SEQ ID NO: 64.
123. The method of claim 122, wherein the antibody is a fully human
antibody.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/713,433, filed Aug. 31, 2005, and U.S.
Provisional Application No. 60/713,478, filed Aug. 31, 2005. U.S.
Provisional Application Nos. 60/713,433 and 60/713,478 are
incorporated by reference herein in their entirety for any
purpose.
FIELD
[0002] Polypeptides are provided. Antibodies or antigen binding
domains are provided which bind such polypeptides. Also provided
are methods of obtaining an antibody that binds tumor necrosis
factor (TNF)-related apoptosis-inducing ligand ("TRAIL") Receptor-2
(TR-2) comprising administering at least one of such polypeptides
to an animal and obtaining an antibody that binds TR-2 from the
animal. Antibodies reactive with TR-2 are provided. Also provided
are cells producing antibodies reactive with TR-2, pharmaceutical
compositions comprising antibodies reactive with TR-2, methods
using antibodies reactive with TR-2, and kits comprising antibodies
reactive with TR-2. Also provided are methods of decreasing or
preventing binding of an antibody to TR-2 by administering such a
polypeptide.
BACKGROUND
[0003] The interaction between TR-2 and its ligand, TRAIL, plays a
role in the induction of apoptosis (see, for example, Almasan et
al., Cytokine & Growth Factor Reviews 14: 337-348 (2003)).
TRAIL, also known as Apo2 ligand, is a homomeric ligand that
interacts with four members of the TNF-receptor superfamily (TRAIL
receptors ("TR") 1 to 4), as well as with the related, soluble,
opsteoprotegerin ("OPG") receptor. Binding of TRAIL to TR-1 or TR-2
at the surface of a cell triggers apoptosis of that cell. After
initial binding of TRAIL to TR-1 or TR-2, intracellular proteins
are recruited to the intracellular death domain of the receptor,
forming a signaling complex. Certain intracellular caspases are
recruited to the complex, where they autoactivate and in turn
activate additional caspases and the intracellular apoptosis
cascade. TR-3 and TR-4 and OPG lack the intracellular domain
responsible for transmitting the apoptosis signal. Thus, binding of
TRAIL to TR-3, TR-4, or OPG does not trigger apoptosis. TR-3 and
TR-4 are also referred to as "decoy" receptors, and their
overexpression has been shown to protect cells from apoptotic
induction by TRAIL. TR-2 is expressed in a variety of cells,
including liver, brain, breast, kidney, colon, lung, spleen,
thymus, peripheral blood lymphocytes, prostate, testis, ovary,
uterus, and various tissues along the gastro-intestinal tract.
(See, for example, Walczak et al., EMBO J. 16: 5386-5397 (1997);
Spierings et al., J. Histochem. Cytochem. 52: 821-831 (2004)).
Though TRAIL and TRAIL receptors are widely expressed, they are
most active in inducing apoptosis in transformed cells. (See, for
example, Daigle et al., Swiss Med. Wkly. 131: 231-237 (2001)).
SUMMARY
[0004] In certain embodiments, an isolated polypeptide is provided
comprising at least one complementarity determining region (CDR)
selected from CDR1a, CDR2a, and CDR3a: [0005] wherein CDR1a
comprises the amino acid sequence a b c d e f g h i j k l, wherein
amino acid a is glycine, amino acid b is selected from glycine,
tyrosine, or phenylalanine; amino acid c is selected from serine or
threonine; amino acid d is selected from isoleucine or
phenylalanine; amino acid e is selected from serine, threonine, or
asparagine; amino acid f is selected from serine, aspartic acid,
tyrosine, asparagine, threonine, or glycine; amino acid g is
selected from glycine, aspartic acid, or tyrosine; amino acid h is
selected from glycine, aspartic acid, tyrosine, asparagine, or
serine; amino acid i is selected from tyrosine, isoleucine,
histidine, methionine, or tryptophan; amino acid j is selected from
asparagine, tyrosine, histidine, serine, or phenylalanine; amino
acid k is tryptophan or is not present; and amino acid l is serine
or is not present; [0006] wherein CDR2a comprises the amino acid
sequence m n o p q r s t u v w x y z a' b' c', wherein amino acid m
is selected from tryptophan, tyrosine, histidine, valine, glutamic
acid, or serine; amino acid n is selected from methionine or
isoleucine; amino acid o is selected from asparagine, tyrosine,
serine, tryptophan, or histidine; amino acid p is selected from
proline, tyrosine, serine, arginine, histidine, or asparagine;
amino acid q is selected from asparagine, serine, or aspartic acid;
amino acid r is selected from serine or glycine; amino acid s is
selected from aspartic acid, serine, threonine, or arginine; amino
acid t is selected from asparagine, threonine, alanine, isoleucine,
or tyrosine; amino acid u is selected from threonine, tyrosine,
leucine, lysine, asparagine, or isoleucine; amino acid v is
selected from glycine, tyrosine, aspartic acid, or cysteine; amino
acid w is selected from tyrosine or asparagine; amino acid x is
selected from alanine or proline; amino acid y is selected from
glutamine, serine, or aspartic acid; amino acid z is selected from
lysine, leucine, or serine; amino acid a' is selected from
phenylalanine, lysine, or valine; amino acid b' is selected from
glutamine, serine, or lysine; and amino acid c' is glycine or is
not present; wherein CDR3a comprises the amino acid sequence d' e'
f' g' h' j' k' m' n' o' p' q' r' s' t' u' v' w', wherein amino acid
d' is selected from tryptophan, aspartic acid, glycine, serine, or
glutamic acid; amino acid e' is selected from asparagine, aspartic
acid, glycine, arginine, serine, valine, or leucine; amino acid f'
is selected from histidine, serine, alanine, tyrosine, proline,
asparagine, glycine or threonine; amino acid g' is selected from
tyrosine, serine, alanine, arginine, tryptophan, glycine or valine;
amino acid h' is selected from glycine, alanine, serine,
asparagine, methionine, tyrosine, tryptophan, cysteine, or aspartic
acid; amino acid i' is selected from serine, tryptophan, glycine,
phenylalanine, aspartic acid, tyrosine, or threonine; amino acid j'
is selected from glycine, threonine, serine, leucine, valine,
asparagine, tryptophan, or tyrosine; amino acid k' is selected from
serine, phenylalanine, aspartic acid, tryptophan, glycine, or
tyrosine, or is not present; amino acid l' is selected from
histidine, aspartic acid, alanine, tryptophan, tyrosine, serine,
phenylalanine, valine, or glycine, or is not present; amino acid m'
is selected from phenylalanine, tyrosine, glutamic acid, proline,
aspartic acid, cysteine, isoleucine, or methionine, or is not
present; amino acid n' is selected from aspartic acid,
phenylalanine, alanine, leucine, or serine, or is not present;
amino acid o' is selected from tyrosine, leucine, aspartic acid,
phenylalanine, proline, or valine, or is not present; amino acid p'
is selected from leucine, aspartic acid, or tyrosine, or is not
present; amino acid q' is selected from serine or tyrosine, or is
not present; amino acid r' is tyrosine or is not present; amino
acid s' is selected from glycine or tyrosine, or is not present;
amino acid t' is selected from glycine or methionine, or is not
present; amino acid u' is selected from methionine or aspartic
acid, or is not present; amino acid v' is selected from aspartic
acid or valine, or is not present; and amino acid w' is valine or
is not present; and wherein the polypeptide, in association with an
antibody light chain, binds TRAIL receptor-2 (TR-2).
[0007] In certain embodiments, an isolated polypeptide is provided
comprising at least one complementarity determining region (CDR)
selected from:
[0008] amino acids 26 to 35 of SEQ ID NO: 2;
[0009] amino acids 50 to 66 of SEQ ID NO: 2;
[0010] amino acids 99 to 110 of SEQ ID NO: 2;
[0011] amino acids 26 to 37 of SEQ ID NO: 4;
[0012] amino acids 52 to 67 of SEQ ID NO: 4;
[0013] amino acids 100 to 109 of SEQ ID NO: 4;
[0014] amino acids 26 to 37 of SEQ ID NO: 6;
[0015] amino acids 52 to 67 of SEQ ID NO: 6;
[0016] amino acids 100 to 109 of SEQ ID NO: 6;
[0017] amino acids 26 to 37 of SEQ ID NO: 8;
[0018] amino acids 52 to 67 of SEQ ID NO: 8;
[0019] amino acids 100 to 109 of SEQ ID NO: 8;
[0020] amino acids 26 to 35 of SEQ ID NO: 10;
[0021] amino acids 50 to 66 of SEQ ID NO: 10;
[0022] amino acids 99 to 110 of SEQ ID NO: 10;
[0023] amino acids 26 to 35 of SEQ ID NO: 12;
[0024] amino acids 50 to 66 of SEQ ID NO: 12;
[0025] amino acids 99 to 111 of SEQ ID NO: 12;
[0026] amino acids 26 to 35 of SEQ ID NO: 14;
[0027] amino acids 50 to 65 of SEQ ID NO: 14;
[0028] amino acids 98 to 111 of SEQ ID NO: 14;
[0029] amino acids 26 to 37 of SEQ ID NO: 16;
[0030] amino acids 52 to 67 of SEQ ID NO: 16;
[0031] amino acids 100 to 109 of SEQ ID NO: 16;
[0032] amino acids 26 to 35 of SEQ ID NO: 18;
[0033] amino acids 50 to 66 of SEQ ID NO: 18;
[0034] amino acids 99 to 105 of SEQ ID NO: 18;
[0035] amino acids 26 to 35 of SEQ ID NO: 20;
[0036] amino acids 50 to 66 of SEQ ID NO: 20;
[0037] amino acids 99 to 118 of SEQ ID NO: 20;
[0038] amino acids 26 to 35 of SEQ ID NO: 22;
[0039] amino acids 50 to 66 of SEQ ID NO: 22;
[0040] amino acids 99 to 118 of SEQ ID NO: 22;
[0041] amino acids 26 to 35 of SEQ ID NO: 24;
[0042] amino acids 50 to 65 of SEQ ID NO: 24;
[0043] amino acids 98 to 108 of SEQ ID NO: 24;
[0044] amino acids 26 to 35 of SEQ ID NO: 26;
[0045] amino acids 50 to 66 of SEQ ID NO: 26;
[0046] amino acids 99 to 110 of SEQ ID NO: 26;
[0047] amino acids 26 to 35 of SEQ ID NO: 28;
[0048] amino acids 50 to 66 of SEQ ID NO: 28;
[0049] amino acids 99 to 117 of SEQ ID NO: 28;
[0050] amino acids 26 to 37 of SEQ ID NO: 30;
[0051] amino acids 52 to 67 of SEQ ID NO: 30;
[0052] amino acids 100 to 111 of SEQ ID NO: 30;
[0053] amino acids 26 to 37 of SEQ ID NO: 32;
[0054] amino acids 52 to 67 of SEQ ID NO: 32;
[0055] amino acids 100 to 111 of SEQ ID NO: 32;
[0056] amino acids 26 to 37 of SEQ ID NO: 34;
[0057] amino acids 52 to 67 of SEQ ID NO: 34; and
[0058] amino acids 100 to 111 of SEQ ID NO: 34;
wherein the polypeptide, in association with an antibody light
chain, binds TR-2.
[0059] In certain embodiments, an isolated polypeptide is provided
comprising at least one complementarity determining region (CDR)
selected from CDR1b, CDR2b, and CDR3b: [0060] wherein CDR1b
comprises the amino acid sequence a1 b1 c1 d1 e1 f1 g1 h1 i1 j1 k1
l1 m1 n1 o1 p1 q1, wherein amino acid a1 is selected from arginine
or lysine; amino acid b1 is selected from threonine, alanine, or
serine; amino acid c1 is serine; amino acid d1 is glutamine; amino
acid e1 is selected from serine or glycine; amino acid f1 is
selected from isoleucine, leucine, or valine; amino acid g1 is
selected from serine, leucine, or arginine; amino acid h1 is
selected from threonine, serine, isoleucine, asparagine, arginine,
histidine, or tyrosine; amino acid i1 is selected from tyrosine,
arginine, tryptophan, aspartic acid, or serine; j1 is selected from
leucine, isoleucine, asparagine, tyrosine, or serine; amino acid k1
is selected from asparagine, glycine, valine, alanine, or leucine;
amino acid l1 is selected from tyrosine, alanine, or asparagine, or
is not present; amino acid m1 is selected from asparagine or
lysine, or is not present; amino acid n1 is selected from tyrosine,
asparagine, or isoleucine, or is not present; amino acid o1 is
selected from leucine or tyrosine, or is not present; amino acid p1
is selected from aspartic acid or leucine, or is not present; and
amino acid q1 is selected from valine, alanine, or threonine, or is
not present; [0061] wherein CDR2b comprises the amino acid sequence
r1 s1 t1 u1 v1 w1 x1, wherein amino acid r1 is selected from
alanine, aspartic acid, leucine, tryptophan, glycine, or valine;
amino acid s1 is selected from threonine, valine, glycine, or
alanine; amino acid t1 is serine; amino acid u1 is selected from
serine, asparagine, or threonine; amino acid v1 is selected from
leucine, phenylalanine, or arginine; amino acid w1 is selected from
glutamine, alanine, or glutamic acid; and amino acid x1 is selected
from serine, arginine, or threonine; [0062] wherein CDR3b comprises
the amino acid sequence y1 z1 a1' b1' c1' d1' e1' f1' g1', wherein
amino acid y1 is selected from glutamine, methionine, leucine, or
histidine; amino acid z1 is selected from glutamine or lysine;
amino acid a1' is selected from serine, threonine, alanine,
histidine, tyrosine, or phenylalanine; amino acid b1' is selected
from tyrosine, leucine, asparagine, or glycine; amino acid c1' is
selected from serine, glutamine, isoleucine, or lysine; amino acid
d1' is selected from threonine, phenylalanine, tyrosine, alanine,
or serine; amino acid e1' is proline; amino acid f1' is selected
from leucine, phenylalanine, tryptophan, serine, or arginine; and
amino acid g1' is selected from threonine or serine; and wherein
the polypeptide, in association with an antibody heavy chain, binds
TR-2.
[0063] In certain embodiments, an isolated polypeptide is provided
comprising at least one complementarity determining region (CDR)
selected from:
[0064] amino acids 24 to 34 of SEQ ID NO: 36;
[0065] amino acids 50 to 56 of SEQ ID NO: 36;
[0066] amino acids 89 to 97 of SEQ ID NO: 36;
[0067] amino acids 24 to 34 of SEQ ID NO: 38;
[0068] amino acids 50 to 56 of SEQ ID NO: 38;
[0069] amino acids 89 to 97 of SEQ ID NO: 38;
[0070] amino acids 24 to 34 of SEQ ID NO: 40;
[0071] amino acids 50 to 56 of SEQ ID NO: 40;
[0072] amino acids 89 to 97 of SEQ ID NO: 40;
[0073] amino acids 24 to 34 of SEQ ID NO: 42;
[0074] amino acids 50 to 56 of SEQ ID NO: 42;
[0075] amino acids 89 to 97 of SEQ ID NO: 42;
[0076] amino acids 24 to 34 of SEQ ID NO: 44;
[0077] amino acids 50 to 56 of SEQ ID NO: 44;
[0078] amino acids 89 to 97 of SEQ ID NO: 44;
[0079] amino acids 24 to 34 of SEQ ID NO: 46;
[0080] amino acids 50 to 56 of SEQ ID NO: 46;
[0081] amino acids 89 to 97 of SEQ ID NO: 46;
[0082] amino acids 24 to 40 of SEQ ID NO: 48;
[0083] amino acids 56 to 62 of SEQ ID NO: 48;
[0084] amino acids 95 to 103 of SEQ ID NO: 48;
[0085] amino acids 24 to 39 of SEQ ID NO: 50;
[0086] amino acids 55 to 61 of SEQ ID NO: 50;
[0087] amino acids 94 to 102 of SEQ ID NO: 50;
[0088] amino acids 24 to 40 of SEQ ID NO: 52;
[0089] amino acids 56 to 62 of SEQ ID NO: 52;
[0090] amino acids 95 to 103 of SEQ ID NO: 52;
[0091] amino acids 24 to 34 of SEQ ID NO: 54;
[0092] amino acids 50 to 56 of SEQ ID NO: 54;
[0093] amino acids 89 to 97 of SEQ ID NO: 54;
[0094] amino acids 24 to 34 of SEQ ID NO: 56,
[0095] amino acids 50 to 56 of SEQ ID NO: 56;
[0096] amino acids 89 to 97 of SEQ ID NO: 56;
[0097] amino acids 24 to 40 of SEQ ID NO: 58;
[0098] amino acids 56 to 62 of SEQ ID NO: 58;
[0099] amino acids 95 to 103 of SEQ ID NO: 58;
[0100] amino acids 24 to 34 of SEQ ID NO: 60;
[0101] amino acids 50 to 56 of SEQ ID NO: 60;
[0102] amino acids 89 to 97 of SEQ ID NO: 60;
[0103] amino acids 24 to 34 of SEQ ID NO: 62;
[0104] amino acids 50 to 56 of SEQ ID NO: 62;
[0105] amino acids 89 to 97 of SEQ ID NO: 62;
[0106] amino acids 24 to 35 of SEQ ID NO: 64;
[0107] amino acids 51 to 57 of SEQ ID NO: 64;
[0108] amino acids 90 to 88 of SEQ ID NO: 64;
[0109] amino acids 24 to 34 of SEQ ID NO: 66;
[0110] amino acids 50 to 57 of SEQ ID NO: 66;
[0111] amino acids 89 to 97 of SEQ ID NO: 66;
[0112] amino acids 24 to 34 of SEQ ID NO: 68;
[0113] amino acids 50 to 56 of SEQ ID NO: 68; and
[0114] amino acids 89 to 97 of SEQ ID NO: 68;
wherein the polypeptide, in association with an antibody heavy
chain, binds TR-2.
[0115] In certain embodiments, an isolated polynucleotide is
provided comprising a sequence encoding a polypeptide comprising at
least one complementarity determining region (CDR) selected from
CDR1a, CDR2a, and CDR3a: [0116] wherein CDR1a comprises the amino
acid sequence a b c d e f g h i j k l, wherein amino acid a is
glycine, amino acid b is selected from glycine, tyrosine, or
phenylalanine; amino acid c is selected from serine or threonine;
amino acid d is selected from isoleucine or phenylalanine; amino
acid e is selected from serine, threonine, or asparagine; amino
acid f is selected from serine, aspartic acid, tyrosine,
asparagine, threonine, or glycine; amino acid g is selected from
glycine, aspartic acid, or tyrosine; amino acid h is selected from
glycine, aspartic acid, tyrosine, asparagine, or serine; amino acid
i is selected from tyrosine, isoleucine, histidine, methionine, or
tryptophan; amino acid j is selected from asparagine, tyrosine,
histidine, serine, or phenylalanine; amino acid k is tryptophan or
is not present; and amino acid l is serine or is not present;
[0117] wherein CDR2a comprises the amino acid sequence m n o p q r
t u v w x y z a' b' c', wherein amino acid m is selected from
tryptophan, tyrosine, histidine, valine, glutamic acid, or serine;
amino acid n is selected from methionine or isoleucine; amino acid
o is selected from asparagine, tyrosine, serine, tryptophan, or
histidine; amino acid p is selected from proline, tyrosine, serine,
arginine, histidine, or asparagine; amino acid q is selected from
asparagine, serine, or aspartic acid; amino acid r is selected from
serine or glycine; amino acid s is selected from aspartic acid,
serine, threonine, or arginine; amino acid t is selected from
asparagine, threonine, alanine, isoleucine, or tyrosine; amino acid
u is selected from threonine, tyrosine, leucine, lysine,
asparagine, or isoleucine; amino acid v is selected from glycine,
tyrosine, aspartic acid, or cysteine; amino acid w is selected from
tyrosine or asparagine; amino acid x is selected from alanine or
proline; amino acid y is selected from glutamine, serine, or
aspartic acid; amino acid z is selected from lysine, leucine, or
serine; amino acid a' is selected from phenylalanine, lysine, or
valine; amino acid b' is selected from glutamine, serine, or
lysine; and amino acid c' is glycine or is not present; [0118]
wherein CDR3a comprises the amino acid sequence d' e' f' g' h' j'
k' m' n' o' p' q' r' s' t' u' v' w', wherein amino acid d' is
selected from tryptophan, aspartic acid, glycine, serine, or
glutamic acid; amino acid e' is selected from asparagine, aspartic
acid, glycine, arginine, serine, valine, or leucine; amino acid f'
is selected from histidine, serine, alanine, tyrosine, proline,
asparagine, glycine or threonine; amino acid g' is selected from
tyrosine, serine, alanine, arginine, tryptophan, glycine or valine;
amino acid h' is selected from glycine, alanine, serine,
asparagine, methionine, tyrosine, tryptophan, cysteine, or aspartic
acid; amino acid i' is selected from serine, tryptophan, glycine,
phenylalanine, aspartic acid, tyrosine, or threonine; amino acid j'
is selected from glycine, threonine, serine, leucine, valine,
asparagine, tryptophan, or tyrosine; amino acid k' is selected from
serine, phenylalanine, aspartic acid, tryptophan, glycine, or
tyrosine, or is not present; amino acid l' is selected from
histidine, aspartic acid, alanine, tryptophan, tyrosine, serine,
phenylalanine, valine, or glycine, or is not present; amino acid m'
is selected from phenylalanine, tyrosine, glutamic acid, proline,
aspartic acid, cysteine, isoleucine, or methionine, or is not
present; amino acid n' is selected from aspartic acid,
phenylalanine, alanine, leucine, or serine, or is not present;
amino acid o' is selected from tyrosine, leucine, aspartic acid,
phenylalanine, proline, or valine, or is not present; amino acid p'
is selected from leucine, aspartic acid, or tyrosine, or is not
present; amino acid q' is selected from serine or tyrosine, or is
not present; amino acid r' is tyrosine or is not present; amino
acid s' is selected from glycine or tyrosine, or is not present;
amino acid t' is selected from glycine or methionine, or is not
present; amino acid u' is selected from methionine or aspartic
acid, or is not present; amino acid v' is selected from aspartic
acid or valine, or is not present; and amino acid w' is valine or
is not present; and wherein the polypeptide, in association with an
antibody light chain, binds TR-2.
[0119] In certain embodiments, an isolated polynucleotide is
provided comprising a sequence encoding a polypeptide comprising at
least one complementarity determining region (CDR) selected from
CDR1b, CDR2b, and CDR3b: [0120] wherein CDR1b comprises the amino
acid sequence a1 b1 c1 d1 e1 f1 g1 h1 i1 j1 k1 l1 m1 n1 o1 p1 q1,
wherein amino acid a1 is selected from arginine or lysine; amino
acid b1 is selected from threonine, alanine, or serine; amino acid
c1 is serine; amino acid d1 is glutamine; amino acid e1 is selected
from serine or glycine; amino acid f1 is selected from isoleucine,
leucine, or valine; amino acid g1 is selected from serine, leucine,
or arginine; amino acid h1 is selected from threonine, serine,
isoleucine, asparagine, arginine, histidine, or tyrosine; amino
acid i1 is selected from tyrosine, arginine, tryptophan, aspartic
acid, or serine; j1 is selected from leucine, isoleucine,
asparagine, tyrosine, or serine; amino acid k1 is selected from
asparagine, glycine, valine, alanine, or leucine; amino acid l1 is
selected from tyrosine, alanine, or asparagine, or is not present;
amino acid m1 is selected from asparagine or lysine, or is not
present; amino acid n1 is selected from tyrosine, asparagine, or
isoleucine, or is not present; amino acid o1 is selected from
leucine or tyrosine, or is not present; amino acid p1 is selected
from aspartic acid or leucine, or is not present; and amino acid q1
is selected from valine, alanine, or threonine, or is not present;
[0121] wherein CDR2b comprises the amino acid sequence r1 s1 t1 u1
v1 w1 x1, wherein amino acid r1 is selected from alanine, aspanic
acid, leucine, tryptophan, glycine, or valine; amino acid s1 is
selected from threonine, valine, glycine, or alanine; amino acid t1
is serine; amino acid u1 is selected from serine, asparagine, or
threonine; amino acid v1 is selected from leucine, phenylalanine,
or arginine; amino acid w1 is selected from glutamine, alanine, or
glutamic acid; and amino acid x1 is selected from serine, arginine,
or threonine; wherein CDR3b comprises the amino acid sequence y1 z1
a1' b1' c1' d1' e1' f1' g1', wherein amino acid y1 is selected from
glutamine, methionine, leucine, or histidine; amino acid z1 is
selected from glutamine or lysine; amino acid a1' is selected from
serine, threonine, alanine, histidine, tyrosine, or phenylalanine;
amino acid b1' is selected from tyrosine, leucine, asparagine, or
glycine; amino acid c1' is selected from serine, glutamine,
isoleucine, or lysine; amino acid d1' is selected from threonine,
phenylalanine, tyrosine, alanine, or serine; amino acid e1' is
proline; amino acid f1' is selected from leucine, phenylalanine,
tryptophan, serine, or arginine; and amino acid g1' is selected
from threonine or serine; and wherein the polypeptide, in
association with an antibody heavy chain, binds TR-2.
[0122] In certain embodiments, an isolated anti-TR-2 antibody
comprising a variable region and a constant region is provided,
wherein the antibody comprises: [0123] (i) a first polypeptide
comprising at least one complementarity determining region (CDR)
selected from CDR1a, CDR2a, and CDR3a, [0124] wherein CDR1a
comprises the amino acid sequence a b c d e f g h i j k l, wherein
amino acid a is glycine, amino acid b is selected from glycine,
tyrosine, or phenylalanine; amino acid c is selected from serine or
threonine; amino acid d is selected from isoleucine or
phenylalanine; amino acid e is selected from serine, threonine, or
asparagine; amino acid f is selected from serine, aspartic acid,
tyrosine, asparagine, threonine, or glycine; amino acid g is
selected from glycine, aspartic acid, or tyrosine; amino acid h is
selected from glycine, aspartic acid, tyrosine, asparagine, or
serine; amino acid i is selected from tyrosine, isoleucine,
histidine, methionine, or tryptophan; amino acid j is selected from
asparagine, tyrosine, histidine, serine, or phenylalanine; amino
acid k is tryptophan or is not present; and amino acid l is serine
or is not present; [0125] wherein CDR2a comprises the amino acid
sequence m n o p q r t u v w x y z a' b' c', wherein amino acid m
is selected from tryptophan, tyrosine, histidine, valine, glutamic
acid, or serine; amino acid n is selected from methionine or
isoleucine; amino acid o is selected from asparagine, tyrosine,
serine, tryptophan, or histidine; amino acid p is selected from
praline, tyrosine, serine, arginine, histidine, or asparagine;
amino acid q is selected from asparagine, serine, or aspartic acid;
amino acid r is selected from serine or glycine; amino acid s is
selected from aspartic acid, serine, threonine, or arginine; amino
acid t is selected from asparagine, threonine, alanine, isoleucine,
or tyrosine; amino acid u is selected from threonine, tyrosine,
leucine, lysine, asparagine, or isoleucine; amino acid v is
selected from glycine, tyrosine, aspartic acid, or cysteine; amino
acid w is selected from tyrosine or asparagine; amino acid x is
selected from alanine or proline; amino acid y is selected from
glutamine, serine, or aspartic acid; amino acid z is selected from
lysine, leucine, or serine; amino acid a' is selected from
phenylalanine, lysine, or valine; amino acid b' is selected from
glutamine, serine, or lysine; and amino acid c' is glycine or is
not present; [0126] wherein CDR3a comprises the amino acid sequence
d' e' f' g' h' j' k' m' n' o' p' q' r' s' t' v' w', wherein amino
acid d' is selected from tryptophan, aspartic acid, glycine,
serine, or glutamic acid; amino acid e' is selected from
asparagine, aspartic acid, glycine, arginine, serine, valine, or
leucine; amino acid f' is selected from histidine, serine, alanine,
tyrosine, proline, asparagine, glycine or threonine; amino acid g'
is selected from tyrosine, serine, alanine, arginine, tryptophan,
glycine or valine; amino acid h' is selected from glycine, alanine,
serine, asparagine, methionine, tyrosine, tryptophan, cysteine, or
aspartic acid; amino acid i' is selected from serine, tryptophan,
glycine, phenylalanine, aspartic acid, tyrosine, or threonine;
amino acid j' is selected from glycine, threonine, serine, leucine,
valine, asparagine, tryptophan, or tyrosine; amino acid k' is
selected from serine, phenylalanine, aspartic acid, tryptophan,
glycine, or tyrosine, or is not present; amino acid l' is selected
from histidine, aspartic acid, alanine, tryptophan, tyrosine,
serine, phenylalanine, valine, or glycine, or is not present; amino
acid m' is selected from phenylalanine, tyrosine, glutamic acid,
proline, aspartic acid, cysteine, isoleucine, or methionine, or is
not present; amino acid n' is selected from aspartic acid,
phenylalanine, alanine, leucine, or serine, or is not present;
amino acid o' is selected from tyrosine, leucine, aspartic acid,
phenylalanine, proline, or valine, or is not present; amino acid p'
is selected from leucine, aspartic acid, or tyrosine, or is not
present; amino acid q' is selected from serine or tyrosine, or is
not present; amino acid r' is tyrosine or is not present; amino
acid s' is selected from glycine or tyrosine, or is not present;
amino acid t' is selected from glycine or methionine, or is not
present; amino acid u' is selected from methionine or aspartic
acid, or is not present; amino acid v' is selected from aspartic
acid or valine, or is not present; and amino acid w' is valine or
is not present; and [0127] wherein the first polypeptide, in
association with an antibody light chain, binds TR-2; and [0128]
(ii) a second polypeptide comprising at least one complementarity
determining region (CDR) selected from CDR1b, CDR2b, and CDR3b
wherein CDR1b comprises the amino acid sequence a1 b1 c1 d1 e1 f1
g1 h1 i1 j1 k1 l1 m1 n1 o1 p1 q1, wherein amino acid a1 is selected
from arginine or lysine; amino acid b1 is selected from threonine,
alanine, or serine; amino acid c1 is serine; amino acid d1 is
glutamine; amino acid e1 is selected from serine or glycine; amino
acid f1 is selected from isoleucine, leucine, or valine; amino acid
g1 is selected from serine, leucine, or arginine; amino acid h1 is
selected from threonine, serine, isoleucine, asparagine, arginine,
histidine, or tyrosine; amino acid i1 is selected from tyrosine,
arginine, tryptophan, aspartic acid, or serine; j1 is selected from
leucine, isoleucine, asparagine, tyrosine, or serine; amino acid k1
is selected from asparagine, glycine, valine, alanine, or leucine;
amino acid l1 is selected from tyrosine, alanine, or asparagine, or
is not present; amino acid m1 is selected from asparagine or
lysine, or is not present; amino acid n1 is selected from tyrosine,
asparagine, or isoleucine, or is not present; amino acid o1 is
selected from leucine or tyrosine, or is not present; amino acid p1
is selected from aspartic acid or leucine, or is not present; and
amino acid q1 is selected from valine, alanine, or threonine, or is
not present; [0129] wherein CDR2b comprises the amino acid sequence
r1 s1 t1 u1 v1 w1 x1, wherein amino acid r1 is selected from
alanine, aspartic acid, leucine, tryptophan, glycine, or valine;
amino acid s1 is selected from threonine, valine, glycine, or
alanine; amino acid t1 is serine; amino acid u1 is selected from
serine, asparagine, or threonine; amino acid v1 is selected from
leucine, phenylalanine, or arginine; amino acid w1 is selected from
glutamine, alanine, or glutamic acid; and amino acid x1 is selected
from serine, arginine, or threonine; [0130] wherein CDR3b comprises
the amino acid sequence y1 z1 a1' c1' d1' e1' f1' wherein amino
acid y1 is selected from glutamine, methionine, leucine, or
histidine; amino acid z1 is selected from glutamine or lysine;
amino acid a1' is selected from serine, threonine, alanine,
histidine, tyrosine, or phenylalanine; amino acid b1' is selected
from tyrosine, leucine, asparagine, or glycine; amino acid c1' is
selected from serine, glutamine, isoleucine, or lysine; amino acid
d1' is selected from threonine, phenylalanine, tyrosine, alanine,
or serine; amino acid e1' is proline; amino acid f1' is selected
from leucine, phenylalanine, tryptophan, serine, or arginine; and
amino acid g1' is selected from threonine or serine; and [0131]
wherein the second polypeptide, in association with an antibody
heavy chain, binds TR-2.
[0132] In certain embodiments, an isolated anti-TR-2 antibody
comprising a variable region and a constant region is provided,
wherein the antibody comprises: [0133] a first polypeptide
comprising complementarity determining regions (CDRs) as set forth
in SEQ ID NO: 2 and a second polypeptide comprising CDRs as set
forth in SEQ ID NO: 36; a first polypeptide comprising
complementarity determining regions (CDRs) as set forth in SEQ ID
NO: 4 and a second polypeptide comprising CDRs as set forth in SEQ
ID NO: 38; a first polypeptide comprising complementarity
determining regions (CDRs) as set forth in SEQ ID NO: 6 and a
second polypeptide comprising CDRs as set forth in SEQ ID NO: 40; a
first polypeptide comprising complementarity determining regions
(CDRs) as set forth in SEQ ID NO: 8 and a second polypeptide
comprising CDRs as set forth in SEQ ID NO: 42; a first polypeptide
comprising complementarity determining regions (CDRs) as set forth
in SEQ ID NO: 10 and a second polypeptide comprising CDRs as set
forth in SEQ ID NO: 44; a first polypeptide comprising
complementarity determining regions (CDRs) as set forth in SEQ ID
NO: 12 and a second polypeptide comprising CDRs as set forth in SEQ
ID NO: 46; a first polypeptide comprising complementarity
determining regions (CDRs) as set forth in SEQ ID NO: 14 and a
second polypeptide comprising CDRs as set forth in SEQ ID NO: 48; a
first polypeptide comprising complementarity determining regions
(CDRs) as set forth in SEQ ID NO: 16 and a second polypeptide
comprising CDRs as set forth in SEQ ID NO: 50; a first polypeptide
comprising complementarity determining regions (CDRs) as set forth
in SEQ ID NO: 18 and a second polypeptide comprising CDRs as set
forth in SEQ ID NO: 52; a first polypeptide comprising
complementarity determining regions (CDRs) as set forth in SEQ ID
NO: 20 and a second polypeptide comprising CDRs as set forth in SEQ
ID NO: 54; a first polypeptide comprising complementarity
determining regions (CDRs) as set forth in SEQ ID NO: 22 and a
second polypeptide comprising CDRs as set forth in SEQ ID NO: 56; a
first polypeptide comprising complementarity determining regions
(CDRs) as set forth in SEQ ID NO: 24 and a second polypeptide
comprising CDRs as set forth in SEQ ID NO: 58; a first polypeptide
comprising complementarity determining regions (CDRs) as set forth
in SEQ ID NO: 26 and a second polypeptide comprising CDRs as set
forth in SEQ ID NO: 60; a first polypeptide comprising
complementarity determining regions (CDRs) as set forth in SEQ ID
NO: 28 and a second polypeptide comprising CDRs as set forth in SEQ
ID NO: 62; a first polypeptide comprising complementarity
determining regions (CDRs) as set forth in SEQ ID NO: 30 and a
second polypeptide comprising CDRs as set forth in SEQ ID NO: 64; a
first polypeptide comprising complementarity determining regions
(CDRs) as set forth in SEQ ID NO: 32 and a second polypeptide
comprising CDRs as set forth in SEQ ID NO: 66; or a first
polypeptide comprising complementarity determining regions (CDRs)
as set forth in SEQ ID NO: 34 and a second polypeptide comprising
CDRs as set forth in SEQ ID NO: 68.
[0134] In certain embodiments, a cell is provided, comprising:
[0135] (a) a first polynucleotide comprising a sequence encoding a
first polypeptide comprising at least one complementarity
determining region (CDR) selected from CDR1a, CDR2a, and CDR3a,
wherein CDR1a comprises the amino acid sequence a b c d e f g h i j
k l, wherein amino acid a is glycine, amino acid b is selected from
glycine, tyrosine, or phenylalanine; amino acid c is selected from
serine or threonine; amino acid d is selected from isoleucine or
phenylalanine; amino acid e is selected from serine, threonine, or
asparagine; amino acid f is selected from serine, aspartic acid,
tyrosine, asparagine, threonine, or glycine; amino acid g is
selected from glycine, aspartic acid, or tyrosine; amino acid h is
selected from glycine, aspartic acid, tyrosine, asparagine, or
serine; amino acid i is selected from tyrosine, isoleucine,
histidine, methionine, or tryptophan; amino acid j is selected from
asparagine, tyrosine, histidine, serine, or phenylalanine; amino
acid k is tryptophan or is not present; and amino acid l is serine
or is not present; [0136] wherein CDR2a comprises the amino acid
sequence m no p q r t u v w x y z a' b' c', wherein amino acid m is
selected from tryptophan, tyrosine, histidine, valine, glutamic
acid, or serine; amino acid n is selected from methionine or
isoleucine; amino acid o is selected from asparagine, tyrosine,
serine, tryptophan, or histidine; amino acid p is selected from
proline, tyrosine, serine, arginine, histidine, or asparagine;
amino acid q is selected from asparagine, serine, or aspartic acid;
amino acid r is selected from serine or glycine; amino acid s is
selected from aspartic acid, serine, threonine, or arginine; amino
acid t is selected from asparagine, threonine, alanine, isoleucine,
or tyrosine; amino acid u is selected from threonine, tyrosine,
leucine, lysine, asparagine, or isoleucine; amino acid v is
selected from glycine, tyrosine, aspartic acid, or cysteine; amino
acid w is selected from tyrosine or asparagine; amino acid x is
selected from alanine or proline; amino acid y is selected from
glutamine, serine, or aspartic acid; amino acid z is selected from
lysine, leucine, or serine; amino acid a' is selected from
phenylalanine, lysine, or valine; amino acid b' is selected from
glutamine, serine, or lysine; and amino acid c' is glycine or is
not present; [0137] wherein CDR3a comprises the amino acid sequence
d' e' f' g' h' j' k' m' n' o' p' q' r' s' t' u' v' w', wherein
amino acid d' is selected from tryptophan, aspartic acid, glycine,
serine, or glutamic acid; amino acid e' is selected from
asparagine, aspartic acid, glycine, arginine, serine, valine, or
leucine; amino acid f' is selected from histidine, serine, alanine,
tyrosine, proline, asparagine, glycine or threonine; amino acid g'
is selected from tyrosine, serine, alanine, arginine, tryptophan,
glycine or valine; amino acid h' is selected from glycine, alanine,
serine, asparagine, methionine, tyrosine, tryptophan, cysteine, or
aspartic acid; amino acid i' is selected from serine, tryptophan,
glycine, phenylalanine, aspartic acid, tyrosine, or threonine;
amino acid j' is selected from glycine, threonine, serine, leucine,
valine, asparagine, tryptophan, or tyrosine; amino acid k' is
selected from serine, phenylalanine, aspartic acid, tryptophan,
glycine, or tyrosine, or is not present; amino acid l' is selected
from histidine, aspartic acid, alanine, tryptophan, tyrosine,
serine, phenylalanine, valine, or glycine, or is not present; amino
acid m' is selected from phenylalanine, tyrosine, glutamic acid,
proline, aspartic acid, cysteine, isoleucine, or methionine, or is
not present; amino acid n' is selected from aspartic acid,
phenylalanine, alanine, leucine, or serine, or is not present;
amino acid o' is selected from tyrosine, leucine, aspartic acid,
phenylalanine, proline, or valine, or is not present; amino acid p'
is selected from leucine, aspartic acid, or tyrosine, or is not
present; amino acid q' is selected from serine or tyrosine, or is
not present; amino acid r' is tyrosine or is not present; amino
acid s' is selected from glycine or tyrosine, or is not present;
amino acid t' is selected from glycine or methionine, or is not
present; amino acid u' is selected from methionine or aspartic
acid, or is not present; amino acid v' is selected from aspartic
acid or valine, or is not present; and amino acid w' is valine or
is not present; wherein the first polypeptide, in association with
an antibody light chain, binds TR-2; and [0138] (b) a second
polynucleotide comprising a sequence encoding a second polypeptide
comprising at least one complementarity determining region (CDR)
selected from CDR1b, CDR2b, and CDR3b, [0139] wherein CDR1b
comprises the amino acid sequence a1 b1 c1 d1 e1 f1 g1 h1 i1 j1 k1
l1 m1 n1 o1 p1 q1, wherein amino acid a1 is selected from arginine
or lysine; amino acid b1 is selected from threonine, alanine, or
serine; amino acid c1 is serine; amino acid d1 is glutamine; amino
acid e1 is selected from serine or glycine; amino acid f1 is
selected from isoleucine, leucine, or valine; amino acid g1 is
selected from serine, leucine, or arginine; amino acid h1 is
selected from threonine, serine, isoleucine, asparagine, arginine,
histidine, or tyrosine; amino acid i1 is selected from tyrosine,
arginine, tryptophan, aspartic acid, or serine; j1 is selected from
leucine, isoleucine, asparagine, tyrosine, or serine; amino acid k1
is selected from asparagine, glycine, valine, alanine, or leucine;
amino acid l1 is selected from tyrosine, alanine, or asparagine, or
is not present; amino acid m1 is selected from asparagine or
lysine, or is not present; amino acid n1 is selected from tyrosine,
asparagine, or isoleucine, or is not present; amino acid o1 is
selected from leucine or tyrosine, or is not present; amino acid p1
is selected from aspartic acid or leucine, or is not present; and
amino acid q1 is selected from valine, alanine, or threonine, or is
not present; [0140] wherein CDR2b comprises the amino acid sequence
r1 s1 t1 u1 v1 w1 x1, wherein amino acid r1 is selected from
alanine, aspanic acid, leucine, tryptophan, glycine, or valine;
amino acid s1 is selected from threonine, valine, glycine, or
alanine; amino acid t1 is serine; amino acid u1 is selected from
serine, asparagine, or threonine; amino acid v1 is selected from
leucine, phenylalanine, or arginine; amino acid w1 is selected from
glutamine, alanine, or glutamic acid; and amino acid x1 is selected
from serine, arginine, or threonine; [0141] wherein CDR3b comprises
the amino acid sequence y1 z1 a1' b1' c1' d1' e1' f1' g1', wherein
amino acid y1 is selected from glutamine, methionine, leucine, or
histidine; amino acid z1 is selected from glutamine or lysine;
amino acid a1' is selected from serine, threonine, alanine,
histidine, tyrosine, or phenylalanine; amino acid b1' is selected
from tyrosine, leucine, asparagine, or glycine; amino acid c1' is
selected from serine, glutamine, isoleucine, or lysine; amino acid
d1' is selected from threonine, phenylalanine, tyrosine, alanine,
or serine; amino acid e1' is praline; amino acid f1' is selected
from leucine, phenylalanine, tryptophan, serine, or arginine; and
amino acid g1' is selected from threonine or serine; wherein the
second polypeptide, in association with an antibody heavy chain,
binds TR-2.
[0142] In certain embodiments, an isolated antibody is provided
that specifically binds to an epitope that is specifically bound by
at least one antibody selected from: Ab A, Ab B, Ab C, Ab D, Ab E,
Ab F, Ab G, Ab H, Ab I, Ab J, Ab K, Ab L, Ab M, Ab N, Ab O, Ab P,
and Ab Q.
[0143] In certain embodiments, a polypeptide is provided comprising
at least one amino acid sequence selected from SEQ ID NO: 94, SEQ
ID NO: 95, and SEQ ID NO: 96.
[0144] In certain embodiments, a polypeptide is provided consisting
essentially of at least one amino acid sequence selected from SEQ
ID NO: 94, SEQ ID NO: 95, and SEQ ID NO: 96.
[0145] In certain embodiments, an antibody or antigen binding
domain is provided which binds at least one amino acid sequence
selected from SEQ ID NO: 94, SEQ ID NO: 95, and SEQ ID NO: 96.
[0146] In certain embodiments, a method of obtaining an antibody
that binds TR-2 is provided comprising administering at least one
polypeptide selected from SEQ ID NO: 94, SEQ ID NO: 95, and SEQ ID
NO: 96 to an animal and obtaining an antibody that binds TR-2 from
the animal.
[0147] In certain embodiments, a method of decreasing or preventing
binding of an antibody to TR-2 by administering a polypeptide
comprising at least one amino acid sequence selected from SEQ ID
NO: 94, SEQ ID NO: 95, and SEQ ID NO: 96 is provided.
[0148] In certain embodiments, a method of decreasing or preventing
binding of an antibody to TR-2 by administering a polypeptide
consisting of at least one amino acid sequence selected from SEQ ID
NO: 94, SEQ ID NO: 95, and SEQ ID NO: 96 is provided.
BRIEF DESCRIPTION OF THE FIGURES
[0149] FIG. 1 shows the immunization schedule used in Example 1 for
a TR-2-His construct in transgenic mice expressing human
immunoglobulin genes, via either footpad inoculation (groups 1, 2,
and 3) or via intraperitoneal injection (groups 4 and 5).
[0150] FIG. 2 shows the results of an ELISA assay to measure the
reactivity of certain blood samples from selected mice described in
FIG. 1 to the antigen TR-2, according to work described in Example
1.
[0151] FIG. 3 shows the nucleotide sequences encoding the heavy
chain (SEQ ID NO: 1) and light chain (SEQ ID NO: 35) variable
regions of anti-TR-2 antibody A, and the amino acid sequences of
the heavy chain (SEQ ID NO: 2) and the light chain (SEQ ID NO: 36)
variable regions of that antibody.
[0152] FIG. 4 shows the nucleotide sequences encoding the heavy
chain (SEQ ID NO: 3) and light chain (SEQ ID NO: 37) variable
regions of anti-TR-2 antibody B, and the amino acid sequences of
the heavy chain (SEQ ID NO: 4) and the light chain (SEQ ID NO: 38)
variable regions of that antibody.
[0153] FIG. 5 shows the nucleotide sequences encoding the heavy
chain (SEQ ID NO: 5) and light chain (SEQ ID NO: 39) variable
regions of anti-TR-2 antibody C, and the amino acid sequences of
the heavy chain (SEQ ID NO: 6) and the light chain (SEQ ID NO: 40)
variable regions of that antibody.
[0154] FIG. 6 shows the nucleotide sequences encoding the heavy
chain (SEQ ID NO: 7) and light chain (SEQ ID NO: 41) variable
regions of anti-TR-2 antibody D, and the amino acid sequences of
the heavy chain (SEQ ID NO: 8) and the light chain (SEQ ID NO: 42)
variable regions of that antibody.
[0155] FIG. 7 shows the nucleotide sequences encoding the heavy
chain (SEQ ID NO: 9) and light chain (SEQ ID NO: 43) variable
regions of anti-TR-2 antibody E, and the amino acid sequences of
the heavy chain (SEQ ID NO: 10) and the light chain (SEQ ID NO: 44)
variable regions of that antibody.
[0156] FIG. 8 shows the nucleotide sequences encoding the heavy
chain (SEQ ID NO: 11) and light chain (SEQ ID NO: 45) variable
regions of anti-TR-2 antibody F, and the amino acid sequences of
the heavy chain (SEQ ID NO: 12) and the light chain (SEQ ID NO: 46)
variable regions of that antibody.
[0157] FIG. 9 shows the nucleotide sequences encoding the heavy
chain (SEQ ID NO: 13) and light chain (SEQ ID NO: 47) variable
regions of anti-TR-2 antibody G, and the amino acid sequences of
the heavy chain (SEQ ID NO: 14) and the light chain (SEQ ID NO: 48)
variable regions of that antibody.
[0158] FIG. 10 shows the nucleotide sequences encoding the heavy
chain (SEQ ID NO: 15) and light chain (SEQ ID NO: 49) variable
regions of anti-TR-2 antibody H, and the amino acid sequences of
the heavy chain (SEQ ID NO: 16) and the light chain (SEQ ID NO: 50)
variable regions of that antibody.
[0159] FIG. 11 shows the nucleotide sequences encoding the heavy
chain (SEQ ID NO: 17) and light chain (SEQ ID NO: 51) variable
regions of anti-TR-2 antibody I, and the amino acid sequences of
the heavy chain (SEQ ID NO: 18) and the light chain (SEQ ID NO: 52)
variable regions of that antibody.
[0160] FIG. 12 shows the nucleotide sequences encoding the heavy
chain (SEQ ID NO: 19) and light chain (SEQ ID NO: 53) variable
regions of anti-TR-2 antibody J, and the amino acid sequences of
the heavy chain (SEQ ID NO: 20) and the light chain (SEQ ID NO: 54)
variable regions of that antibody.
[0161] FIG. 13 shows the nucleotide sequences encoding the heavy
chain (SEQ ID NO: 21) and light chain (SEQ ID NO: 55) variable
regions of anti-TR-2 antibody K, and the amino acid sequences of
the heavy chain (SEQ ID NO: 22) and the light chain (SEQ ID NO: 56)
variable regions of that antibody.
[0162] FIG. 14 shows the nucleotide sequences encoding the heavy
chain (SEQ ID NO: 23) and light chain (SEQ ID NO: 57) variable
regions of anti-TR-2 antibody L, and the amino acid sequences of
the heavy chain (SEQ ID NO: 24) and the light chain (SEQ ID NO: 58)
variable regions of that antibody.
[0163] FIG. 15 shows the nucleotide sequences encoding the heavy
chain (SEQ ID NO: 25) and light chain (SEQ ID NO: 59) variable
regions of anti-TR-2 antibody M, and the amino acid sequences of
the heavy chain (SEQ ID NO: 26) and the light chain (SEQ ID NO: 60)
variable regions of that antibody.
[0164] FIG. 16 shows the nucleotide sequences encoding the heavy
chain (SEQ ID NO: 27) and light chain (SEQ ID NO: 61) variable
regions of anti-TR-2 antibody N, and the amino acid sequences of
the heavy chain (SEQ ID NO: 28) and the light chain (SEQ ID NO: 62)
variable regions of that antibody.
[0165] FIG. 17 shows the nucleotide sequences encoding the heavy
chain (SEQ ID NO: 29) and light chain (SEQ ID NO: 63) variable
regions of anti-TR-2 antibody O, and the amino acid sequences of
the heavy chain (SEQ ID NO: 30) and the light chain (SEQ ID NO: 64)
variable regions of that antibody.
[0166] FIG. 18 shows the nucleotide sequences encoding the heavy
chain (SEQ ID NO: 31) and light chain (SEQ ID NO: 65) variable
regions of anti-TR-2 antibody P, and the amino acid sequences of
the heavy chain (SEQ ID NO: 32) and the light chain (SEQ ID NO: 66)
variable regions of that antibody.
[0167] FIG. 19 shows the nucleotide sequences encoding the heavy
chain (SEQ ID NO: 33) and light chain (SEQ ID NO: 67) variable
regions of anti-TR-2 antibody Q, and the amino acid sequences of
the heavy chain (SEQ ID NO: 34) and the light chain (SEQ ID NO: 68)
variable regions of that antibody.
[0168] FIG. 20 is an alignment of the amino acid sequences of the
heavy chain variable regions for anti-TR-2 antibodies A to Q (SEQ
ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,
and 34). Framework regions 1 through 3 (FR1, FR2, and FR3) and
complementarity determining regions 1 through 3 (CDR1, CDR2, and
CDR3) for each sequence are shown.
[0169] FIG. 21 is an alignment of the amino acid sequences of the
light chain variable regions for anti-TR-2 antibodies A to Q (SEQ
ID NOs: 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64,
66, and 68). Framework regions 1 through 3 (FR1, FR2, and FR3) and
complementarity determining regions 1 through 3 (CDR1, CDR2, and
CDR3) for each sequence are shown.
[0170] FIG. 22 is a table showing the classification of certain
human anti-TR-2 antibodies into one of four reactivity groups
according to the ability of each to bind to the truncated and
chimeric N-avidin TR-2 proteins, according to work described in
Example 5.
[0171] FIG. 23 shows schematic representations of the thirteen
truncations of human N-avidin-TR-2 used in epitope mapping,
according to the work described in Example 6.
[0172] FIG. 24 is a bar graph showing the binding of certain human
anti-TR-2 antibodies to the N-avidin-TR-2 truncations according to
work described in Example 6.
[0173] FIG. 25 shows schematic representations of N-avidin-cyno
TR-2 truncations and N-avidin-cyno/human TR-2 chimeras used in
epitope mapping, according to work described in Example 6.
[0174] FIG. 26 is an alignment of the human TR-2, cyno TR-2 (short
form), and mouse TR-2 sequences, according to work described in
Example 6.
[0175] FIG. 27 is a bar graph showing the binding of certain human
anti-TR-2 antibodies to the N-avidin-TR-2 truncations, chimeras,
and domain replacements according to work described in Example
6.
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
[0176] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described. All documents or portions of documents cited in
this application, including but not limited to patents, patent
applications, articles, books, and treatises, are expressly
incorporated by reference herein in their entirety for any
purpose.
DEFINITIONS
[0177] Standard techniques may be used for recombinant DNA,
oligonucleotide synthesis, and tissue culture and transformation
(e.g., electroporation, lipofection). Enzymatic reactions and
purification techniques may be performed according to
manufacturer's specifications or as commonly accomplished in the
art or as described herein. The foregoing techniques and procedures
may be generally performed according to conventional methods well
known in the art and as described in various general and more
specific references that are cited and discussed throughout the
present specification. See e.g., Sambrook et al. Molecular Cloning:
A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y. (1989)). Unless specific definitions are
provided, the nomenclatures utilized in connection with, and the
laboratory procedures and techniques of, analytical chemistry,
synthetic organic chemistry, and medicinal and pharmaceutical
chemistry described herein are those well known and commonly used
in the art. Standard techniques may be used for chemical syntheses,
chemical analyses, pharmaceutical preparation, formulation,
delivery, and treatment of patients.
[0178] In this application, the use of the singular includes the
plural unless specifically stated otherwise. In this application,
the use of "or" means "and/or" unless stated otherwise.
Furthermore, the use of the term "including", as well as other
forms, such as "includes" and "included", is not limiting. Also,
terms such as "element" or "component" encompass both elements and
components comprising one unit and elements and components that
comprise more than one subunit unless specifically stated
otherwise.
[0179] As utilized in accordance with the present disclosure, the
following terms, unless otherwise indicated, shall be understood to
have the following meanings:
[0180] The term "isolated polynucleotide" as used herein shall mean
a polynucleotide of genomic, cDNA, or synthetic origin or some
combination thereof, which by virtue of its origin the "isolated
polynucleotide" (1) is not associated with all or a portion of a
polynucleotide in which the "isolated polynucleotide" is found in
nature, (2) is linked to a polynucleotide which it is not linked to
in nature, or (3) does not occur in nature as part of a larger
sequence.
[0181] The terms "polynucleotide" and "oligonucleotide" are used
interchangeably, and as referred to herein mean a polymeric form of
nucleotides of at least 10 bases in length. In certain embodiments,
the bases may comprise at least one of ribonucleotides,
deoxyribonucleotides, and a modified form of either type of
nucleotide. The term includes single and double stranded forms of
DNA. The term "polynucleotide" also encompasses sequences that
comprise one or more of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17,
19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51,
53, 55, 57, 59, 61, 63, 65, and 67. In certain embodiments,
polynucleotides have nucleotide sequences that are about 90
percent, or about 95 percent, or about 96 percent, or about 97
percent, or about 98 percent, or about 99 percent identical to
nucleotide sequences shown in FIGS. 3-19. In certain embodiments,
polynucleotides complementary to specific polynucleotides that
encode certain polypeptides described herein are provided.
[0182] In certain embodiments, a polynucleotide comprises a
sequence encoding a polypeptide comprising at least one
complementarity determining region (CDR) selected from CDR1a,
CDR2a, and CDR3a, wherein CDR1a comprises the amino acid sequence a
b c d e f g h i j k l, wherein amino acid a is glycine, amino acid
b is selected from glycine, tyrosine, or phenylalanine; amino acid
c is selected from serine or threonine; amino acid d is selected
from isoleucine or phenylalanine; amino acid e is selected from
serine, threonine, or asparagine; amino acid f is selected from
serine, aspartic acid, tyrosine, asparagine, threonine, or glycine;
amino acid g is selected from glycine, aspartic acid, or tyrosine;
amino acid h is selected from glycine, aspartic acid, tyrosine,
asparagine, or serine; amino acid i is selected from tyrosine,
isoleucine, histidine, methionine, or tryptophan; amino acid j is
selected from asparagine, tyrosine, histidine, serine, or
phenylalanine; amino acid k is tryptophan or is not present; and
amino acid l is serine or is not present; wherein CDR2a comprises
the amino acid sequence m n o p q r t u v w x y z a' b' c', wherein
amino acid m is selected from tryptophan, tyrosine, histidine,
valine, glutamic acid, or serine; amino acid n is selected from
methionine or isoleucine; amino acid o is selected from asparagine,
tyrosine, serine, tryptophan, or histidine; amino acid p is
selected from proline, tyrosine, serine, arginine, histidine, or
asparagine; amino acid q is selected from asparagine, serine, or
aspartic acid; amino acid r is selected from serine or glycine;
amino acid s is selected from aspartic acid, serine, threonine, or
arginine; amino acid t is selected from asparagine, threonine,
alanine, isoleucine, or tyrosine; amino acid u is selected from
threonine, tyrosine, leucine, lysine, asparagine, or isoleucine;
amino acid v is selected from glycine, tyrosine, aspartic acid, or
cysteine; amino acid w is selected from tyrosine or asparagine;
amino acid x is selected from alanine or proline; amino acid y is
selected from glutamine, serine, or aspartic acid; amino acid z is
selected from lysine, leucine, or serine; amino acid a' is selected
from phenylalanine, lysine, or valine; amino acid b' is selected
from glutamine, serine, or lysine; and amino acid c' is glycine or
is not present; wherein CDR3a comprises the amino acid sequence d'
e' f' g' h' j' k' m' n' o' p' q' r' s' t' u' w', wherein amino acid
d' is selected from tryptophan, aspartic acid, glycine, serine, or
glutamic acid; amino acid e' is selected from asparagine, aspartic
acid, glycine, arginine, serine, valine, or leucine; amino acid f'
is selected from histidine, serine, alanine, tyrosine, proline,
asparagine, glycine or threonine; amino acid g' is selected from
tyrosine, serine, alanine, arginine, tryptophan, glycine or valine;
amino acid h' is selected from glycine, alanine, serine,
asparagine, methionine, tyrosine, tryptophan, cysteine, or aspartic
acid; amino acid i' is selected from serine, tryptophan, glycine,
phenylalanine, aspartic acid, tyrosine, or threonine; amino acid j'
is selected from glycine, threonine, serine, leucine, valine,
asparagine, tryptophan, or tyrosine; amino acid k' is selected from
serine, phenylalanine, aspartic acid, tryptophan, glycine, or
tyrosine, or is not present; amino acid l' is selected from
histidine, aspartic acid, alanine, tryptophan, tyrosine, serine,
phenylalanine, valine, or glycine, or is not present; amino acid m'
is selected from phenylalanine, tyrosine, glutamic acid, proline,
aspartic acid, cysteine, isoleucine, or methionine, or is not
present; amino acid n' is selected from aspartic acid,
phenylalanine, alanine, leucine, or serine, or is not present;
amino acid o' is selected from tyrosine, leucine, aspartic acid,
phenylalanine, proline, or valine, or is not present; amino acid p'
is selected from leucine, aspartic acid, or tyrosine, or is not
present; amino acid q' is selected from serine or tyrosine, or is
not present; amino acid r' is tyrosine or is not present; amino
acid s' is selected from glycine or tyrosine, or is not present;
amino acid t' is selected from glycine or methionine, or is not
present; amino acid u' is selected from methionine or aspartic
acid, or is not present; amino acid v' is selected from aspartic
acid or valine, or is not present; and amino acid w' is valine or
is not present; and wherein the polypeptide, in association with an
antibody light chain, binds TR-2.
[0183] In certain embodiments, a polynucleotide comprises a
sequence encoding CDR2a, wherein CDR2a comprises the amino acid
sequence m n o p q r s t u v w x y z a' b' c', wherein amino acid m
is selected from tryptophan, tyrosine, histidine, valine, glutamic
acid, or serine; amino acid n is selected from methionine or
isoleucine; amino acid o is selected from asparagine, tyrosine,
serine, tryptophan, or histidine; amino acid p is selected from
proline, tyrosine, serine, arginine, histidine, or asparagine;
amino acid q is selected from asparagine, serine, or aspartic acid;
amino acid r is selected from serine or glycine; amino acid s is
selected from aspartic acid, serine, threonine, or arginine; amino
acid t is selected from asparagine, threonine, alanine, isoleucine,
or tyrosine; amino acid u is selected from threonine, tyrosine,
leucine, lysine, asparagine, or isoleucine; amino acid v is
selected from glycine, tyrosine, aspartic acid, or cysteine; amino
acid w is selected from tyrosine or asparagine; amino acid x is
selected from alanine or proline; amino acid y is selected from
glutamine, serine, or aspartic acid; amino acid z is selected from
lysine, leucine, or serine; amino acid a' is selected from
phenylalanine, lysine, or valine; amino acid b' is selected from
glutamine, serine, or lysine; and amino acid c' is glycine or is
not present.
[0184] In certain embodiments, a polynucleotide comprises a
sequence encoding CDR3a comprising the amino acid sequence d' e' f'
g' h' k' m' n' o' p' q' r' s' t' u' v' w', wherein amino acid d' is
selected from tryptophan, aspartic acid, glycine, serine, or
glutamic acid; amino acid e' is selected from asparagine, aspartic
acid, glycine, arginine, serine, valine, or leucine; amino acid f'
is selected from histidine, serine, alanine, tyrosine, proline,
asparagine, glycine or threonine; amino acid g' is selected from
tyrosine, serine, alanine, arginine, tryptophan, glycine or valine;
amino acid h' is selected from glycine, alanine, serine,
asparagine, methionine, tyrosine, tryptophan, cysteine, or aspartic
acid; amino acid i' is selected from serine, tryptophan, glycine,
phenylalanine, aspartic acid, tyrosine, or threonine; amino acid j'
is selected from glycine, threonine, serine, leucine, valine,
asparagine, tryptophan, or tyrosine; amino acid k' is selected from
serine, phenylalanine, aspartic acid, tryptophan, glycine, or
tyrosine, or is not present; amino acid l' is selected from
histidine, aspartic acid, alanine, tryptophan, tyrosine, serine,
phenylalanine, valine, or glycine, or is not present; amino acid m'
is selected from phenylalanine, tyrosine, glutamic acid, proline,
aspartic acid, cysteine, isoleucine, or methionine, or is not
present; amino acid n' is selected from aspartic acid,
phenylalanine, alanine, leucine, or serine, or is not present;
amino acid o' is selected from tyrosine, leucine, aspartic acid,
phenylalanine, proline, or valine, or is not present; amino acid p'
is selected from leucine, aspartic acid, or tyrosine, or is not
present; amino acid q' is selected from serine or tyrosine, or is
not present; amino acid r' is tyrosine or is not present; amino
acid s' is selected from glycine or tyrosine, or is not present;
amino acid t' is selected from glycine or methionine, or is not
present; amino acid u' is selected from methionine or aspartic
acid, or is not present; amino acid v' is selected from aspartic
acid or valine, or is not present; and amino acid w' is valine or
is not present.
[0185] In certain embodiments, a polynucleotide comprises a
sequence encoding a polypeptide comprising at least two
complementarity determining regions (CDR) selected from CDR1a,
CDR2a, and CDR3a, wherein the polypeptide, in association with an
antibody light chain, binds TR-2. In certain embodiments, a
polynucleotide comprises a sequence encoding a polypeptide
comprising CDR1a, CDR2a, and CDR3a, wherein the polypeptide, in
association with an antibody light chain, binds TR-2.
[0186] In certain embodiments, a polynucleotide comprises a
sequence encoding a polypeptide comprising an antibody heavy chain
variable region. In certain embodiments, a polynucleotide comprises
a sequence encoding a polypeptide comprising a human antibody heavy
chain variable region. In certain embodiments, a polynucleotide
comprises a sequence encoding a polypeptide comprising a heavy
chain constant region. In certain embodiments, a polynucleotide
comprises a sequence encoding a polypeptide comprising a human
heavy chain constant region. In certain embodiments, a
polynucleotide comprises a sequence encoding a polypeptide
comprising an amino acid sequence as set forth in SEQ ID NO: 2, SEQ
ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12,
SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID
NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30,
SEQ ID NO: 32, or SEQ ID NO: 34. In certain embodiments, a
polynucleotide comprises a sequence encoding a polypeptide
comprising a non-human heavy chain constant region. In certain
embodiments, a polynucleotide comprises a sequence encoding a
polypeptide comprising a heavy chain constant region of a species
other than human.
[0187] In certain embodiments, a polynucleotide comprises a
sequence encoding a polypeptide comprising at least one
complementarity determining region (CDR) selected from amino acids
26 to 35 of SEQ ID NO: 2; amino acids 50 to 66 of SEQ ID NO: 2;
amino acids 99 to 110 of SEQ ID NO: 2; amino acids 26 to 37 of SEQ
ID NO: 4; amino acids 52 to 67 of SEQ ID NO: 4; amino acids 100 to
109 of SEQ ID NO: 4; amino acids 26 to 37 of SEQ ID NO: 6; amino
acids 52 to 67 of SEQ ID NO: 6; amino acids 100 to 109 of SEQ ID
NO: 6; amino acids 26 to 37 of SEQ ID NO: 8; amino acids 52 to 67
of SEQ ID NO: 8; amino acids 100 to 109 of SEQ ID NO: 8; amino
acids 26 to 35 of SEQ ID NO: 10; amino acids 50 to 66 of SEQ ID NO:
10; amino acids 99 to 110 of SEQ ID NO: 10; amino acids 26 to 35 of
SEQ ID NO: 12; amino acids 50 to 66 of SEQ ID NO: 12; amino acids
99 to 111 of SEQ ID NO: 12; amino acids 26 to 35 of SEQ ID NO: 14;
amino acids 50 to 65 of SEQ ID NO: 14; amino acids 98 to 111 of SEQ
ID NO: 14; amino acids 26 to 37 of SEQ ID NO: 16; amino acids 52 to
67 of SEQ ID NO: 16; amino acids 100 to 109 of SEQ ID NO: 16; amino
acids 26 to 35 of SEQ ID NO: 18; amino acids 50 to 66 of SEQ ID NO:
18; amino acids 99 to 105 of SEQ ID NO: 18; amino acids 26 to 35 of
SEQ ID NO: 20; amino acids 50 to 66 of SEQ ID NO: 20; amino acids
99 to 118 of SEQ ID NO: 20; amino acids 26 to 35 of SEQ ID NO: 22;
amino acids 50 to 66 of SEQ ID NO: 22; amino acids 99 to 118 of SEQ
ID NO: 22; amino acids 26 to 35 of SEQ ID NO: 24; amino acids 50 to
65 of SEQ ID NO: 24; amino acids 98 to 108 of SEQ ID NO: 24; amino
acids 26 to 35 of SEQ ID NO: 26; amino acids 50 to 66 of SEQ ID NO:
26; amino acids 99 to 110 of SEQ ID NO: 26; amino acids 26 to 35 of
SEQ ID NO: 28; amino acids 50 to 66 of SEQ ID NO: 28; amino acids
99 to 117 of SEQ ID NO: 28; amino acids 26 to 37 of SEQ ID NO: 30;
amino acids 52 to 67 of SEQ ID NO: 30; amino acids 100 to 111 of
SEQ ID NO: 30; amino acids 26 to 37 of SEQ ID NO: 32; amino acids
52 to 67 of SEQ ID NO: 32; amino acids 100 to 111 of SEQ ID NO: 32;
amino acids 26 to 37 of SEQ ID NO: 34; amino acids 52 to 67 of SEQ
ID NO: 34; and amino acids 100 to 111 of SEQ ID NO: 34, wherein the
polypeptide, in association with an antibody light chain, binds
TR-2. In certain embodiments, a polynucleotide comprises a sequence
encoding a polypeptide comprising at least two of the CDRs of SEQ
ID NOS. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,
or 34. In certain embodiments, a polynucleotide comprises a
sequence encoding a polypeptide comprising three of the CDRs of SEQ
ID NOS. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,
or 34.
[0188] In certain embodiments, a polynucleotide comprises a
sequence encoding a polypeptide comprising amino acids 26 to 35 of
SEQ ID NO: 2, amino acids 50 to 66 of SEQ ID NO: 2, and amino acids
99 to 110 of SEQ ID NO: 2. In certain embodiments, a polynucleotide
comprises a sequence encoding a polypeptide comprising amino acids
26 to 37 of SEQ ID NO: 4, amino acids 52 to 67 of SEQ ID NO: 4, and
amino acids 100 to 109 of SEQ ID NO: 4. In certain embodiments, a
polynucleotide comprises a sequence encoding a polypeptide
comprising amino acids 26 to 37 of SEQ ID NO: 6, amino acids 52 to
67 of SEQ ID NO: 6, and amino acids 100 to 109 of SEQ ID NO: 6. In
certain embodiments, a polynucleotide comprises a sequence encoding
a polypeptide comprising amino acids 26 to 37 of SEQ ID NO: 8,
amino acids 52 to 67 of SEQ ID NO: 8, and amino acids 100 to 109 of
SEQ ID NO: 8. In certain embodiments, a polynucleotide comprises a
sequence encoding a polypeptide comprising amino acids 26 to 35 of
SEQ ID NO: 10, amino acids 50 to 66 of SEQ ID NO: 10, and amino
acids 99-110 of SEQ ID NO: 10. In certain embodiments, a
polynucleotide comprises a sequence encoding a polypeptide
comprising amino acids 26 to 35 of SEQ ID NO: 12, amino acids 50 to
66 of SEQ ID NO: 12, and amino acids 99-111 of SEQ ID NO: 12. In
certain embodiments, a polynucleotide comprises a sequence encoding
a polypeptide comprising amino acids 26 to 35 of SEQ ID NO: 14,
amino acids 50 to 65 of SEQ ID NO: 14, and amino acids 98 to 111 of
SEQ ID NO: 14. In certain embodiments, a polynucleotide comprises a
sequence encoding a polypeptide comprising amino acids 26 to 37 of
SEQ ID NO: 16, amino acids 52 to 67 of SEQ ID NO: 16, and amino
acids 100 to 109 of SEQ ID NO: 16. In certain embodiments, a
polynucleotide comprises a sequence encoding a polypeptide
comprising amino acids 26 to 35 of SEQ ID NO: 18, amino acids 50 to
66 of SEQ ID NO: 18, and amino acids 99 to 105 of SEQ ID NO: 18. In
certain embodiments, a polynucleotide comprises a sequence encoding
a polypeptide comprising amino acids 26 to 35 of SEQ ID NO: 20,
amino acids 50 to 66 of SEQ ID NO: 20, and amino acids 99 to 118 of
SEQ ID NO: 20. In certain embodiments, a polynucleotide comprises a
sequence encoding a polypeptide comprising amino acids 26 to 35 of
SEQ ID NO: 22, amino acids 50 to 66 of SEQ ID NO: 22, and amino
acids 99 to 118 of SEQ ID NO: 22. In certain embodiments, a
polynucleotide comprises a sequence encoding a polypeptide
comprising amino acids 26 to 35 of SEQ ID NO: 24, amino acids 50 to
65 of SEQ ID NO: 24, and amino acids 98 to 108 of SEQ ID NO: 24. In
certain embodiments, a polynucleotide comprises a sequence encoding
a polypeptide comprising amino acids 26 to 35 of SEQ ID NO: 26,
amino acids 50 to 66 of SEQ ID NO: 26, and amino acids 99 to 110 of
SEQ ID NO: 26. In certain embodiments, a polynucleotide comprises a
sequence encoding a polypeptide comprising amino acids 26 to 35 of
SEQ ID NO: 28, amino acids 50 to 66 of SEQ ID NO: 28, and amino
acids 99 to 117 of SEQ ID NO: 28. In certain embodiments, a
polynucleotide comprises a sequence encoding a polypeptide
comprising amino acids 26 to 37 of SEQ ID NO: 30, amino acids 52 to
67 of SEQ ID NO: 30, and amino acids 100 to 111 of SEQ ID NO: 30.
In certain embodiments, a polynucleotide comprises a sequence
encoding a polypeptide comprising amino acids 26 to 37 of SEQ ID
NO: 32, amino acids 52 to 67 of SEQ ID NO: 32, and amino acids 100
to 111 of SEQ ID NO: 32. In certain embodiments, a polynucleotide
comprises a sequence encoding a polypeptide comprising amino acids
26 to 37 of SEQ ID NO: 34, amino acids 52 to 67 of SEQ ID NO: 34,
and amino acids 100 to 111 of SEQ ID NO: 34.
[0189] In certain embodiments, a polynucleotide comprises a
sequence encoding a polypeptide comprising at least one
complementarity determining region (CDR) selected from CDR1b,
CDR2b, and CDR3b, wherein CDR1b comprises the amino acid sequence
a1 b1 c1 d1 e1 f1 g1 h1 j1 k1 l1 m1 n1 o1 p1 q1, wherein amino acid
a1 is selected from arginine or lysine; amino acid b1 is selected
from threonine, alanine, or serine; amino acid c1 is serine; amino
acid d1 is glutamine; amino acid e1 is selected from serine or
glycine; amino acid f1 is selected from isoleucine, leucine, or
valine; amino acid g1 is selected from serine, leucine, or
arginine; amino acid h1 is selected from threonine, serine,
isoleucine, asparagine, arginine, histidine, or tyrosine; amino
acid i1 is selected from tyrosine, arginine, tryptophan, aspartic
acid, or serine; j1 is selected from leucine, isoleucine,
asparagine, tyrosine, or serine; amino acid k1 is selected from
asparagine, glycine, valine, alanine, or leucine; amino acid l1 is
selected from tyrosine, alanine, or asparagine, or is not present;
amino acid m1 is selected from asparagine or lysine, or is not
present; amino acid n1 is selected from tyrosine, asparagine, or
isoleucine, or is not present; amino acid o1 is selected from
leucine or tyrosine, or is not present; amino acid p1 is selected
from aspartic acid or leucine, or is not present; and amino acid q1
is selected from valine, alanine, or threonine, or is not present;
wherein CDR2b comprises the amino acid sequence r1 s1 t1 p1 v1 w1
x1, wherein amino acid r1 is selected from alanine, aspartic acid,
leucine, tryptophan, glycine, or valine; amino acid s1 is selected
from threonine, valine, glycine, or alanine; amino acid t1 is
serine; amino acid u1 is selected from serine, asparagine, or
threonine; amino acid v1 is selected from leucine, phenylalanine,
or arginine; amino acid w1 is selected from glutamine, alanine, or
glutamic acid; and amino acid x1 is selected from serine, arginine,
or threonine; wherein CDR3b comprises the amino acid sequence y1 z1
a1' b1' c1' d1' e1' f1' g1', wherein amino acid y1 is selected from
glutamine, methionine, leucine, or histidine; amino acid z1 is
selected from glutamine or lysine; amino acid a1' is selected from
serine, threonine, alanine, histidine, tyrosine, or phenylalanine;
amino acid b1' is selected from tyrosine, leucine, asparagine, or
glycine; amino acid c1' is selected from serine, glutamine,
isoleucine, or lysine; amino acid d1' is selected from threonine,
phenylalanine, tyrosine, alanine, or serine; amino acid e1' is
proline; amino acid f1' is selected from leucine, phenylalanine,
tryptophan, serine, or arginine; and amino acid g1' is selected
from threonine or serine; and wherein the polypeptide, in
association with an antibody heavy chain, binds TR-2.
[0190] In certain embodiments, a polynucleotide comprises a
sequence encoding a polypeptide comprising at least two
complementarity determining regions (CDR) selected from CDR1b,
CDR2b, and CDR3b, wherein the polypeptide, in association with an
antibody heavy chain, binds TR-2. In certain embodiments, a
polynucleotide comprises a sequence encoding a polypeptide
comprising CDR1b, CDR2b, and CDR3b, wherein the polypeptide, in
association with an antibody heavy chain, binds TR-2.
[0191] In certain embodiments, a polynucleotide comprises a
sequence encoding a polypeptide comprising an antibody light chain
variable region. In certain embodiments, a polynucleotide comprises
a sequence encoding a polypeptide comprising a human antibody light
chain variable region. In certain embodiments, a polynucleotide
comprises a sequence encoding a polypeptide comprising a light
chain constant region. In certain embodiments, a polynucleotide
comprises a sequence encoding a polypeptide comprising a human
light chain constant region. In certain embodiments, a
polynucleotide comprises a sequence encoding a polypeptide
comprising an amino acid sequence as set forth in SEQ ID NO: 36,
SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID
NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54,
SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID
NO: 64, SEQ ID NO: 66, or SEQ ID NO: 68. In certain embodiments, a
polynucleotide comprises a sequence encoding a polypeptide
comprising a non-human light chain constant region. In certain
embodiments, a polynucleotide comprises a sequence encoding a
polypeptide comprising a light chain constant region of a species
other than human.
[0192] In certain embodiments, a polynucleotide comprises a
sequence encoding a polypeptide comprising at least one
complementarity determining region (CDR) selected from amino acids
24 to 34 of SEQ ID NO: 36; amino acids 50 to 56 of SEQ ID NO: 36;
amino acids 89 to 97 of SEQ ID NO: 36; amino acids 24 to 34 of SEQ
ID NO: 38; amino acids 50 to 56 of SEQ ID NO: 38; amino acids 89 to
97 of SEQ ID NO: 38; amino acids 24 to 34 of SEQ ID NO: 40; amino
acids 50 to 56 of SEQ ID NO: 40; amino acids 89 to 97 of SEQ ID NO:
40; amino acids 24 to 34 of SEQ ID NO: 42; amino acids 50 to 56 of
SEQ ID NO: 42; amino acids 89 to 97 of SEQ ID NO: 42; amino acids
24 to 34 of SEQ ID NO: 44; amino acids 50 to 56 of SEQ ID NO: 44;
amino acids 89 to 97 of SEQ ID NO: 44; amino acids 24 to 34 of SEQ
ID NO: 46; amino acids 50 to 56 of SEQ ID NO: 46; amino acids 89 to
97 of SEQ ID NO: 46; amino acids 24 to 40 of SEQ ID NO: 48; amino
acids 56 to 62 of SEQ ID NO: 48; amino acids 95 to 103 of SEQ ID
NO: 48; amino acids 24 to 39 of SEQ ID NO: 50; amino acids 55 to 61
of SEQ ID NO: 50; amino acids 94 to 102 of SEQ ID NO: 50; amino
acids 24 to 40 of SEQ ID NO: 52; amino acids 56 to 62 of SEQ ID NO:
52; amino acids 95 to 103 of SEQ ID NO: 52; amino acids 24 to 34 of
SEQ ID NO: 54; amino acids 50 to 56 of SEQ ID NO: 54; amino acids
89 to 97 of SEQ ID NO: 54; amino acids 24 to 34 of SEQ ID NO: 56;
amino acids 50 to 56 of SEQ ID NO: 56; amino acids 89 to 97 of SEQ
ID NO: 56; amino acids 24 to 40 of SEQ ID NO: 58; amino acids 56 to
62 of SEQ ID NO: 58; amino acids 95 to 103 of SEQ ID NO: 58; amino
acids 24 to 34 of SEQ ID NO: 60; amino acids 50 to 56 of SEQ ID NO:
60; amino acids 89 to 97 of SEQ ID NO: 60; amino acids 24 to 34 of
SEQ ID NO: 62; amino acids 50 to 56 of SEQ ID NO: 62; amino acids
89 to 97 of SEQ ID NO: 62; amino acids 24 to 35 of SEQ ID NO: 64;
amino acids 51 to 57 of SEQ ID NO: 64; amino acids 90 to 88 of SEQ
ID NO: 64; amino acids 24 to 34 of SEQ ID NO: 66; amino acids 50 to
57 of SEQ ID NO: 66; amino acids 89 to 97 of SEQ ID NO: 66; amino
acids 24 to 34 of SEQ ID NO: 68; amino acids 50 to 56 of SEQ ID NO:
68; and amino acids 89 to 97 of SEQ ID NO: 68, wherein the
polypeptide, in association with an antibody heavy chain, binds
TR-2. In certain embodiments, a polynucleotide comprises a sequence
encoding a polypeptide comprising at least two of the CDRs of SEQ
ID NOS. 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64,
or 68. In certain embodiments, a polynucleotide comprises a
sequence encoding a polypeptide comprising three of the CDRs of SEQ
ID NOS. 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64,
or 68.
[0193] In certain embodiments, a polynucleotide comprises a
sequence encoding a polypeptide comprising amino acids 24 to 34 of
SEQ ID NO: 36, amino acids 50 to 56 of SEQ ID NO: 36, and amino
acids 89-97 of SEQ ID NO: 36. In certain embodiments, a
polynucleotide comprises a sequence encoding a polypeptide
comprising amino acids 24 to 34 of SEQ ID NO: 38, amino acids 50 to
56 of SEQ ID NO: 38, and amino acids 89 to 97 of SEQ ID NO: 38. In
certain embodiments, a polynucleotide comprises a sequence encoding
a polypeptide comprising amino acids 24 to 34 of SEQ ID NO: 40,
amino acids 50 to 56 of SEQ ID NO: 40, and amino acids 89 to 97 of
SEQ ID NO: 40. In certain embodiments, a polynucleotide comprises a
sequence encoding a polypeptide comprising amino acids 24 to 34 of
SEQ ID NO: 42, amino acids 50 to 56 of SEQ ID NO: 42, and amino
acids 89 to 97 of SEQ ID NO: 42. In certain embodiments, a
polynucleotide comprises a sequence encoding a polypeptide
comprising amino acids 24 to 34 of SEQ ID NO: 44, amino acids 50 to
56 of SEQ ID NO: 44, and amino acids 89-97 of SEQ ID NO: 44. In
certain embodiments, a polynucleotide comprises a sequence encoding
a polypeptide comprising amino acids 24 to 34 of SEQ ID NO: 46,
amino acids 50 to 56 of SEQ ID NO: 46, and amino acids 89 to 97 of
SEQ ID NO: 46. In certain embodiments, a polynucleotide comprises a
sequence encoding a polypeptide comprising amino acids 24 to 40 of
SEQ ID NO: 48, amino acids 56 to 62 of SEQ ID NO: 48, and amino
acids 95 to 103 of SEQ ID NO: 48. In certain embodiments, a
polynucleotide comprises a sequence encoding a polypeptide
comprising amino acids 24 to 39 of SEQ ID NO: 50, amino acids 55 to
61 of SEQ ID NO: 50, and amino acids 94 to 102 of SEQ ID NO: 50. In
certain embodiments, a polynucleotide comprises a sequence encoding
a polypeptide comprising amino acids 24 to 40 of SEQ ID NO: 52,
amino acids 56 to 62 of SEQ ID NO: 52, and amino acids 95 to 103 of
SEQ ID NO: 52. In certain embodiments, a polynucleotide comprises a
sequence encoding a polypeptide comprising 24 to 34 of SEQ ID NO:
54, amino acids 50 to 56 of SEQ ID NO: 54, and amino acids 89 to 97
of SEQ ID NO: 54. In certain embodiments, a polynucleotide
comprises a sequence encoding a polypeptide comprising amino acids
24 to 34 of SEQ ID NO: 56, amino acids 50 to 56 of SEQ ID NO: 56,
and amino acids 89 to 97 of SEQ ID NO: 56, In certain embodiments,
a polynucleotide comprises a sequence encoding a polypeptide
comprising amino acids 24 to 40 of SEQ ID NO: 58, amino acids 56 to
62 of SEQ ID NO: 58, and amino acids 95 to 103 of SEQ ID NO: 58. In
certain embodiments, a polynucleotide comprises a sequence encoding
a polypeptide comprising amino acids 24 to 34 of SEQ ID NO: 60,
amino acids 50 to 56 of SEQ ID NO: 60, and amino acids 89-97 of SEQ
ID NO: 60. In certain embodiments, a polynucleotide comprises a
sequence encoding a polypeptide comprising amino acids 24 to 34 of
SEQ ID NO: 62, amino acids 50 to 56 of SEQ ID NO: 62, and amino
acids 89 to 97 of SEQ ID NO: 62. In certain embodiments, a
polynucleotide comprises a sequence encoding a polypeptide
comprising amino acids 24 to 35 of SEQ ID NO: 64, amino acids 51 to
57 of SEQ ID NO: 64, and amino acids 90 to 88 of SEQ ID NO: 64. In
certain embodiments, a polynucleotide comprises a sequence encoding
a polypeptide comprising amino acids 24 to 34 of SEQ ID NO: 66,
amino acids 50 to 57 of SEQ ID NO: 66, and amino acids 89 to 97 of
SEQ ID NO: 66. In certain embodiments, a polynucleotide comprises a
sequence encoding a polypeptide comprising amino acids 24 to 34 of
SEQ ID NO: 68, amino acids 50 to 56 of SEQ ID NO: 68, and amino
acids 89 to 97 of SEQ ID NO: 68.
[0194] In certain embodiments, this application discusses certain
polynucleotides encoding antibody heavy and light chains. In
certain embodiments, this application discusses certain
polynucleotides encoding an antibody heavy chain variable region.
In certain embodiments, this application discusses certain
polynucleotides encoding a human antibody heavy chain variable
region. In certain embodiments, this application discusses certain
polynucleotides encoding antibody light chain variable regions. In
certain embodiments, this application discusses certain
polynucleotides encoding a human antibody light chain variable
region. In certain embodiments, this application discusses certain
polynucleotides encoding an antibody heavy chain constant region.
In certain embodiments, this application discusses certain
polynucleotides encoding a human antibody heavy chain constant
region. In certain embodiments, this application discusses certain
polynucleotides encoding an antibody heavy chain constant region of
a species other than human. In certain embodiments, this
application discusses certain polynucleotides encoding antibody
light chain constant regions. In certain embodiments, this
application discusses certain polynucleotides encoding a human
antibody light chain constant region. In certain embodiments, this
application discusses certain polynucleotides encoding an antibody
light chain constant region of a species other than human. In
certain embodiments, this application discusses certain
polynucleotides encoding a single-chain antibody.
[0195] In certain embodiments, these antibody heavy and light chain
polynucleotides and polypeptides are human antibody heavy and light
chain polynucleotides and polypeptides. In certain embodiments a
polynucleotide comprises a nucleotide sequence as set forth in SEQ
ID NOS. SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,
27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59,
61, 63, 65, or 67. In certain embodiments, a polynucleotide
comprises a nucleotide sequence that has one or more deletions,
additions, and/or substitutions of one or more nucleotides of those
sequences. In certain embodiments, a polynucleotide comprises a
nucleotide sequence encoding an amino acid sequence comprising an
amino acid sequence as set forth in SEQ ID NOS: 2, 4, 6, 8, 10, 12,
14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,
48, 50, 52, 54, 56, 58, 60, 62, 64, 66, or 68. In certain
embodiments, variable region sequences comprising complementarity
determining regions (CDRs), e.g., CDR1 through CDR3, are provided.
In certain embodiments, variable region polynucleotides and
polypeptides are human variable region polynucleotides and
polypeptides.
[0196] The term "naturally occurring nucleotides" includes
deoxyribonucleotides and ribonucleotides. Deoxyribonucleotides
include, but are not limited to, adenosine, guanine, cytosine, and
thymidine. Ribonucleotides include, but are not limited to,
adenosine, cytosine, thymidine, and uracil. The term "modified
nucleotides" includes, but is not limited to, nucleotides with
modified or substituted sugar groups and the like. The term
"polynucleotide linkages" includes, but is not limited to,
polynucleotide linkages such as phosphorothioate,
phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,
phosphoroanilothioate, phoshoraniladate, phosphoroamidate, and the
like. See, e.g., LaPlanche et al. Nucl. Acids Res. 14:9081 (1986);
Stec et al. J. Am. Chem. Soc. 106:6077 (1984); Stein et al. Nucl.
Acids Res. 16:3209 (1988); Zon et al. Anti-Cancer Drug Design 6:539
(1991); Zon et al. Oligonucleotides and Analogues: A Practical
Approach, pp. 87-108 (F. Eckstein, Ed., Oxford University Press,
Oxford England (1991)); Stec et al. U.S. Pat. No. 5,151,510;
Uhlmann and Peyman Chemical Reviews 90:543 (1990). In certain
embodiments, a polynucleotide can include a label for
detection.
[0197] The term "isolated polypeptide" refers to any polypeptide
that (1) is free of at least some proteins with which it would
normally be found, (2) is essentially free of other proteins from
the same source, e.g., from the same species, (3) is expressed by a
cell from a different species, or (4) does not occur in nature.
[0198] The terms "polypeptide," "peptide," and "protein" are used
interchangeably herein and refer to a polymer of two or more amino
acids joined to each other by peptide bonds or modified peptide
bonds, i.e., peptide isosteres. The terms apply to amino acid
polymers containing naturally occurring amino acids as well as
amino acid polymers in which one or more amino acid residues is a
non-naturally occurring amino acid or a chemical analogue of a
naturally occurring amino acid. An amino acid polymer may contain
one or more amino acid residues that has been modified by one or
more natural processes, such as post-translational processing,
and/or one or more amino acid residues that has been modified by
one or more chemical modification techniques known in the art.
[0199] A "fragment" of a reference polypeptide refers to a
contiguous stretch of amino acids from any portion of the reference
polypeptide. A fragment may be of any length that is less than the
length of the reference polypeptide.
[0200] A "variant" of a reference polypeptide refers to a
polypeptide having one or more amino acid substitutions, deletions,
or insertions relative to the reference polypeptide. In certain
embodiments, a variant of a reference polypeptide has an altered
post-translational modification site (i.e., a glycosylation site).
In certain embodiments, both a reference polypeptide and a variant
of a reference polypeptide are specific binding agents. In certain
embodiments, both a reference polypeptide and a variant of a
reference polypeptide are antibodies.
[0201] Variants of a reference polypeptide include, but are not
limited to, glycosylation variants. Glycosylation variants include
variants in which the number and/or type of glycosylation sites
have been altered as compared to the reference polypeptide. In
certain embodiments, glycosylation variants of a reference
polypeptide comprise a greater or a lesser number of N-linked
glycosylation sites than the reference polypeptide. In certain
embodiments, an N-linked glycosylation site is characterized by the
sequence Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue
designated as X may be any amino acid residue except proline. In
certain embodiments, glycosylation variants of a reference
polypeptide comprise a rearrangement of N-linked carbohydrate
chains wherein one or more N-linked glycosylation sites (typically
those that are naturally occurring) are eliminated and one or more
new N-linked sites are created.
[0202] Variants of a reference polypeptide include, but are not
limited to, cysteine variants. In certain embodiments, cysteine
variants include variants in which one or more cysteine residues of
the reference polypeptide are replaced by one or more non-cysteine
residues; and/or one or more non-cysteine residues of the reference
polypeptide are replaced by one or more cysteine residues. Cysteine
variants may be useful, in certain embodiments, when a particular
polypeptide must be refolded into a biologically active
conformation, e.g., after the isolation of insoluble inclusion
bodies. In certain embodiments, cysteine variants of a reference
polypeptide have fewer cysteine residues than the reference
polypeptide. In certain embodiments, cysteine variants of a
reference polypeptide have an even number of cysteines to minimize
interactions resulting from unpaired cysteines. In certain
embodiments, cysteine variants have more cysteine residues than the
native protein.
[0203] A "derivative" of a reference polypeptide refers to: a
polypeptide: (1) having one or more modifications of one or more
amino acid residues of the reference polypeptide; and/or (2) in
which one or more peptidyl linkages has been replaced with one or
more non-peptidyl linkages; and/or (3) in which the N-terminus
and/or the C-terminus has been modified. Certain exemplary
modifications include, but are not limited to, acetylation,
acylation, ADP-ribosylation, amidation, biotinylation, covalent
attachment of flavin, covalent attachment of a heme moiety,
covalent attachment of a nucleotide or nucleotide derivative,
covalent attachment of a lipid or lipid derivative, covalent
attachment of phosphotidylinositol, cross-linking, cyclization,
disulfide bond formation, demethylation, formation of covalent
cross-links, formation of cystine, formation of pyroglutamate,
formylation, gamma-carboxylation, glycosylation, GPI anchor
formation, hydroxylation, iodination, methylation, myristoylation,
oxidation, proteolytic processing, phosphorylation, prenylation,
racemization, selenoylation, sulfation, transfer-RNA mediated
addition of amino acids to proteins such as arginylation, and
ubiquitination. In certain embodiments, both a reference
polypeptide and a derivative of a reference polypeptide are
specific binding agents. In certain embodiments, both a reference
polypeptide and a derivative of a reference polypeptide are
antibodies.
[0204] Polypeptides include, but are not limited to, amino acid
sequences modified either by natural processes, such as
post-translational processing, or by chemical modification
techniques that are well known in the art. In certain embodiments,
modifications may occur anywhere in a polypeptide, including the
peptide backbone, the amino acid side-chains and the amino or
carboxyl termini. In certain such embodiments, the modifications
may be present to the same or varying degrees at several sites in a
given polypeptide. In certain embodiments, a given polypeptide
contains many types of modifications such as deletions, additions,
and/or substitutions of one or more amino acids of a native
sequence. In certain embodiments, polypeptides may be branched
and/or cyclic. Cyclic, branched and branched cyclic polypeptides
may result from post-translational natural processes (including,
but not limited to, ubiquitination) or may be made by synthetic
methods. The term "polypeptide" also encompasses sequences that
comprise the amino acid sequences of the heavy chain and/or light
chain of an antibody selected from Ab A, Ab B, Ab C, Ab D, Ab E, Ab
F, Ab G, Ab H, Ab I, Ab J, Ab K, Ab L, Ab M, Ab N, Ab O, Ab P, and
Ab Q, as described below (see SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14,
16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48,
50, 52, 54, 56, 58, 60, 62, 64, 66, and 68). The term "polypeptide"
also encompasses sequences that have one or more deletions,
additions, and/or substitutions of one or more amino acids of those
sequences. In certain embodiments, certain polypeptide sequences
comprise at least one complementarity determining region (CDR).
[0205] In certain embodiments, a polypeptide comprises at least one
complementarity determining region (CDR) selected from CDR1a,
CDR2a, and CDR3a wherein CDR1a comprises the amino acid sequence a
b c d e f g h i j k l, wherein amino acid a is glycine, amino acid
b is selected from glycine, tyrosine, or phenylalanine; amino acid
c is selected from serine or threonine; amino acid d is selected
from isoleucine or phenylalanine; amino acid e is selected from
serine, threonine, or asparagine; amino acid f is selected from
serine, aspartic acid, tyrosine, asparagine, threonine, or glycine;
amino acid g is selected from glycine, aspartic acid, or tyrosine;
amino acid h is selected from glycine, aspartic acid, tyrosine,
asparagine, or serine; amino acid i is selected from tyrosine,
isoleucine, histidine, methionine, or tryptophan; amino acid j is
selected from asparagine, tyrosine, histidine, serine, or
phenylalanine; amino acid k is tryptophan or is not present; and
amino acid l is serine or is not present; wherein CDR2a comprises
the amino acid sequence m n o p q r s t u v w x y z a' b' c',
wherein amino acid m is selected from tryptophan, tyrosine,
histidine, valine, glutamic acid, or serine; amino acid n is
selected from methionine or isoleucine; amino acid a is selected
from asparagine, tyrosine, serine, tryptophan, or histidine; amino
acid p is selected from proline, tyrosine, serine, arginine,
histidine, or asparagine; amino acid q is selected from asparagine,
serine, or aspartic acid; amino acid r is selected from serine or
glycine; amino acid s is selected from aspartic acid, serine,
threonine, or arginine; amino acid t is selected from asparagine,
threonine, alanine, isoleucine, or tyrosine; amino acid u is
selected from threonine, tyrosine, leucine, lysine, asparagine, or
isoleucine; amino acid v is selected from glycine, tyrosine,
aspartic acid, or cysteine; amino acid w is selected from tyrosine
or asparagine; amino acid x is selected from alanine or proline;
amino acid y is selected from glutamine, serine, or aspartic acid;
amino acid z is selected from lysine, leucine, or serine; amino
acid a' is selected from phenylalanine, lysine, or valine; amino
acid b' is selected from glutamine, serine, or lysine; and amino
acid c' is glycine or is not present; wherein CDR3a comprises the
amino acid sequence d' e' f' g' h' j' k' m' n' o' p' q' r' s' t' u'
v' w', wherein amino acid d' is selected from tryptophan, aspartic
acid, glycine, serine, or glutamic acid; amino acid e' is selected
from asparagine, aspartic acid, glycine, arginine, serine, valine,
or leucine; amino acid f' is selected from histidine, serine,
alanine, tyrosine, proline, asparagine, glycine or threonine; amino
acid g' is selected from tyrosine, serine, alanine, arginine,
tryptophan, glycine or valine; amino acid h' is selected from
glycine, alanine, serine, asparagine, methionine, tyrosine,
tryptophan, cysteine, or aspartic acid; amino acid i' is selected
from serine, tryptophan, glycine, phenylalanine, aspartic acid,
tyrosine, or threonine; amino acid j' is selected from glycine,
threonine, serine, leucine, valine, asparagine, tryptophan, or
tyrosine; amino acid k' is selected from serine, phenylalanine,
aspartic acid, tryptophan, glycine, or tyrosine, or is not present;
amino acid l' is selected from histidine, aspartic acid, alanine,
tryptophan, tyrosine, serine, phenylalanine, valine, or glycine, or
is not present; amino acid m' is selected from phenylalanine,
tyrosine, glutamic acid, proline, aspartic acid, cysteine,
isoleucine, or methionine, or is not present; amino acid n' is
selected from aspartic acid, phenylalanine, alanine, leucine, or
serine, or is not present; amino acid o' is selected from tyrosine,
leucine, aspartic acid, phenylalanine, proline, or valine, or is
not present; amino acid p' is selected from leucine, aspartic acid,
or tyrosine, or is not present; amino acid q' is selected from
serine or tyrosine, or is not present; amino acid r' is tyrosine or
is not present; amino acid s' is selected from glycine or tyrosine,
or is not present; amino acid t' is selected from glycine or
methionine, or is not present; amino acid u' is selected from
methionine or aspartic acid, or is not present; amino acid v' is
selected from aspartic acid or valine, or is not present; and amino
acid w' is valine or is not present; and wherein the polypeptide,
in association with an antibody light chain, binds TR-2.
[0206] In certain embodiments, a polypeptide comprises at least two
complementarity determining regions (CDR) selected from CDR1a,
CDR2a, and CDR3a, wherein the polypeptide, in association with an
antibody light chain, binds TR-2. In certain embodiments, a
polypeptide comprises CDR1a, CDR2a, and CDR3a, wherein the
polypeptide, in association with an antibody light chain, binds
TR-2.
[0207] In certain embodiments, a polypeptide comprises an antibody
heavy chain variable region. In certain embodiments, a polypeptide
comprises a human antibody heavy chain variable region. In certain
embodiments, a polypeptide comprises a heavy chain constant region.
In certain embodiments, a polypeptide comprises a human heavy chain
constant region. In certain embodiments, a polypeptide comprises an
amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ
ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:
14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ
ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO:
32, or SEQ ID NO: 34. In certain embodiments, a polypeptide
comprises a non-human heavy chain constant region. In certain
embodiments, a polypeptide comprises a heavy chain constant region
of a species other than human.
[0208] In certain embodiments, a polypeptide comprises at least one
complementarity determining region (CDR) selected from amino acids
26 to 35 of SEQ ID NO: 2; amino acids 50 to 66 of SEQ ID NO: 2;
amino acids 99 to 110 of SEQ ID NO: 2; amino acids 26 to 37 of SEQ
ID NO: 4; amino acids 52 to 67 of SEQ ID NO: 4; amino acids 100 to
109 of SEQ ID NO: 4; amino acids 26 to 37 of SEQ ID NO: 6; amino
acids 52 to 67 of SEQ ID NO: 6; amino acids 100 to 109 of SEQ ID
NO: 6; amino acids 26 to 37 of SEQ ID NO: 8; amino acids 52 to 67
of SEQ ID NO: 8; amino acids 100 to 109 of SEQ ID NO: 8; amino
acids 26 to 35 of SEQ ID NO: 10, amino acids 50 to 66 of SEQ ID NO:
10; amino acids 99-110 of SEQ ID NO: 10; amino acids 26 to 35 of
SEQ ID NO: 12; amino acids 50 to 66 of SEQ ID NO: 12; amino acids
99-111 of SEQ ID NO: 12; amino acids 26 to 35 of SEQ ID NO: 14;
amino acids 50 to 65 of SEQ ID NO: 14; amino acids 98 to 111 of SEQ
ID NO: 14; amino acids 26 to 37 of SEQ ID NO: 16; amino acids 52 to
67 of SEQ ID NO: 16; amino acids 100 to 109 of SEQ ID NO: 16; amino
acids 26 to 35 of SEQ ID NO: 18; amino acids 50 to 66 of SEQ ID NO:
18; amino acids 99 to 105 of SEQ ID NO: 18; amino acids 26 to 35 of
SEQ ID NO: 20; amino acids 50 to 66 of SEQ ID NO: 20; amino acids
99 to 118 of SEQ ID NO: 20; amino acids 26 to 35 of SEQ ID NO: 22;
amino acids 50 to 66 of SEQ ID NO: 22; amino acids 99 to 118 of SEQ
ID NO: 22; amino acids 26 to 35 of SEQ ID NO: 24; amino acids 50 to
65 of SEQ ID NO: 24; amino acids 98 to 108 of SEQ ID NO: 24; amino
acids 26 to 35 of SEQ ID NO: 26; amino acids 50 to 66 of SEQ ID NO:
26; amino acids 99 to 110 of SEQ ID NO: 26; amino acids 26 to 35 of
SEQ ID NO: 28; amino acids 50 to 66 of SEQ ID NO: 28; amino acids
99 to 117 of SEQ ID NO: 28; amino acids 26 to 37 of SEQ ID NO: 30;
amino acids 52 to 67 of SEQ ID NO: 30; amino acids 100 to 111 of
SEQ ID NO: 30; amino acids 26 to 37 of SEQ ID NO: 32; amino acids
52 to 67 of SEQ ID NO: 32; amino acids 100 to 111 of SEQ ID NO: 32;
amino acids 26 to 37 of SEQ ID NO: 34; amino acids 52 to 67 of SEQ
ID NO: 34; and amino acids 100 to 111 of SEQ ID NO: 34, wherein the
polypeptide, in association with an antibody light chain, binds
TR-2. In certain embodiments, a polypeptide comprises at least two
of the CDRs of SEQ ID NOS. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,
24, 26, 28, 30, 32, or 34. In certain embodiments, a polypeptide
comprises at least three of the CDRs of SEQ ID NOS. 2, 4, 6, 8, 10,
12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34.
[0209] In certain embodiments, a polypeptide comprises amino acids
26 to 35 of SEQ ID NO: 2, amino acids 50 to 66 of SEQ ID NO: 2, and
amino acids 99 to 110 of SEQ ID NO: 2. In certain embodiments, a
polypeptide comprises amino acids 26 to 37 of SEQ ID NO: 4, amino
acids 52 to 67 of SEQ ID NO: 4, and amino acids 100 to 109 of SEQ
ID NO: 4. In certain embodiments, a polypeptide comprises amino
acids 26 to 37 of SEQ ID NO: 6, amino acids 52 to 67 of SEQ ID NO:
6, and amino acids 100 to 109 of SEQ ID NO: 6. In certain
embodiments, a polypeptide comprises amino acids 26 to 37 of SEQ ID
NO: 8, amino acids 52 to 67 of SEQ ID NO: 8, and amino acids 100 to
109 of SEQ ID NO: 8. In certain embodiments, a polypeptide
comprises amino acids 26 to 35 of SEQ ID NO: 10, amino acids 50 to
66 of SEQ ID NO: 10, and amino acids 99-110 of SEQ ID NO: 10. In
certain embodiments, a polypeptide comprises amino acids 26 to 35
of SEQ ID NO: 12, amino acids 50 to 66 of SEQ ID NO: 12, and amino
acids 99-111 of SEQ ID NO: 12. In certain embodiments, a
polypeptide comprises amino acids 26 to 35 of SEQ ID NO: 14, amino
acids 50 to 65 of SEQ ID NO: 14, and amino acids 98 to 111 of SEQ
ID NO: 14. In certain embodiments, a polypeptide comprises amino
acids 26 to 37 of SEQ ID NO: 16, amino acids 52 to 67 of SEQ ID NO:
16, and amino acids 100 to 109 of SEQ ID NO: 16. In certain
embodiments, a polypeptide comprises amino acids 26 to 35 of SEQ ID
NO: 18, amino acids 50 to 66 of SEQ ID NO: 18, and amino acids 99
to 105 of SEQ ID NO: 18. In certain embodiments, a polypeptide
comprises amino acids 26 to 35 of SEQ ID NO: 20, amino acids 50 to
66 of SEQ ID NO: 20, and amino acids 99 to 118 of SEQ ID NO: 20. In
certain embodiments, a polypeptide comprises amino acids 26 to 35
of SEQ ID NO: 22, amino acids 50 to 66 of SEQ ID NO: 22, and amino
acids 99 to 118 of SEQ ID NO: 22. In certain embodiments, a
polypeptide comprises amino acids 26 to 35 of SEQ ID NO: 24, amino
acids 50 to 65 of SEQ ID NO: 24, and amino acids 98 to 108 of SEQ
ID NO: 24. In certain embodiments, a polypeptide comprises amino
acids 26 to 35 of SEQ ID NO: 26, amino acids 50 to 66 of SEQ ID NO:
26, and amino acids 99 to 110 of SEQ ID NO: 26. In certain
embodiments, a polypeptide comprises amino acids 26 to 35 of SEQ ID
NO: 28, amino acids 50 to 66 of SEQ ID NO: 28, and amino acids 99
to 117 of SEQ ID NO: 28. In certain embodiments, a polypeptide
comprises amino acids 26 to 37 of SEQ ID NO: 30, amino acids 52 to
67 of SEQ ID NO: 30, and amino acids 100 to 111 of SEQ ID NO: 30.
In certain embodiments, a polypeptide comprises amino acids 26 to
37 of SEQ ID NO: 32, amino acids 52 to 67 of SEQ ID NO: 32, and
amino acids 100 to 111 of SEQ ID NO: 32. In certain embodiments, a
polypeptide comprises amino acids 26 to 37 of SEQ ID NO: 34, amino
acids 52 to 67 of SEQ ID NO: 34, and amino acids 100 to 111 of SEQ
ID NO: 34.
[0210] In certain embodiments, a polypeptide comprises at least one
complementarity determining region (CDR) selected from CDR1b,
CDR2b, and CDR3b, wherein CDR1b comprises a1 b1 c1 d1 e1 f1 g1 h1
i1 j1 k1 l1 m1 n1 o1 p1 q1, wherein amino acid a1 is selected from
arginine or lysine; amino acid b1 is selected from threonine,
alanine, or serine; amino acid c1 is serine; amino acid d1 is
glutamine; amino acid e1 is selected from serine or glycine; amino
acid f1 is selected from isoleucine, leucine, or valine; amino acid
g1 is selected from serine, leucine, or arginine; amino acid h1 is
selected from threonine, serine, isoleucine, asparagine, arginine,
histidine, or tyrosine; amino acid i1 is selected from tyrosine,
arginine, tryptophan, aspartic acid, or serine; j1 is selected from
leucine, isoleucine, asparagine, tyrosine, or serine; amino acid k1
is selected from asparagine, glycine, valine, alanine, or leucine;
amino acid l1 is selected from tyrosine, alanine, or asparagine, or
is not present; amino acid m1 is selected from asparagine or
lysine, or is not present; amino acid n1 is selected from tyrosine,
asparagine, or isoleucine, or is not present; amino acid o1 is
selected from leucine or tyrosine, or is not present; amino acid p1
is selected from aspartic acid or leucine, or is not present; and
amino acid q1 is selected from valine, alanine, or threonine, or is
not present; wherein CDR2b comprises the amino acid r1 s1 t1 u1 v1
w1 x1, wherein amino acid r1 is selected from alanine, aspartic
acid, leucine, tryptophan, glycine, or valine; amino acid s1 is
selected from threonine, valine, glycine, or alanine; amino acid t1
is serine; amino acid u1 is selected from serine, asparagine, or
threonine; amino acid v1 is selected from leucine, phenylalanine,
or arginine; amino acid w1 is selected from glutamine, alanine, or
glutamic acid; and amino acid x1 is selected from serine, arginine,
or threonine; wherein CDR3b comprises the amino acid sequence y1 z1
a1' b1' c1' d1' e1' f1' g1', wherein amino acid y1 is selected from
glutamine, methionine, leucine, or histidine; amino acid z1 is
selected from glutamine or lysine; amino acid a1' is selected from
serine, threonine, alanine, histidine, tyrosine, or phenylalanine;
amino acid b1' is selected from tyrosine, leucine, asparagine, or
glycine; amino acid c1' is selected from serine, glutamine,
isoleucine, or lysine; amino acid d1' is selected from threonine,
phenylalanine, tyrosine, alanine, or serine; amino acid e1' is
proline; amino acid f1' is selected from leucine, phenylalanine,
tryptophan, serine, or arginine; and amino acid g1' is selected
from threonine or serine; and wherein the polypeptide, in
association with an antibody heavy chain, binds TR-2.
[0211] In certain embodiments, a polypeptide comprises at least two
complementarity determining regions (CDR) selected from CDR1b,
CDR2b, and CDR3b, wherein the polypeptide, in association with an
antibody heavy chain, binds TR-2. In certain embodiments, a
polypeptide comprises CDR1b, CDR2b, and CDR3b, wherein the
polypeptide, in association with an antibody heavy chain, binds
TR-2.
[0212] In certain embodiments, a polypeptide comprises an antibody
light chain variable region. In certain embodiments, a polypeptide
comprises a human antibody light chain variable region. In certain
embodiments, a polypeptide comprises a light chain constant region.
In certain embodiments, a polypeptide comprises a human light chain
constant region. In certain embodiments, a polypeptide comprises an
amino acid sequence as set forth in SEQ ID NO: 36, SEQ ID NO: 38,
SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID
NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56,
SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID
NO: 66, or SEQ ID NO: 68. In certain embodiments, a polypeptide
comprises a non-human light chain constant region. In certain
embodiments, a polypeptide comprises a light chain constant region
of a species other than human.
[0213] In certain embodiments, a polypeptide which comprises at
least one complementarity determining region (CDR) selected from
amino acids 24 to 34 of SEQ ID NO: 36; amino acids 50 to 56 of SEQ
ID NO: 36; amino acids 89-97 of SEQ ID NO: 36; amino acids 24 to 34
of SEQ ID NO: 38; amino acids 50 to 56 of SEQ ID NO: 38; amino
acids 89 to 97 of SEQ ID NO: 38; amino acids 24 to 34 of SEQ ID NO:
40; amino acids 50 to 56 of SEQ ID NO: 40; amino acids 89 to 97 of
SEQ ID NO: 40; amino acids 24 to 34 of SEQ ID NO: 42; amino acids
50 to 56 of SEQ ID NO: 42; amino acids 89 to 97 of SEQ ID NO: 42;
amino acids 24 to 34 of SEQ ID NO: 44; amino acids 50 to 56 of SEQ
ID NO: 44; amino acids 89-97 of SEQ ID NO: 44; amino acids 24 to 34
of SEQ ID NO: 46; amino acids 50 to 56 of SEQ ID NO: 46; amino
acids 89 to 97 of SEQ ID NO: 46; amino acids 24 to 40 of SEQ ID NO:
48; amino acids 56 to 62 of SEQ ID NO: 48; amino acids 95 to 103 of
SEQ ID NO: 48; amino acids 24 to 39 of SEQ ID NO: 50; amino acids
55 to 61 of SEQ ID NO: 50; amino acids 94 to 102 of SEQ ID NO: 50;
amino acids 24 to 40 of SEQ ID NO: 52; amino acids 56 to 62 of SEQ
ID NO: 52; amino acids 95 to 103 of SEQ ID NO: 52; 24 to 34 of SEQ
ID NO: 54; amino acids 50 to 56 of SEQ ID NO: 54; amino acids 89 to
97 of SEQ ID NO: 54; amino acids 24 to 34 of SEQ ID NO: 56, amino
acids 50 to 56 of SEQ ID NO: 56; amino acids 89 to 97 of SEQ ID NO:
56; amino acids 24 to 40 of SEQ ID NO: 58; amino acids 56 to 62 of
SEQ ID NO: 58; amino acids 95 to 103 of SEQ ID NO: 58; amino acids
24 to 34 of SEQ ID NO: 60; amino acids 50 to 56 of SEQ ID NO: 60;
amino acids 89-97 of SEQ ID NO: 60; amino acids 24 to 34 of SEQ ID
NO: 62; amino acids 50 to 56 of SEQ ID NO: 62; amino acids 89 to 97
of SEQ ID NO: 62; amino acids 24 to 35 of SEQ ID NO: 64; amino
acids 51 to 57 of SEQ ID NO: 64; amino acids 90 to 88 of SEQ ID NO:
64; amino acids 24 to 34 of SEQ ID NO: 66; amino acids 50 to 57 of
SEQ ID NO: 66; amino acids 89 to 97 of SEQ ID NO: 66; amino acids
24 to 34 of SEQ ID NO: 68; amino acids 50 to 56 of SEQ ID NO: 68;
and amino acids 89 to 97 of SEQ ID NO: 68, wherein the polypeptide,
in association with an antibody heavy chain, binds TR-2. In certain
embodiments, a polypeptide comprises at least two of the CDRs of
SEQ ID NOS. 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,
64, 66, or 68. In certain embodiments, a polypeptide comprises at
least three of the CDRs of SEQ ID NOS. 36, 38, 40, 42, 44, 46, 48,
50, 52, 54, 56, 58, 60, 62, 64, 66, or 68.
[0214] In certain embodiments, a polypeptide comprises amino acids
24 to 34 of SEQ ID NO: 36, amino acids 50 to 56 of SEQ ID NO: 36,
and amino acids 89-97 of SEQ ID NO: 36. In certain embodiments, a
polypeptide comprises amino acids 24 to 34 of SEQ ID NO: 38, amino
acids 50 to 56 of SEQ ID NO: 38, and amino acids 89 to 97 of SEQ ID
NO: 38. In certain embodiments, a polypeptide comprises amino acids
24 to 34 of SEQ ID NO: 40, amino acids 50 to 56 of SEQ ID NO: 40,
and amino acids 89 to 97 of SEQ ID NO: 40. In certain embodiments,
a polypeptide comprises amino acids 24 to 34 of SEQ ID NO: 42,
amino acids 50 to 56 of SEQ ID NO: 42, and amino acids 89 to 97 of
SEQ ID NO: 42. In certain embodiments, a polypeptide comprises
amino acids 24 to 34 of SEQ ID NO: 44, amino acids 50 to 56 of SEQ
ID NO: 44, and amino acids 89-97 of SEQ ID NO: 44. In certain
embodiments, a polypeptide comprises amino acids 24 to 34 of SEQ ID
NO: 46, amino acids 50 to 56 of SEQ ID NO: 46, and amino acids 89
to 97 of SEQ ID NO: 46. In certain embodiments, a polypeptide
comprises amino acids 24 to 40 of SEQ ID NO: 48, amino acids 56 to
62 of SEQ ID NO: 48, and amino acids 95 to 103 of SEQ ID NO: 48. In
certain embodiments, a polypeptide comprises amino acids 24 to 39
of SEQ ID NO: 50, amino acids 55 to 61 of SEQ ID NO: 50, and amino
acids 94 to 102 of SEQ ID NO: 50. In certain embodiments, a
polypeptide comprises amino acids 24 to 40 of SEQ ID NO: 52, amino
acids 56 to 62 of SEQ ID NO: 52, and amino acids 95 to 103 of SEQ
ID NO: 52. In certain embodiments, a polypeptide comprises amino
acids 24 to 34 of SEQ ID NO: 54, amino acids 50 to 56 of SEQ ID NO:
54, and amino acids 89 to 97 of SEQ ID NO: 54. In certain
embodiments, a polypeptide comprises amino acids 24 to 34 of SEQ ID
NO: 56, amino acids 50 to 56 of SEQ ID NO: 56, and amino acids 89
to 97 of SEQ ID NO: 56, In certain embodiments, a polypeptide
comprises amino acids 24 to 40 of SEQ ID NO: 58, amino acids 56 to
62 of SEQ ID NO: 58, and amino acids 95 to 103 of SEQ ID NO: 58. In
certain embodiments, a polypeptide comprises amino acids 24 to 34
of SEQ ID NO: 60, amino acids 50 to 56 of SEQ ID NO: 60, and amino
acids 89-97 of SEQ ID NO: 60. In certain embodiments, a polypeptide
comprises amino acids 24 to 34 of SEQ ID NO: 62, amino acids 50 to
56 of SEQ ID NO: 62, and amino acids 89 to 97 of SEQ ID NO: 62. In
certain embodiments, a polypeptide comprises amino acids 24 to 35
of SEQ ID NO: 64, amino acids 51 to 57 of SEQ ID NO: 64, and amino
acids 90 to 88 of SEQ ID NO: 64. In certain embodiments, a
polypeptide comprises amino acids 24 to 34 of SEQ ID NO: 66, amino
acids 50 to 57 of SEQ ID NO: 66, and amino acids 89 to 97 of SEQ ID
NO: 66. In certain embodiments, a polypeptide comprises amino acids
24 to 34 of SEQ ID NO: 68, amino acids 50 to 56 of SEQ ID NO: 68,
and amino acids 89 to 97 of SEQ ID NO: 68.
[0215] The term "naturally-occurring" as applied to an object means
that an object can be found in nature. For example, a polypeptide
or polynucleotide that is present in an organism (including
viruses) that can be isolated from a source in nature and which has
not been intentionally modified by man in the laboratory or
otherwise is naturally-occurring.
[0216] The term "operably linked" as used herein refers to
components that are in a relationship permitting them to function
in their intended manner. For example, in the context of a
polynucleotide sequence, a control sequence may be "operably
linked" to a coding sequence when the control sequence and coding
sequence are in association with each other in such a way that
expression of the coding sequence is achieved under conditions
compatible with the functioning of the control sequence.
[0217] The term "control sequence" refers to polynucleotide
sequences which may effect the expression and processing of coding
sequences with which they are in association. The nature of such
control sequences may differ depending upon the host organism.
Certain exemplary control sequences for prokaryotes include, but
are not limited to, promoters, ribosomal binding sites, and
transcription termination sequences. Certain exemplary control
sequences for eukaryotes include, but are not limited to,
promoters, enhancers, and transcription termination sequences. In
certain embodiments, "control sequences" can include leader
sequences and/or fusion partner sequences.
[0218] In certain embodiments, a first polynucleotide coding
sequence is operably linked to a second polynucleotide coding
sequence when the first and second polynucleotide coding sequences
are transcribed into a single contiguous mRNA that can be
translated into a single contiguous polypeptide.
[0219] In the context of polypeptides, two or more polypeptides are
"operably linked" if each linked polypeptide is able to function in
its intended manner. A polypeptide that is able to function in its
intended manner when operably linked to another polypeptide may or
may not be able to function in its intended manner when not
operably linked to another polypeptide. For example, in certain
embodiments, a first polypeptide may be unable to function in its
intended manner when unlinked, but may be stabilized by being
linked to a second polypeptide such that it becomes able to
function in its intended manner. Alternatively, in certain
embodiments, a first polypeptide may be able to function in its
intended manner when unlinked, and may retain that ability when
operably linked to a second polypeptide.
[0220] As used herein, two or more polypeptides are "fused" when
the two or more polypeptides are linked by translating them as a
single contiguous polypeptide sequence or by synthesizing them as a
single contiguous polypeptide sequence. In certain embodiments, two
or more fused polypeptides may have been translated in vivo from
two or more operably linked polynucleotide coding sequences. In
certain embodiments, two or more fused polypeptides may have been
translated in vitro from two or more operably linked polynucleotide
coding sequences.
[0221] As used herein, two or more polypeptides are "operably
fused" if each linked polypeptide is able to function in its
intended manner.
[0222] In certain embodiments, a first polypeptide that contains
two or more distinct polypeptide units is considered to be linked
to a second polypeptide so long as at least one of the distinct
polypeptide units of the first polypeptide is linked to the second
polypeptide. As a non-limiting example, in certain embodiments, an
antibody is considered linked to a second polypeptide in all of the
following instances: (a) the second polypeptide is linked to one of
the heavy chain polypeptides of the antibody; (b) the second
polypeptide is linked to one of the light chain polypeptides of the
antibody; (c) a first molecule of the second polypeptide is linked
to one of the heavy chain polypeptides of the antibody and a second
molecule of the second polypeptide is linked to one of the light
chain polypeptides of the antibody; and (d) first and second
molecules of the second polypeptide are linked to the first and
second heavy chain polypeptides of the antibody and third and
fourth molecules of the second polypeptide are linked to first and
second light chain polypeptides of the antibody.
[0223] In certain embodiments, the language "a first polypeptide
linked to a second polypeptide" encompasses situations where: (a)
only one molecule of a first polypeptide is linked to only one
molecule of a second polypeptide; (b) only one molecule of a first
polypeptide is linked to more than one molecule of a second
polypeptide; (c) more than one molecule of a first polypeptide is
linked to only one molecule of a second polypeptide; and (d) more
than one molecule of a first polypeptide is linked to more than one
molecule of a second polypeptide. In certain embodiments, when a
linked molecule comprises more than one molecule of a first
polypeptide and only one molecule of a second polypeptide, all or
fewer than all of the molecules of the first polypeptide may be
covalently or noncovalently linked to the second polypeptide. In
certain embodiments, when a linked molecule comprises more than one
molecule of a first polypeptide, one or more molecules of the first
polypeptide may be covalently or noncovalently linked to other
molecules of the first polypeptide.
[0224] As used herein, a "flexible linker" refers to any linker
that is not predicted, according to its chemical structure, to be
fixed in three-dimensional space. One skilled in the art can
predict whether a particular linker is flexible in its intended
context. In certain embodiments, a peptide linker comprising 3 or
more amino acids is a flexible linker.
[0225] As used herein, the twenty conventional amino acids and
their abbreviations follow conventional usage. See Immunology--A
Synthesis (2nd Edition, E. S. Golub and D. R. Gren, Eds., Sinauer
Associates, Sunderland, Mass. (1991)). In certain embodiments, one
or more unconventional amino acids may be incorporated into a
polypeptide. The term "unconventional amino acid" refers to any
amino acid that is not one of the twenty conventional amino acids.
The term "non-naturally occurring amino acids" refers to amino
acids that are not found in nature. Non-naturally occurring amino
acids are a subset of unconventional amino acids. Unconventional
amino acids include, but are not limited to, stereoisomers (e.g.,
D-amino acids) of the twenty conventional amino acids, unnatural
amino acids such as .alpha.-, .alpha.-disubstituted amino acids,
N-alkyl amino acids, lactic acid, homoserine, homocysteine,
4-hydroxyproline, .gamma.-carboxyglutamate,
.epsilon.-N,N,N-trimethyllysine, .epsilon.-N-acetyllysine,
O-phosphoserine, N-acetylserine, N-formylmethionine,
3-methylhistidine, 5-hydroxylysine, .sigma.-N-methylarginine, and
other similar amino acids and imino acids (e.g., 4-hydroxyproline)
known in the art. In the polypeptide notation used herein, the
left-hand direction is the amino terminal direction and the
right-hand direction is the carboxy-terminal direction, in
accordance with standard usage and convention.
[0226] In certain embodiments, conservative amino acid
substitutions include substitution with one or more unconventional
amino acid residues. In certain embodiments, unconventional amino
acid residues are incorporated by chemical peptide synthesis rather
than by synthesis in biological systems.
[0227] The term "acidic residue" refers to an amino acid residue in
D- or L-form that comprises at least one acidic group when
incorporated into a polypeptide between two other amino acid
residues that are the same or different. In certain embodiments, an
acidic residue comprises a sidechain that comprises at least one
acidic group. Exemplary acidic residues include, but are not
limited to, aspartic acid (D) and glutamic acid (E). In certain
embodiments, an acidic residue may be an unconventional amino
acid.
[0228] The term "aromatic residue" refers to an amino acid residue
in D- or L-form that comprises at least one aromatic group. In
certain embodiments, an aromatic residue comprises a sidechain that
comprises at least one aromatic group. Exemplary aromatic residues
include, but are not limited to, phenylalanine (F), tyrosine (Y),
and tryptophan (W). In certain embodiments, an aromatic residue may
be an unconventional amino acid.
[0229] The term "basic residue" refers to an amino acid residue in
F- or L-form that may comprise at least one basic group when
incorporated into a polypeptide next to one or more amino acid
residues that are the same or different. In certain embodiments, a
basic residue comprises a sidechain that comprises at least one
basic group. Exemplary basic residues include, but are not limited
to, histidine (H), lysine (K), and arginine (R). In certain
embodiments, a basic residue may be an unconventional amino
acid.
[0230] The term "neutral hydrophilic residue" refers to an amino
acid residue in D- or L-form that comprises at least one
hydrophilic and/or polar group, but does not comprise an acidic or
basic group when incorporated into a polypeptide next to one or
more amino acid residues that are the same or different. Exemplary
neutral hydrophilic residues include, but are not limited to,
alanine (A), cysteine (C), serine (S), threonine (T), asparagine
(N), and glutamine (Q). In certain embodiments, a neutral
hydrophilic residue may be an unconventional amino acid.
[0231] The terms "lipophilic residue" and "Laa" refer to an amino
acid residue in D- or L-form having at least one uncharged,
aliphatic and/or aromatic group. In certain embodiments, a
lipophilic residue comprises a side chain that comprises at least
one uncharged, aliphatic, and/or aromatic group. Exemplary
lipophilic sidechains include, but are not limited to, alanine (A),
phenylalanine (F), isoleucine (I), leucine (L), norleucine (Nile),
methionine (M), valine (V), tryptophan (W), and tyrosine (Y). In
certain embodiments, a lipophilic residue may be an unconventional
amino acid.
[0232] The term "amphiphilic residue" refers to an amino acid
residue in D- or L-form that is capable of being either a
hydrophilic or lipophilic residue. An exemplary amphiphilic residue
includes, but is not limited to, alanine (A). In certain
embodiments, an amphiphilic residue may be an unconventional amino
acid.
[0233] The term "nonfunctional residue" refers to an amino acid
residue in ID- or L-form that lacks acidic, basic, and aromatic
groups when incorporated into a polypeptide next to one or more
amino acid residues that are the same or different. Exemplary
nonfunctional amino acid residues include, but are not limited to,
methionine (M), glycine (G), alanine (A), valine (V), isoleucine
(I), leucine (L), and norleucine (Nle). In certain embodiments, a
nonfunctional residue may be an unconventional amino acid.
[0234] In certain embodiments, glycine (G) and proline (P) are
considered amino acid residues that can influence polypeptide chain
orientation.
[0235] In certain embodiments, a conservative substitution may
involve replacing a member of one residue type with a member of the
same residue type. As a non-limiting example, in certain
embodiments, a conservative substitution may involve replacing an
acidic residue, such as D, with a different acidic residue, such as
E. In certain embodiments, a non-conservative substitution may
involve replacing a member of one residue type with a member of a
different residue type. As a non-limiting example, in certain
embodiments, a non-conservative substitution may involve replacing
an acidic residue, such as D, with a basic residue, such as K. In
certain embodiments, a cysteine residue is substituted with another
amino acid residue to prevent disulfide bond formation with that
position in the polypeptide.
[0236] In making conservative or non-conservative substitutions,
according to certain embodiments, the hydropathic index of amino
acids may be considered. Each amino acid has been assigned a
hydropathic index on the basis of its hydrophobicity and charge
characteristics. The hydropathic indices of the 20
naturally-occurring amino acids are: isoleucine (+4.5); valine
(+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine
(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4);
threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine
(-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5);
glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine
(-3.9); and arginine (-4.5).
[0237] The importance of the hydropathic amino acid index in
conferring interactive biological function on a protein is
understood in the art. Kyte et al., J. Mol. Biol., 157:105-131
(1982). It is known in certain instances that certain amino acids
may be substituted for other amino acids having a similar
hydropathic index or score and still retain a similar biological
activity. In making changes based upon the hydropathic index, in
certain embodiments, the substitution of amino acids whose
hydropathic indices are within .+-.2 is included. In certain
embodiments, those which are within .+-.1 are included, and in
certain embodiments, those within .+-.0.5 are included.
[0238] It is also understood in the art that the substitution of
like amino acids can be made effectively on the basis of
hydrophilicity, particularly where the biologically functional
protein or peptide thereby created is intended for use in
immunological embodiments, as in the present case. In certain
embodiments, the greatest local average hydrophilicity of a
protein, as governed by the hydrophilicity of its adjacent amino
acids, correlates with its immunogenicity and antigenicity, i.e.,
with a biological property of the polypeptide.
[0239] The following hydrophilicity values have been assigned to
these amino acid residues: arginine (+3.0); lysine (+3.0);
aspartate (+3.0.+-.1); glutamate (+3.0.+-.1); serine (+0.3);
asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4);
proline (-0.5.+-.1); alanine (-0.5); histidine (-0.5); cysteine
(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8);
isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5) and
tryptophan (-3.4). In making changes based upon similar
hydrophilicity values, in certain embodiments, the substitution of
amino acids whose hydrophilicity values are within .+-.2 is
included, in certain embodiments, those which are within .+-.1 are
included, and in certain embodiments, those within .+-.0.5 are
included. In certain instances, one may also identify epitopes from
primary amino acid sequences on the basis of hydrophilicity. These
regions are also referred to as "epitopic core regions."
[0240] Exemplary amino acid substitutions are set forth in Table
1.
TABLE-US-00001 TABLE 1 Amino Acid Substitutions More specific
Original Exemplary exemplary Residues Substitutions Substitutions
Ala Val, Leu, Ile Val Arg Lys, Gln, Asn Lys Asn Gln Gln Asp Glu Glu
Cys Ser, Ala Ser Gln Asn Asn Glu Asp Asp Gly Pro, Ala Ala His Asn,
Gln, Lys, Arg Arg Ile Leu, Val, Met, Ala, Leu Phe, Norleucine Leu
Norleucine, Ile, Ile Val, Met, Ala, Phe Lys Arg, 1,4
Diamino-butyric Arg Acid, Gln, Asn Met Leu, Phe, Ile Leu Phe Leu,
Val, Ile, Ala, Leu Tyr Pro Ala Gly Ser Thr, Ala, Cys Thr Thr Ser
Ser Trp Tyr, Phe Tyr Tyr Trp, Phe, Thr, Ser Phe Val Ile, Met, Leu,
Phe, Leu Ala, Norleucine
[0241] Similarly, as used herein, unless specified otherwise, the
left-hand end of single-stranded polynucleotide sequences is the 5'
end; the left-hand direction of double-stranded polynucleotide
sequences is referred to as the 5' direction. The direction of 5'
to 3' addition of nascent RNA transcripts is referred to herein as
the transcription direction; sequence regions on the DNA strand
having the same sequence as the RNA and which are 5' to the 5' end
of the RNA transcript are referred to herein as "upstream
sequences"; sequence regions on the DNA strand having the same
sequence as the RNA and which are 3' to the 3' end of the RNA
transcript are referred to herein as "downstream sequences."
[0242] In certain embodiments, conservative amino acid
substitutions encompass non-naturally occurring amino acid
residues, which are typically incorporated by chemical peptide
synthesis rather than by synthesis in biological systems. Those
non-naturally occurring amino acid residues include, but are not
limited to, peptidomimetics and other reversed or inverted forms of
amino acid moieties.
[0243] A skilled artisan will be able to determine suitable
substitution variants of a reference polypeptide as set forth
herein using well-known techniques. In certain embodiments, one
skilled in the art may identify suitable areas of the molecule that
may be changed without destroying activity by targeting regions not
believed to be important for activity. In certain embodiments, one
can identify residues and portions of the molecules that are
conserved among similar polypeptides. In certain embodiments, even
areas that may be important for biological activity, including, but
not limited to, the CDRs of an antibody, or that may be important
for structure may be subject to conservative amino acid
substitutions without destroying the biological activity or without
adversely affecting the polypeptide structure.
[0244] Additionally, in certain embodiments, one skilled in the art
can review structure-function studies identifying residues in
similar polypeptides that are important for activity and/or
structure. In view of such a comparison, in certain embodiments,
one can predict the importance of amino acid residues in a
polypeptide that correspond to amino acid residues which are
important for activity or structure in similar polypeptides. In
certain embodiments, one skilled in the art may opt for chemically
similar amino acid substitutions for such predicted important amino
acid residues.
[0245] In certain embodiments, one skilled in the art can also
analyze the three-dimensional structure and amino acid sequence in
relation to that structure in similar polypeptides. In view of such
information, one skilled in the art may predict the alignment of
amino acid residues of an antibody with respect to its three
dimensional structure. In certain embodiments, one skilled in the
art may choose not to make radical changes to amino acid residues
predicted to be on the surface of the protein, since such residues
may be involved in important interactions with other molecules.
Moreover, in certain embodiments, one skilled in the art may
generate test variants containing a single amino acid substitution
at each desired amino acid residue. In certain embodiments, the
variants can then be screened using activity assays known to those
skilled in the art. For example, in certain embodiments, the
variants can be screened for their ability to bind to TR-2. In
certain embodiments, such variants could be used to gather
information about suitable variants. For example, in certain
embodiments, if one discovered that a change to a particular amino
acid residue resulted in destroyed, undesirably reduced, or
unsuitable activity, variants with such a change may be avoided. In
other words, based on information gathered from such routine
experiments, one skilled in the art can readily determine the amino
acids where further substitutions should be avoided, either alone
or in combination with other mutations.
[0246] A number of scientific publications have been devoted to the
prediction of secondary structure. See Moult J., Curr. Op. in
Biotech., 7(4):422-427 (1996), Chou et al., Biochemistry,
13(2):222-245 (1974); Chou et al., Biochemistry, 113(2):211-222
(1974); Chou et al., Adv. Enzymol. Relat. Areas Mol. Biol.,
47:45-148 (1978); Chou et al., Ann. Rev. Biochem., 47:251-276 and
Chou et al., Biophys. J., 26:367-384 (1979). Moreover, computer
programs are currently available to assist with predicting
secondary structure. One method of predicting secondary structure
is based upon homology modeling. For example, two polypeptides or
proteins which have a sequence identity of greater than 30%, or
similarity greater than 40% often have similar structural
topologies. The recent growth of the protein structural database
(PDB) has provided enhanced predictability of secondary structure,
including the potential number of folds within a polypeptide's or
protein's structure. See Holm et al., Nucl. Acid. Res.,
27(1):244-247 (1999). It has been suggested that there are a
limited number of folds in a given polypeptide or protein and that
once a critical number of structures have been resolved, structural
prediction will become dramatically more accurate. See, e.g.,
Brenner et al., Curr. Op. Struct. Biol., 7(3):369-376 (1997).
[0247] Additional exemplary methods of predicting secondary
structure include, but are not limited to, "threading" (Jones, D.,
Curr. Opin. Struct. Biol., 7(3):377-87 (1997); Sippl et al.,
Structure, 4(1):15-19 (1996)), "profile analysis" (Bowie et at.,
Science, 253:164-170 (1991); Gribskov et al., Meth. Enzym.,
183:146-159 (1990); Gribskov et al., Proc. Nat. Acad. Sci.,
84(13):4355-4358 (1987)), and "evolutionary linkage" (See Holm,
supra (1999), and Brenner, supra (1997).).
[0248] In certain embodiments, the identity and similarity of
related polypeptides can be readily calculated by known methods.
Such methods include, but are not limited to, those described in
Computational Molecular Biology, Lesk, A. M., ed., Oxford
University Press, New York (1988); Biocomputing: Informatics and
Genome Projects, Smith, D. W., ed., Academic Press, New York
(1993); Computer Analysis of Sequence Data, Part 1, Griffin, A. M.,
and Griffin, H. G., eds., Humana Press, New Jersey (1994); Sequence
Analysis in Molecular Biology, von Neije, G., Academic Press
(1987); Sequence Analysis Primer, Gribskov, M. and Devereux, J.,
eds., M. Stockton Press, New York (1991); and Carillo et al., SIAM
J. Applied Math., 48:1073 (1988). In certain embodiments,
polypeptides have amino acid sequences that are about 90 percent,
or about 95 percent, or about 96 percent, or about 97 percent, or
about 98 percent, or about 99 percent identical to amino acid
sequences shown in FIGS. 3-19.
[0249] In certain embodiments, methods to determine identity are
designed to give the largest match between the sequences tested. In
certain embodiments, certain methods to determine identity are
described in publicly available computer programs. Certain computer
program methods to determine identity between two sequences
include, but are not limited to, the GCG program package, including
GAP (Devereux et al., Nucl. Acid. Res., 12:387 (1984); Genetics
Computer Group, University of Wisconsin, Madison, Wis., BLASTP,
BLASTN, and FASTA (Altschul et al., J. Mol. Biol., 215:403-410
(1990)). The BLASTX program is publicly available from the National
Center for Biotechnology Information (NCBI) and other sources
(BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894;
Altschul et al., supra (1990)). In certain embodiments, the Smith
Waterman algorithm, which is known in the art, may also be used to
determine identity.
[0250] Certain alignment schemes for aligning two amino acid
sequences may result in the matching of only a short region of the
two sequences, and this small aligned region may have very high
sequence identity even though there is no significant relationship
between the two full-length sequences. Accordingly, in certain
embodiments, the selected alignment method (GAP program) will
result in an alignment that spans at least 50 contiguous amino
acids of the target polypeptide.
[0251] For example, using the computer algorithm GAP (Genetics
Computer Group, University of Wisconsin, Madison, Wis.), two
polypeptides for which the percent sequence identity is to be
determined are aligned for optimal matching of their respective
amino acids (the "matched span", as determined by the algorithm).
In certain embodiments, a gap opening penalty (which is calculated
as 3.times. the average diagonal; the "average diagonal" is the
average of the diagonal of the comparison matrix being used; the
"diagonal" is the score or number assigned to each perfect amino
acid match by the particular comparison matrix) and a gap extension
penalty (which is usually 1/10 times the gap opening penalty), as
well as a comparison matrix such as PAM 250 or BLOSUM 62 are used
in conjunction with the algorithm. In certain embodiments, a
standard comparison matrix is also used by the algorithm. See,
e.g., Dayhoff et al., Atlas of Protein Sequence and Structure,
5(3)(1978) for the PAM 250 comparison matrix; Henikoff et al.,
Proc. Natl. Acad. Sci USA, 89:10915-10919 (1992) for the BLOSUM 62
comparison matrix.
[0252] In certain embodiments, the parameters for a polypeptide
sequence comparison include the following:
[0253] Algorithm: Needleman et al., J. Mol. Biol., 48:443-453
(1970);
[0254] Comparison matrix: BLOSUM 62 from Henikoff et al., supra
(1992);
[0255] Gap Penalty: 12
[0256] Gap Length Penalty: 4
[0257] Threshold of Similarity: 0
[0258] In certain embodiments, the GAP program may be useful with
the above parameters. In certain embodiments, the aforementioned
parameters are the default parameters for polypeptide comparisons
(along with no penalty for end gaps) using the GAP algorithm.
[0259] According to certain embodiments, amino acid substitutions
are those which: (1) reduce susceptibility to proteolysis, (2)
reduce susceptibility to oxidation, (3) alter binding affinity for
forming protein complexes, (4) alter binding affinities, and/or (4)
confer or modify other physicochemical or functional properties on
such polypeptides. According to certain embodiments, single or
multiple amino acid substitutions (in certain embodiments,
conservative amino acid substitutions) may be made in the
naturally-occurring sequence (in certain embodiments, in the
portion of the polypeptide outside the domain(s) forming
intermolecular contacts).
[0260] In certain embodiments, a conservative amino acid
substitution typically may not substantially change the structural
characteristics of the parent sequence (e.g., a replacement amino
acid should not tend to break a helix that occurs in the parent
sequence, or disrupt other types of secondary structure that
characterizes the parent sequence). Examples of art-recognized
polypeptide secondary and tertiary structures are described, e.g.,
in Proteins. Structures and Molecular Principles (Creighton, Ed.,
W.H. Freeman and Company, New York (1984)); Introduction to Protein
Structure (C. Branden and J. Tooze, eds., Garland Publishing, New
York, N.Y. (1991)); and Thornton et al. Nature 354:105 (1991).
[0261] The term "polypeptide fragment" as used herein refers to a
polypeptide that has an amino-terminal and/or carboxy-terminal
deletion. In certain embodiments, fragments are at least 5 to 500
amino acids long. It will be appreciated that in certain
embodiments, fragments are at least 5, 6, 8, 10, 14, 20, 50, 70,
100, 150, 200, 250, 300, 350, 400, 450, or 500 amino acids
long.
[0262] Peptide analogs are commonly used in the pharmaceutical
industry as non-peptide drugs with properties analogous to those of
the template peptide. These types of non-peptide compound are
termed "peptide mimetics" or "peptidomimetics." Fauchere, J. Adv.
Drug Res. 15:29 (1986); Veber and Freidinger TINS p. 392 (1985);
and Evans et al. J. Med. Chem. 30:1229 (1987). Such compounds are
often developed with the aid of computerized molecular modeling.
Peptide mimetics that are structurally similar to therapeutically
useful peptides may be used to produce a similar therapeutic or
prophylactic effect. Generally, peptidomimetics are structurally
similar to a paradigm polypeptide (i.e., a polypeptide that has a
biochemical property or pharmacological activity), such as a human
antibody, but have one or more peptide linkages optionally replaced
by a linkage selected from:--CH.sub.2 NH--, --CH.sub.2 S--,
--CH.sub.2 --CH.sub.2--, --CH.dbd.CH-(cis and trans),
--COCH.sub.2--, --CH(OH)CH.sub.2--, and --CH.sub.2 SO--, by methods
well known in the art. Systematic substitution of one or more amino
acids of a consensus sequence with a D-amino acid of the same type
(e.g., D-lysine in place of L-lysine) may be used in certain
embodiments to generate more stable peptides. In addition,
constrained peptides comprising a consensus sequence or a
substantially identical consensus sequence variation may be
generated by methods known in the art (Rizo and Gierasch Ann. Rev.
Biochem. 61:387 (1992)); for example, and not limitation, by adding
internal cysteine residues capable of forming intramolecular
disulfide bridges which cyclize the peptide.
[0263] The term "specific binding agent" refers to a natural or
non-natural molecule that specifically binds to a target. Examples
of specific binding agents include, but are not limited to,
proteins, peptides, nucleic acids, carbohydrates, lipids, and small
molecule compounds. In certain embodiments, a specific binding
agent is an antibody. In certain embodiments, a specific binding
agent is an antigen binding region.
[0264] The term "specifically binds" refers to the ability of a
specific binding agent to bind to a target with greater affinity
than it binds to a non-target. In certain embodiments, specific
binding refers to binding to a target with an affinity that is at
least 10, 50, 100, 250, 500, or 1000 times greater than the
affinity for a non-target. In certain embodiments, affinity is
determined by an affinity ELISA assay. In certain embodiments,
affinity is determined by a BIAcore assay. In certain embodiments,
affinity is determined by a kinetic method. In certain embodiments,
affinity is determined by an equilibrium/solution method.
[0265] The term "specific binding agent to TR-2" refers to a
specific binding agent that specifically binds any portion of TR-2.
In certain embodiments, a specific binding agent to TR-2 is an
antibody to TR-2. In certain embodiments, a specific binding agent
is an antigen binding region.
[0266] "Antibody" or "antibody peptide(s)" both refer to an intact
antibody, or a fragment thereof. In certain embodiments, the
antibody fragment may be a binding fragment that competes with the
intact antibody for specific binding. The term "antibody" also
encompasses polyclonal antibodies and monoclonal antibodies. In
certain embodiments, binding fragments are produced by recombinant
DNA techniques. In certain embodiments, binding fragments are
produced by enzymatic or chemical cleavage of intact antibodies. In
certain embodiments, binding fragments are produced by recombinant
DNA techniques. Binding fragments include, but are not limited to,
Fab, Fab', F(ab').sub.2, Fv, and single-chain antibodies.
Non-antigen binding fragments include, but are not limited to, Fc
fragments. In certain embodiments, an antibody specifically binds
to an epitope that is specifically bound by at least one antibody
selected from Ab A, Ab B, Ab C, Ab D, Ab E, Ab F, Ab G, Ab H, Ab I,
Ab J, Ab K, Ab L, Ab M, Ab N, Ab O, Ab P, and Ab Q. The term
"antibody" also encompasses anti-idiotypic antibodies that
specifically bind to the variable region of another antibody. In
certain embodiments, an anti-idiotypic antibody specifically binds
to the variable region of an anti-TR-2 antibody. In certain
embodiments, anti-idiotypic antibodies may be used to detect the
presence of a particular anti-TR-2 antibody in a sample or to block
the activity of an anti-TR-2 antibody.
[0267] The term "anti-TR-2 antibody" as used herein means an
antibody that specifically binds to TR-2. In certain embodiments,
an anti-TR-2 antibody binds to a TR-2 epitope to which at least one
antibody selected from Ab A to Q binds. In various embodiments,
TR-2 may be the TR-2 of any species, including, but not limited to,
human, cynomolgus monkeys, mice, and rabbits. Certain assays for
determining the specificity of an antibody are well known to the
skilled artisan and include, but are not limited to, ELISA,
ELISPOT, western blots, BIAcore assays, solution affinity binding
assays, T cell costimulation assays, and T cell migration
assays.
[0268] The term "isolated antibody" as used herein means an
antibody which (1) is free of at least some proteins with which it
would normally be found, (2) is essentially free of other proteins
from the same source, e.g., from the same species, (3) is expressed
by a cell from a different species, or (4) does not occur in
nature.
[0269] The term "polyclonal antibody" refers to a heterogeneous
mixture of antibodies that bind to different epitopes of the same
antigen.
[0270] The term "monoclonal antibodies" refers to a collection of
antibodies encoded by the same nucleic acid molecule. In certain
embodiments, monoclonal antibodies are produced by a single
hybridoma or other cell line, or by a transgenic mammal. Monoclonal
antibodies typically recognize the same epitope. The term
"monoclonal" is not limited to any particular method for making an
antibody.
[0271] The term "CDR grafted antibody" refers to an antibody in
which the CDR from one antibody is inserted into the framework of
another antibody. In certain embodiments, the antibody from which
the CDR is derived and the antibody from which the framework is
derived are of different species. In certain embodiments, the
antibody from which the CDR is derived and the antibody from which
the framework is derived are of different isotypes.
[0272] The term "multi-specific antibody" refers to an antibody
wherein two or more variable regions bind to different epitopes.
The epitopes may be on the same or different targets. In certain
embodiments, a multi-specific antibody is a "bi-specific antibody,"
which recognizes two different epitopes on the same or different
antigens.
[0273] The term "catalytic antibody" refers to an antibody in which
one or more catalytic moieties is attached. In certain embodiments,
a catalytic antibody is a cytotoxic antibody, which comprises a
cytotoxic moiety.
[0274] The term "humanized antibody" refers to an antibody in which
all or part of an antibody framework region is derived from a
human, but all or part of one or more CDR regions is derived from
another species, for example a mouse.
[0275] The terms "human antibody" and "fully human antibody" are
used interchangeably and refer to an antibody in which both the CDR
and the framework comprise substantially human sequences. In
certain embodiments, fully human antibodies are produced in
non-human mammals, including, but not limited to, mice, rats, and
lagomorphs. In certain embodiments, fully human antibodies are
produced in hybridoma cells. In certain embodiments, fully human
antibodies are produced recombinantly.
[0276] In certain embodiments, an anti-TR-2 antibody comprises:
[0277] (i) a first polypeptide comprising at least one
complementarity determining region (CDR) selected from CDR1a,
CDR2a, and CDR3a [0278] wherein CDR1a comprises the amino acid
sequence a b c d e f g h i j k l, wherein amino acid a is glycine,
amino acid b is selected from glycine, tyrosine, or phenylalanine;
amino acid c is selected from serine or threonine; amino acid d is
selected from isoleucine or phenylalanine; amino acid e is selected
from serine, threonine, or asparagine; amino acid f is selected
from serine, aspartic acid, tyrosine, asparagine, threonine, or
glycine; amino acid g is selected from glycine, aspartic acid, or
tyrosine; amino acid h is selected from glycine, aspartic acid,
tyrosine, asparagine, or serine; amino acid i is selected from
tyrosine, isoleucine, histidine, methionine, or tryptophan; amino
acid j is selected from asparagine, tyrosine, histidine, serine, or
phenylalanine; amino acid k is tryptophan or is not present; and
amino acid l is serine or is not present; [0279] wherein CDR2a
comprises the amino acid sequence m n o p q r t u v w x y z a' b'
c', wherein amino acid m is selected from tryptophan, tyrosine,
histidine, valine, glutamic acid, or serine; amino acid n is
selected from methionine or isoleucine; amino acid o is selected
from asparagine, tyrosine, serine, tryptophan, or histidine; amino
acid p is selected from proline, tyrosine, serine, arginine,
histidine, or asparagine; amino acid q is selected from asparagine,
serine, or aspartic acid; amino acid r is selected from serine or
glycine; amino acid s is selected from aspartic acid, serine,
threonine, or arginine; amino acid t is selected from asparagine,
threonine, alanine, isoleucine, or tyrosine; amino acid u is
selected from threonine, tyrosine, leucine, lysine, asparagine, or
isoleucine; amino acid v is selected from glycine, tyrosine,
aspartic acid, or cysteine; amino acid w is selected from tyrosine
or asparagine; amino acid x is selected from alanine or proline;
amino acid y is selected from glutamine, serine, or aspartic acid;
amino acid z is selected from lysine, leucine, or serine; amino
acid a' is selected from phenylalanine, lysine, or valine; amino
acid b' is selected from glutamine, serine, or lysine; and amino
acid c' is glycine or is not present; [0280] wherein CDR3a
comprises the amino acid sequence d' e' f' g' h' j' k' m' n' o' p'
q' r' s' t' u' v' w', wherein amino acid d' is selected from
tryptophan, aspartic acid, glycine, serine, or glutamic acid; amino
acid e' is selected from asparagine, aspartic acid, glycine,
arginine, serine, valine, or leucine; amino acid f' is selected
from histidine, serine, alanine, tyrosine, proline, asparagine,
glycine or threonine; amino acid g' is selected from tyrosine,
serine, alanine, arginine, tryptophan, glycine or valine; amino
acid h' is selected from glycine, alanine, serine, asparagine,
methionine, tyrosine, tryptophan, cysteine, or aspartic acid; amino
acid i' is selected from serine, tryptophan, glycine,
phenylalanine, aspartic acid, tyrosine, or threonine; amino acid j'
is selected from glycine, threonine, serine, leucine, valine,
asparagine, tryptophan, or tyrosine; amino acid k' is selected from
serine, phenylalanine, aspartic acid, tryptophan, glycine, or
tyrosine, or is not present; amino acid l' is selected from
histidine, aspartic acid, alanine, tryptophan, tyrosine, serine,
phenylalanine, valine, or glycine, or is not present; amino acid m'
is selected from phenylalanine, tyrosine, glutamic acid, proline,
aspartic acid, cysteine, isoleucine, or methionine, or is not
present; amino acid n' is selected from aspartic acid,
phenylalanine, alanine, leucine, or serine, or is not present;
amino acid o' is selected from tyrosine, leucine, aspartic acid,
phenylalanine, proline, or valine, or is not present; amino acid p'
is selected from leucine, aspartic acid, or tyrosine, or is not
present; amino acid q' is selected from serine or tyrosine, or is
not present; amino acid r' is tyrosine or is not present; amino
acid s' is selected from glycine or tyrosine, or is not present;
amino acid t' is selected from glycine or methionine, or is not
present; amino acid u' is selected from methionine or aspartic
acid, or is not present; amino acid v' is selected from aspartic
acid or valine, or is not present; and amino acid w' is valine or
is not present; and [0281] wherein the first polypeptide, in
association with an antibody light chain, binds TR-2; and [0282]
(ii) a second polypeptide comprising at least one complementarity
determining region (CDR) selected from CDR1b, CDR2b, and CDR3b
[0283] wherein CDR1b comprises the amino acid sequence a1 b1 c1 d1
e1 f1 g1 h1 i1 j1 k1 l1 m1 n1 o1 p1 q1, wherein amino acid a1 is
selected from arginine or lysine; amino acid b1 is selected from
threonine, alanine, or serine; amino acid c1 is serine; amino acid
d1 is glutamine; amino acid e1 is selected from serine or glycine;
amino acid f1 is selected from isoleucine, leucine, or valine;
amino acid g1 is selected from serine, leucine, or arginine; amino
acid h1 is selected from threonine, serine, isoleucine, asparagine,
arginine, histidine, or tyrosine; amino acid i1 is selected from
tyrosine, arginine, tryptophan, aspartic acid, or serine; j1 is
selected from leucine, isoleucine, asparagine, tyrosine, or serine;
amino acid k1 is selected from asparagine, glycine, valine,
alanine, or leucine; amino acid l1 is selected from tyrosine,
alanine, or asparagine, or is not present; amino acid m1 is
selected from asparagine or lysine, or is not present; amino acid
n1 is selected from tyrosine, asparagine, or isoleucine, or is not
present; amino acid o1 is selected from leucine or tyrosine, or is
not present; amino acid p1 is selected from aspartic acid or
leucine, or is not present; and amino acid q1 is selected from
valine, alanine, or threonine, or is not present; [0284] wherein
CDR2b comprises the amino acid sequence r1 s1 t1 u1 v1 w1 x1,
wherein amino acid r1 is selected from alanine, aspartic acid,
leucine, tryptophan, glycine, or valine; amino acid s1 is selected
from threonine, valine, glycine, or alanine; amino acid t1 is
serine; amino acid u1 is selected from serine, asparagine, or
threonine; amino acid v1 is selected from leucine, phenylalanine,
or arginine; amino acid w1 is selected from glutamine, alanine, or
glutamic acid; and amino acid x1 is selected from serine, arginine,
or threonine; [0285] wherein CDR3b comprises the amino acid
sequence y1 z1 a1' b1' c1' d1' e1' f1' g1', wherein amino acid y1
is selected from glutamine, methionine, leucine, or histidine;
amino acid z1 is selected from glutamine or lysine; amino acid a1'
is selected from serine, threonine, alanine, histidine, tyrosine,
or phenylalanine; amino acid b1' is selected from tyrosine,
leucine, asparagine, or glycine; amino acid c1' is selected from
serine, glutamine, isoleucine, or lysine; amino acid d1' is
selected from threonine, phenylalanine, tyrosine, alanine, or
serine; amino acid e1' is proline; amino acid f1' is selected from
leucine, phenylalanine, tryptophan, serine, or arginine; and amino
acid g1' is selected from threonine or serine; and wherein the
second polypeptide, in association with an antibody heavy chain,
binds TR-2.
[0286] In certain embodiments, an anti-TR-2 antibody comprises: a
first polypeptide comprising complementarity determining regions
(CDRs) as set forth in SEQ ID NO: 2 and a second polypeptide
comprising CDRs as set forth in SEQ ID NO: 36. In certain
embodiments, an anti-TR-2 antibody comprises: a first polypeptide
comprising complementarity determining regions (CDRs) as set forth
in SEQ ID NO: 4 and a second polypeptide comprising CDRs as set
forth in SEQ ID NO: 38. In certain embodiments, an anti-TR-2
antibody comprises: a first polypeptide comprising complementarity
determining regions (CDRs) as set forth in SEQ ID NO: 6 and a
second polypeptide comprising CDRs as set forth in SEQ ID NO: 40.
In certain embodiments, an anti-TR-2 antibody comprises: a first
polypeptide comprising complementarity determining regions (CDRs)
as set forth in SEQ ID NO: 8 and a second polypeptide comprising
CDRs as set forth in SEQ ID NO: 42. In certain embodiments, an
anti-TR-2 antibody comprises: a first polypeptide comprising
complementarity determining regions (CDRs) as set forth in SEQ ID
NO: 10 and a second polypeptide comprising CDRs as set forth in SEQ
ID NO: 44. In certain embodiments, an anti-TR-2 antibody comprises:
a first polypeptide comprising complementarity determining regions
(CDRs) as set forth in SEQ ID NO: 12 and a second polypeptide
comprising CDRs as set forth in SEQ ID NO: 46. In certain
embodiments, an anti-TR-2 antibody comprises: a first polypeptide
comprising complementarity determining regions (CDRs) as set forth
in SEQ ID NO: 14 and a second polypeptide comprising CDRs as set
forth in SEQ ID NO: 48. In certain embodiments, an anti-TR-2
antibody comprises: a first polypeptide comprising complementarity
determining regions (CDRs) as set forth in SEQ ID NO: 16 and a
second polypeptide comprising CDRs as set forth in SEQ ID NO: 50.
In certain embodiments, an anti-TR-2 antibody comprises: a first
polypeptide comprising complementarity determining regions (CDRs)
as set forth in SEQ ID NO: 18 and a second polypeptide comprising
CDRs as set forth in SEQ ID NO: 52. In certain embodiments, an
anti-TR-2 antibody comprises: a first polypeptide comprising
complementarity determining regions (CDRs) as set forth in SEQ ID
NO: 20 and a second polypeptide comprising CDRs as set forth in SEQ
ID NO: 54. In certain embodiments, an anti-TR-2 antibody comprises:
a first polypeptide comprising complementarity determining regions
(CDRs) as set forth in SEQ ID NO: 22 and a second polypeptide
comprising CDRs as set forth in SEQ ID NO: 56. In certain
embodiments, an anti-TR-2 antibody comprises: a first polypeptide
comprising complementarity determining regions (CDRs) as set forth
in SEQ ID NO: 24 and a second polypeptide comprising CDRs as set
forth in SEQ ID NO: 58. In certain embodiments, an anti-TR-2
antibody comprises: a first polypeptide comprising complementarity
determining regions (CDRs) as set forth in SEQ ID NO: 26 and a
second polypeptide comprising CDRs as set forth in SEQ ID NO: 60.
In certain embodiments, an anti-TR-2 antibody comprises: a first
polypeptide comprising complementarity determining regions (CDRs)
as set forth in SEQ ID NO: 28 and a second polypeptide comprising
CDRs as set forth in SEQ ID NO: 62. In certain embodiments, an
anti-TR-2 antibody comprises: a first polypeptide comprising
complementarity determining regions (CDRs) as set forth in SEQ ID
NO: 30 and a second polypeptide comprising CDRs as set forth in SEQ
ID NO: 64. In certain embodiments, an anti-TR-2 antibody comprises:
a first polypeptide comprising complementarity determining regions
(CDRs) as set forth in SEQ ID NO: 32 and a second polypeptide
comprising CDRs as set forth in SEQ ID NO: 66. In certain
embodiments, an anti-TR-2 antibody comprises: a first polypeptide
comprising complementarity determining regions (CDRs) as set forth
in SEQ ID NO: 34 and a second polypeptide comprising CDRs as set
forth in SEQ ID NO: 68. In certain embodiments, an anti-TR-2
antibody comprises a first polypeptide as set forth in paragraph
[079] above and a second polypeptide as set forth in paragraph
above. In certain embodiments, an anti-TR-2 antibody comprises a
first polypeptide as set forth in paragraph [080] above and a
second polypeptide as set forth in paragraph [085] above. In
certain embodiments, an anti-TR-2 antibody is a human antibody. In
certain embodiments, an anti-TR-2 antibody comprises a detectable
label. In certain embodiments, an anti-TR-2 antibody is a chimeric
antibody.
[0287] "Chimeric antibody" refers to an antibody that has an
antibody variable region of a first species fused to another
molecule, for example, an antibody constant region of another
second species. See, e.g., U.S. Pat. No. 4,816,567 and Morrison et
al., Proc Natl Acad Sci (USA), 81:6851-6855 (1985). In certain
embodiments, the first species may be different from the second
species. In certain embodiments, the first species may be the same
as the second species. In certain embodiments, chimeric antibodies
may be made through mutagenesis or CDR grafting to match a portion
of the known sequence of anti-TR-2 antibody variable regions. CDR
grafting typically involves grafting the CDRs from an antibody with
desired specificity onto the framework regions (FRs) of another
antibody.
[0288] A bivalent antibody other than a "multispecific" or
"multifunctional" antibody, in certain embodiments, typically is
understood to have each of its binding sites be identical.
[0289] An antibody substantially inhibits adhesion of a ligand to a
receptor when an excess of antibody reduces the quantity of
receptor bound to the ligand by at least about 20%, 40%, 60%, 80%,
85%, or more (as measured in an in vitro competitive binding
assay).
[0290] The term "epitope" refers to a portion of a molecule capable
of being bound by a specific binding agent. Exemplary epitopes may
comprise any polypeptide determinant capable of specific binding to
an immunoglobulin and/or T-cell receptor. Exemplary epitope
determinants include, but are not limited to, chemically active
surface groupings of molecules, for example, but not limited to,
amino acids, sugar side chains, phosphoryl groups, and sulfonyl
groups. In certain embodiments, epitope determinants may have
specific three dimensional structural characteristics, and/or
specific charge characteristics. In certain embodiments, an epitope
is a region of an antigen that is bound by an antibody. Epitopes
may be contiguous or non-contiguous. In certain embodiments,
epitopes may be mimetic in that they comprise a three dimensional
structure that is similar to an epitope used to generate the
antibody, yet comprise none or only some of the amino acid residues
found in that epitope used to generate the antibody.
[0291] The term "inhibiting and/or neutralizing epitope" refers to
an epitope, which when bound by a specific binding agent results in
a decrease in a biological activity in vivo, in vitro, and/or in
situ. In certain embodiments, a neutralizing epitope is located on
or is associated with a biologically active region of a target.
[0292] The term "activating epitope" refers to an epitope, which
when bound by a specific binding agent results in activation or
maintenance of a biological activity in vivo, in vitro, and/or in
situ. In certain embodiments, an activating epitope is located on
or is associated with a biologically active region of a target.
[0293] In certain embodiments, an epitope is specifically bound by
at least one antibody selected from Ab A, Ab B, Ab C, Ab D, Ab E,
Ab F, Ab G, Ab H, Ab I, Ab J, Ab K, Ab Ab M, Ab N, Ab O, Ab P, and
Ab Q. In certain such embodiments, the epitope is substantially
pure. In certain such embodiments, the epitope is at a
concentration of at least 1 nM. In certain such embodiments, the
epitope is at a concentration of between 1 nM and 5 nM. In certain
such embodiments, the epitope is at a concentration of between 5 nM
and 10 nM. In certain such embodiments, the epitope is at a
concentration of between 10 nM and 15 nM.
[0294] In certain embodiments, an antibody specifically binds to an
epitope that is specifically bound by at least one antibody
selected from Ab A, Ab B, Ab C, Ab D, Ab E, Ab F, Ab G, Ab H, Ab I,
Ab J, Ab k, Ab Ab M, Ab N, Ab O, Ab P, and Ab Q, and is
substantially pure. In certain such embodiments, the antibody is at
a concentration of at least 1 nM. In certain such embodiments, the
antibody is at a concentration of between 1 nM and 5 nM. In certain
such embodiments, the antibody is at a concentration of between 5
nM and 10 nM. In certain such embodiments, the antibody is at a
concentration of between 10 nM and 15 nM.
[0295] In certain embodiments, an antibody specifically binds to
amino acids 1 to 85 of mature human TR-2, and is substantially
pure. In certain such embodiments, the antibody is at a
concentration of at least 1 nM. In certain such embodiments, the
antibody is at a concentration of between 1 nM and 5 nM. In certain
such embodiments, the antibody is at a concentration of between 5
nM and 10 nM. In certain such embodiments, the antibody is at a
concentration of between 10 nM and 15 nM.
[0296] In certain embodiments, an antibody competes for binding to
an epitope with at least one antibody selected from Ab A, Ab B, Ab
C, Ab D, Ab E, Ab F, Ab G, Ab H, Ab I, Ab J, Ab K, Ab L, Ab M, Ab
N, Ab O, Ab P, and Ab Q. In certain such embodiments, the antibody
is substantially pure. In certain such embodiments, the antibody is
at a concentration of at least 1 nM. In certain such embodiments,
the antibody is at a concentration of between 1 nM and 5 nM. In
certain such embodiments, the antibody is at a concentration of
between 5 nM and 10 nM. In certain such embodiments, the antibody
is at a concentration of between 10 nM and 15 nM.
[0297] In certain embodiments, an antibody competes for binding to
amino acids 1 to 85 of mature human TR-2 with at least one antibody
selected from Ab A, Ab B, Ab C, Ab D, Ab E, Ab F, Ab G, Ab H, Ab I,
Ab J, Ab K, Ab L, Ab M, Ab N, Ab O, Ab P, and Ab Q. In certain such
embodiments, the antibody is substantially pure. In certain such
embodiments, the antibody is at a concentration of at least 1 nM.
In certain such embodiments, the antibody is at a concentration of
between 1 nM and 5 nM. In certain such embodiments, the antibody is
at a concentration of between 5 nM and 10 nM. In certain such
embodiments, the antibody is at a concentration of between 10 nM
and 15 nM.
[0298] The term "agent" is used herein to denote a chemical
compound, a mixture of chemical compounds, a biological
macromolecule, or an extract made from biological materials.
[0299] As used herein, the term "label" refers to any molecule that
can be detected. In a certain embodiment, an antibody may be
labeled by incorporation of a radiolabeled amino acid. In a certain
embodiment, biotin moieties that can be detected by marked avidin
(e.g., streptavidin containing a fluorescent marker or enzymatic
activity that can be detected by optical or colorimetric methods)
may be attached to the antibody. In certain embodiments, a label
may be incorporated into or attached to another reagent which in
turn binds to the antibody of interest. In certain embodiments, a
label may be incorporated into or attached to an antibody that in
turn specifically binds the antibody of interest. In certain
embodiments, the label or marker can also be therapeutic. Various
methods of labeling polypeptides and glycoproteins are known in the
art and may be used. Certain general classes of labels include, but
are not limited to, enzymatic, fluorescent, chemiluminescent, and
radioactive labels. Certain examples of labels for polypeptides
include, but are not limited to, the following: radioisotopes or
radionuclides (e.g., .sup.3H, .sup.14C, .sup.15N, .sup.35S,
.sup.90Y, .sup.99Tc, .sup.111In, .sup.125I, .sup.131I), fluorescent
labels (e.g., fluorescein isothocyanate (FITC), rhodamine,
lanthanide phosphors, phycoerythrin (PE)), enzymatic labels (e.g.,
horseradish peroxidase, .beta.-galactosidase, luciferase, alkaline
phosphatase, glucose oxidase, glucose-6-phosphate dehydrogenase,
alcohol dehydrogenase, malate dehydrogenase, penicillinase,
luciferase), chemiluminescent labels, biotinyl groups, and
predetermined polypeptide epitopes recognized by a secondary
reporter (e.g., leucine zipper pair sequences, binding sites for
secondary antibodies, metal binding domains, epitope tags). In
certain embodiments, labels are attached by spacer arms of various
lengths to reduce potential steric hindrance.
[0300] The term "sample", as used herein, includes, but is not
limited to, any quantity of a substance from a living thing or
formerly living thing. Such living things include, but are not
limited to, humans, mice, monkeys, rats, rabbits, and other
animals. Such substances include, but are not limited to, blood,
serum, urine, cells, organs, tissues, bone, bone marrow, lymph
nodes, and skin.
[0301] The term "pharmaceutical agent or drug" as used herein
refers to a chemical compound or composition capable of inducing a
desired therapeutic effect when properly administered to a
patient.
[0302] The term "modulator," as used herein, is a compound that
changes or alters the activity or function of a molecule. For
example, a modulator may cause an increase or decrease in the
magnitude of a certain activity or function of a molecule compared
to the magnitude of the activity or function observed in the
absence of the modulator. In certain embodiments, a modulator is an
inhibitor, which decreases the magnitude of at least one activity
or function of a molecule. Certain exemplary activities and
functions of a molecule include, but are not limited to, binding
affinity, enzymatic activity, and signal transduction. Certain
exemplary inhibitors include, but are not limited to, proteins,
peptides, antibodies, peptibodies, carbohydrates, and small organic
molecules. Exemplary peptibodies are described, e.g., in WO
01/83525.
[0303] As used herein, "substantially pure" means an object species
is the predominant species present (i.e., on a molar basis it is
more abundant than any other individual species in the
composition). In certain embodiments, a substantially purified
fraction is a composition wherein the object species comprises at
least about 50 percent (on a molar basis) of all macromolecular
species present. In certain embodiments, a substantially pure
composition will comprise more than about 80%, 85%, 90%, 95%, or
99% of all macromolar species present in the composition. In
certain embodiments, the object species is purified to essential
homogeneity (contaminant species cannot be detected in the
composition by conventional detection methods) wherein the
composition consists essentially of a single macromolecular
species.
[0304] The term "patient" includes human and animal subjects.
[0305] According to certain embodiments, a cell line expressing
anti-TR-2 antibodies is provided.
[0306] In certain embodiments, chimeric antibodies that comprise at
least a portion of a human sequence and another species' sequence
are provided. In certain embodiments, such a chimeric antibody may
result in a reduced immune response in a host than an antibody
without that host's antibody sequences. For example, in certain
instances, an animal of interest may be used as a model for a
particular human disease. To study the effect of an antibody on
that disease in the animal host, one could use an antibody from a
different species. But, in certain instances, such antibodies from
another species, may elicit an immune response to the antibodies
themselves in the host animal, thus impeding evaluation of these
antibodies. In certain embodiments, replacing part of the amino
acid sequence of an anti-TR-2 antibody with antibody amino acid
sequence from the host animal may decrease the magnitude of the
host animal's anti-antibody response.
[0307] In certain embodiments, a chimeric antibody comprises a
heavy chain and a light chain, wherein the variable regions of the
light chain and the heavy chain are from a first species and the
constant regions of the light chain and the heavy chain are from a
second species. In certain embodiments, the antibody heavy chain
constant region is an antibody heavy chain constant region of a
species other than human. In certain embodiments, the antibody
light chain constant region is an antibody light chain constant
region of a species other than human. In certain embodiments, the
antibody heavy chain constant region is a human antibody heavy
chain constant region, and the antibody heavy chain variable region
is an antibody heavy chain variable region of a species other than
human. In certain embodiments, the antibody light chain constant
region is a human antibody light chain constant region, and the
antibody light chain variable region is an antibody light chain
variable region of a species other than human. Exemplary antibody
constant regions include, but are not limited to, a human antibody
constant region, a cynomolgus monkey antibody constant region, a
mouse antibody constant region, and a rabbit antibody constant
region. Exemplary antibody variable regions include, but are not
limited to, a human antibody variable region, a mouse antibody
variable region, a pig antibody variable region, a guinea pig
antibody variable region, a cynomolgus monkey antibody variable
region, and a rabbit antibody variable region. In certain
embodiments, the framework regions of the variable region in the
heavy chain and light chain may be replaced with framework regions
derived from other antibody sequences.
[0308] Certain exemplary chimeric antibodies may be produced by
methods well known to those of ordinary skill in the art. In
certain embodiments, the polynucleotide of the first species
encoding the heavy chain variable region and the polynucleotide of
the second species encoding the heavy chain constant region can be
fused. In certain embodiments, the polynucleotide of the first
species encoding the light chain variable region and the nucleotide
sequence of the second species encoding the light chain constant
region can be fused. In certain embodiments, these fused nucleotide
sequences can be introduced into a cell either in a single
expression vector (e.g., a plasmid) or in multiple expression
vectors. In certain embodiments, a cell comprising at least one
expression vector may be used to make polypeptide. In certain
embodiments, these fused nucleotide sequences can be introduced
into a cell either in separate expression vectors or in a single
expression vector. In certain embodiments, the host cell expresses
both the heavy chain and the light chain, which combine to produce
an antibody. In certain embodiments, a cell comprising at least one
expression vector may be used to make an antibody. Exemplary
methods for producing and expressing antibodies are discussed
below.
[0309] In certain embodiments, conservative modifications to the
heavy and light chains of an anti-TR-2 antibody (and corresponding
modifications to the encoding nucleotides) will produce antibodies
having functional and chemical characteristics similar to those of
the original antibody. In contrast, in certain embodiments,
substantial modifications in the functional and/or chemical
characteristics of an anti-TR-2 antibody may be accomplished by
selecting substitutions in the amino acid sequence of the heavy and
light chains that differ significantly in their effect on
maintaining (a) the structure of the molecular backbone in the area
of the substitution, for example, as a sheet or helical
conformation, (b) the charge or hydrophobicity of the molecule at
the target site, or (c) the bulk of the side chain.
[0310] Certain desired amino acid substitutions (whether
conservative or non-conservative) can be determined by those
skilled in the art at the time such substitutions are desired. In
certain embodiments, amino acid substitutions can be used to
identify important residues of the anti-TR-2 antibodies, such as
those which may increase or decrease the affinity of the antibodies
to TR-2 or the effector function of the antibodies.
[0311] In certain embodiments, the effects of an anti-TR-2 antibody
may be evaluated by measuring a reduction in the amount of symptoms
of the disease. In certain embodiments, the disease of interest may
be caused by a pathogen. In certain embodiments, a disease may be
established in an animal host by other methods including
introduction of a substance (such as a carcinogen) and genetic
manipulation. In certain embodiments, effects may be evaluated by
detecting one or more adverse events in the animal host. The term
"adverse event" includes, but is not limited to, an adverse
reaction in an animal host that receives an antibody that is not
present in an animal host that does not receive the antibody. In
certain embodiments, adverse events include, but are not limited
to, a fever, an immune response to an antibody, inflammation,
and/or death of the animal host.
[0312] Various antibodies specific to an antigen may be produced in
a number of ways. In certain embodiments, an antigen containing an
epitope of interest may be introduced into an animal host (e.g., a
mouse), thus producing antibodies specific to that epitope. In
certain instances, antibodies specific to an epitope of interest
may be obtained from biological samples taken from hosts that were
naturally exposed to the epitope. In certain instances,
introduction of human immunoglobulin (Ig) loci into mice in which
the endogenous Ig genes have been inactivated offers the
opportunity to obtain human monoclonal antibodies (MAbs).
Naturally Occurring Antibody Structure
[0313] Naturally occurring antibody structural units typically
comprise a tetramer. Each such tetramer typically is composed of
two identical pairs of polypeptide chains, each pair having one
full-length "light" chain (in certain embodiments, about 25 kDa)
and one full-length "heavy" chain (in certain embodiments, about
50-70 kDa). The term "heavy chain" includes any polypeptide having
sufficient variable region sequence to confer specificity for a
particular antigen. A full-length heavy chain includes a variable
region domain, V.sub.H, and three constant region domains,
C.sub.H1, C.sub.H2, and C.sub.H3. The V.sub.H domain is at the
amino-terminus of the polypeptide, and the C.sub.H3 domain is at
the carboxy-terminus. The term "heavy chain", as used herein,
encompasses a full-length antibody heavy chain and fragments
thereof.
[0314] The term "light chain" includes any polypeptide having
sufficient variable region sequence to confer specificity for a
particular antigen. A full-length light chain includes a variable
region domain, V.sub.L, and a constant region domain, C.sub.L. Like
the heavy chain, the variable region domain of the light chain is
at the amino-terminus of the polypeptide. The term "light chain",
as used herein, encompasses a full-length light chain and fragments
thereof.
[0315] The amino-terminal portion of each chain typically includes
a variable region (V.sub.H in the heavy chain and V.sub.L in the
light chain) of about 100 to 110 or more amino acids that typically
is responsible for antigen recognition. The carboxy-terminal
portion of each chain typically defines a constant region (C.sub.H
domains in the heavy chain and C.sub.L in the light chain) that may
be responsible for effector function. Antibody effector functions
include activation of complement and stimulation of
opsonophagocytosis. Human light chains are typically classified as
kappa and lambda light chains. Heavy chains are typically
classified as mu, delta, gamma, alpha, or epsilon, and define the
antibody's isotype as 10/1, IgD, IgG, IgA, and IgE, respectively.
IgG has several subclasses, including, but not limited to, IgG1,
IgG2, IgG3, and IgG4. IgM has subclasses including, but not limited
to, IgM1 and IgM2. IgA is similarly subdivided into subclasses
including, but not limited to, IgA1 and IgA2. Within full-length
light and heavy chains, typically, the variable and constant
regions are joined by a "J" region of about 12 or more amino acids,
with the heavy chain also including a "D" region of about 10 more
amino acids. See, e.g., Fundamental Immunology Ch. 7 (Paul, W.,
ed., 2nd ed. Raven Press, N.Y. (1989)). The variable regions of
each light/heavy chain pair typically form the antigen binding
site.
[0316] The variable regions typically exhibit the same general
structure of relatively conserved framework regions (FR) joined by
three hypervariable regions, also called complementarity
determining regions or CDRs. The CDRs from the heavy and light
chains of each pair typically are aligned by the framework regions,
which may enable binding to a specific epitope. From N-terminal to
C-terminal, both light and heavy chain variable regions typically
comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The
assignment of amino acids to each domain is typically in accordance
with the definitions of Kabat Sequences of Proteins of
Immunological Interest (National Institutes of Health, Bethesda,
Md. (1987 and 1991)), or Chothia & desk J. Mol. Biol.
196:901-917 (1987); Chothia et al. Nature 342:878-883 (1989).
[0317] As discussed above, there are several types of antibody
fragments. A Fab fragment is comprised of one light chain and the
C.sub.H1 and variable regions of one heavy chain. The heavy chain
of a Fab molecule cannot form a disulfide bond with another heavy
chain molecule. A Fab' fragment contains one light chain and one
heavy chain that contains more of the constant region, between the
C.sub.H1 and C.sub.H2 domains, such that an interchain disulfide
bond can be formed between two heavy chains to form a F(ab')2
molecule. A Fab fragment is similar to a F(ab')2 molecule, except
the constant region in the heavy chains of the molecule extends to
the end of the C.sub.H2 domain. The Fv region comprises the
variable regions from both the heavy and light chains, but lacks
the constant regions. Single-chain antibodies are Fv molecules in
which the heavy and light chain variable regions have been
connected by a flexible linker to form a single polypeptide chain
which forms an antigen-binding region. Exemplary single chain
antibodies are discussed in detail, e.g., in WO 88/01649 and U.S.
Pat. Nos. 4,946,778 and 5,260,203. A Fc fragment contains the
C.sub.H2 and C.sub.H3 domains of the heavy chain and contains more
of the constant region, between the C.sub.H1 and C.sub.H2 domains,
such that an interchain disulfide bond can be formed between two
heavy chains.
[0318] In certain embodiments, functional domains, C.sub.H1,
C.sub.H2, C.sub.H3, and intervening sequences can be shuffled to
create a different antibody constant region. For example, in
certain embodiments, such hybrid constant regions can be optimized
for half-life in serum, for assembly and folding of the antibody
tetramer, and/or for improved effector function. In certain
embodiments, modified antibody constant regions may be produced by
introducing single point mutations into the amino acid sequence of
the constant region and testing the resulting antibody for improved
qualities, e.g., one or more of those listed above.
[0319] In certain embodiments, an antibody of one isotype is
converted to a different isotype by isotype switching without
losing its specificity for a particular target molecule. Methods of
isotype switching include, but are not limited to, direct
recombinant techniques (see e.g., U.S. Pat. No. 4,816,397) and
cell-cell fusion techniques (see e.g., U.S. Pat. No. 5,916,771),
among others. In certain embodiments, an antibody can be converted
from one subclass to another subclass using techniques described
above or otherwise known in the art without losing its specificity
for a particular target molecule, including, but not limited to,
conversion from an IgG2 subclass to an IgG1, IgG3, or IgG4
subclass.
Bispecific or Bifunctional Antibodies
[0320] A bispecific or bifunctional antibody typically is an
artificial hybrid antibody having two different heavy/light chain
pairs and two different binding sites. Bispecific antibodies may be
produced by a variety of methods including, but not limited to,
fusion of hybridomas or linking of Fab' fragments. See, e.g.,
Songsivilai & Lachmann Clin. Exp. Immunol. 79: 315-321 (1990),
Kostelny et al. J. Immunol. 148:1547-1553 (1992).
Certain Preparation of Antibodies
[0321] In certain embodiments, antibodies can be expressed in cell
lines other than hybridoma cell lines. In certain embodiments,
sequences encoding particular antibodies, including chimeric
antibodies, can be used for transformation of a suitable mammalian
host cell. According to certain embodiments, transformation can be
by any known method for introducing polynucleotides into a host
cell, including, for example packaging the polynucleotide in a
virus (or into a viral vector) and transducing a host cell with the
virus or by transfecting a vector using procedures known in the
art, as exemplified by U.S. Pat. Nos. 4,399,216; 4,912,040;
4,740,461; and 4,959,455.
[0322] In certain embodiments, an expression vector comprises any
of the polynucleotide sequences discussed herein. In certain
embodiments, a method of making a polypeptide comprising producing
the polypeptide in a cell comprising any of the above expression
vectors in conditions suitable to express the polynucleotide
contained therein to produce the polypeptide is provided.
[0323] In certain embodiments, an expression vector comprises a
polynucleotide comprising a sequence encoding a polypeptide
comprising at least one complementarity determining region (CDR)
selected from CDR1a, CDR2a, and CDR3a, wherein CDR1a comprises the
amino acid sequence a b c d e f g h i j k l, wherein amino acid a
is glycine, amino acid b is selected from glycine, tyrosine, or
phenylalanine; amino acid c is selected from serine or threonine;
amino acid d is selected from isoleucine or phenylalanine; amino
acid e is selected from serine, threonine, or asparagine; amino
acid f is selected from serine, aspartic acid, tyrosine,
asparagine, threonine, or glycine; amino acid g is selected from
glycine, aspartic acid, or tyrosine; amino acid h is selected from
glycine, aspartic acid, tyrosine, asparagine, or serine; amino acid
i is selected from tyrosine, isoleucine, histidine, methionine, or
tryptophan; amino acid j is selected from asparagine, tyrosine,
histidine, serine, or phenylalanine; amino acid k is tryptophan or
is not present; and amino acid l is serine or is not present;
wherein CDR2a comprises the amino acid sequence m n o p q r s t u v
w x y z a' b' c', wherein amino acid m is selected from tryptophan,
tyrosine, histidine, valine, glutamic acid, or serine; amino acid n
is selected from methionine or isoleucine; amino acid o is selected
from asparagine, tyrosine, serine, tryptophan, or histidine; amino
acid p is selected from proline, tyrosine, serine, arginine,
histidine, or asparagine; amino acid q is selected from asparagine,
serine, or aspartic acid; amino acid r is selected from serine or
glycine; amino acid s is selected from aspartic acid, serine,
threonine, or arginine; amino acid t is selected from asparagine,
threonine, alanine, isoleucine, or tyrosine; amino acid u is
selected from threonine, tyrosine, leucine, lysine, asparagine, or
isoleucine; amino acid v is selected from glycine, tyrosine,
aspartic acid, or cysteine; amino acid w is selected from tyrosine
or asparagine; amino acid x is selected from alanine or proline;
amino acid y is selected from glutamine, serine, or aspartic acid;
amino acid z is selected from lysine, leucine, or serine; amino
acid a' is selected from phenylalanine, lysine, or valine; amino
acid b' is selected from glutamine, serine, or lysine; and amino
acid c' is glycine or is not present; wherein CDR3a comprises the
amino acid sequence d' e' f' g' h' j' k' m' n' o' p' q' r' s' t' u'
v' w', wherein amino acid d' is selected from tryptophan, aspartic
acid, glycine, serine, or glutamic acid; amino acid e' is selected
from asparagine, aspartic acid, glycine, arginine, serine, valine,
or leucine; amino acid f' is selected from histidine, serine,
alanine, tyrosine, proline, asparagine, glycine or threonine; amino
acid g' is selected from tyrosine, serine, alanine, arginine,
tryptophan, glycine or valine; amino acid h' is selected from
glycine, alanine, serine, asparagine, methionine, tyrosine,
tryptophan, cysteine, or aspartic acid; amino acid i' is selected
from serine, tryptophan, glycine, phenylalanine, aspartic acid,
tyrosine, or threonine; amino acid j' is selected from glycine,
threonine, serine, leucine, valine, asparagine, tryptophan, or
tyrosine; amino acid k' is selected from serine, phenylalanine,
aspartic acid, tryptophan, glycine, or tyrosine, or is not present;
amino acid l' is selected from histidine, aspartic acid, alanine,
tryptophan, tyrosine, serine, phenylalanine, valine, or glycine, or
is not present; amino acid m' is selected from phenylalanine,
tyrosine, glutamic acid, proline, aspartic acid, cysteine,
isoleucine, or methionine, or is not present; amino acid n' is
selected from aspartic acid, phenylalanine, alanine, leucine, or
serine, or is not present; amino acid o' is selected from tyrosine,
leucine, aspartic acid, phenylalanine, proline, or valine, or is
not present; amino acid p' is selected from leucine, aspartic acid,
or tyrosine, or is not present; amino acid q' is selected from
serine or tyrosine, or is not present; amino acid r' is tyrosine or
is not present; amino acid s' is selected from glycine or tyrosine,
or is not present; amino acid t' is selected from glycine or
methionine, or is not present; amino acid u' is selected from
methionine or aspartic acid, or is not present; amino acid v' is
selected from aspartic acid or valine, or is not present; and amino
acid w' is valine or is not present; and wherein the polypeptide,
in association with an antibody light chain, binds TR-2. In certain
embodiments, a method of making a polypeptide comprising producing
the polypeptide in a cell comprising the above expression vector in
conditions suitable to express the polynucleotide contained therein
to produce the polypeptide is provided.
[0324] In certain embodiments, an expression vector comprises a
polynucleotide comprising a sequence encoding a polypeptide
comprising at least one complementarity determining region (CDR)
selected from CDR1b, CDR2b, and CDR3b, wherein CDR1b comprises the
amino acid sequence a1 b1 c1 d1 e1 f1 g1 h1 i1 j1 k1 l1 m1 n1 o1 p1
q1, wherein amino acid a1 is selected from arginine or lysine;
amino acid b1 is selected from threonine, alanine, or serine; amino
acid c1 is serine; amino acid d1 is glutamine; amino acid e1 is
selected from serine or glycine; amino acid f1 is selected from
isoleucine, leucine, or valine; amino acid g1 is selected from
serine, leucine, or arginine; amino acid h1 is selected from
threonine, serine, isoleucine, asparagine, arginine, histidine, or
tyrosine; amino acid i1 is selected from tyrosine, arginine,
tryptophan, aspartic acid, or serine; j1 is selected from leucine,
isoleucine, asparagine, tyrosine, or serine; amino acid k1 is
selected from asparagine, glycine, valine, alanine, or leucine;
amino acid l1 is selected from tyrosine, alanine, or asparagine, or
is not present; amino acid m1 is selected from asparagine or
lysine, or is not present; amino acid n1 is selected from tyrosine,
asparagine, or isoleucine, or is not present; amino acid o1 is
selected from leucine or tyrosine, or is not present; amino acid p1
is selected from aspartic acid or leucine, or is not present; and
amino acid q1 is selected from valine, alanine, or threonine, or is
not present; wherein CDR2b comprises the amino acid sequence r1 s1
t1 u1 v1 w1 x1, wherein amino acid r1 is selected from alanine,
aspartic acid, leucine, tryptophan, glycine, or valine; amino acid
s1 is selected from threonine, valine, glycine, or alanine; amino
acid t1 is serine; amino acid u1 is selected from serine,
asparagine, or threonine; amino acid v1 is selected from leucine,
phenylalanine, or arginine; amino acid w1 is selected from
glutamine, alanine, or glutamic acid; and amino acid x1 is selected
from serine, arginine, or threonine; wherein CDR3b comprises the
amino acid sequence y1 z1 a1' c1' d1' e1' f1' g1', wherein amino
acid y1 is selected from glutamine, methionine, leucine, or
histidine; amino acid z1 is selected from glutamine or lysine;
amino acid a1' is selected from serine, threonine, alanine,
histidine, tyrosine, or phenylalanine; amino acid b1' is selected
from tyrosine, leucine, asparagine, or glycine; amino acid c1' is
selected from serine, glutamine, isoleucine, or lysine; amino acid
d1' is selected from threonine, phenylalanine, tyrosine, alanine,
or serine; amino acid e1' is proline; amino acid f1' is selected
from leucine, phenylalanine, tryptophan, serine, or arginine; and
amino acid g1' is selected from threonine or serine; and wherein
the polypeptide, in association with an antibody heavy chain, binds
TR-2. In certain embodiments, a method of making a polypeptide
comprising producing the polypeptide in a cell comprising the above
expression vector in conditions suitable to express the
polynucleotide contained therein to produce the polypeptide is
provided. In certain embodiments, a cell comprising at least one of
the above expression vectors is provided. In certain embodiments, a
method of making an polypeptide comprising producing the
polypeptide in a cell comprising the above expression vector in
conditions suitable to express the polynucleotide contained therein
to produce the polypeptide is provided.
[0325] In certain embodiments, an expression vector expresses an
anti-TR-2 antibody heavy chain. In certain embodiments, an
expression vector expresses an anti-TR-2 antibody light chain. In
certain embodiments, an expression vector expresses both an
anti-TR-2 antibody heavy chain and an anti-TR-2 antibody light
chain. In certain embodiments, a method of making an anti-TR-2
antibody comprising producing the antibody in a cell comprising at
least one of the expression vectors described herein in conditions
suitable to express the polynucleotides contained therein to
produce the antibody is provided.
[0326] In certain embodiments, the transfection procedure used may
depend upon the host to be transformed. Certain methods for
introduction of heterologous polynucleotides into mammalian cells
are known in the art and include, but are not limited to,
dextran-mediated transfection, calcium phosphate precipitation,
polybrene mediated transfection, protoplast fusion,
electroporation, encapsulation of the polynucleotide(s) in
liposomes, and direct microinjection of the DNA into nuclei.
[0327] Certain mammalian cell lines available as hosts for
expression are known in the art and include, but are not limited
to, many immortalized cell lines available from the American Type
Culture Collection (ATCC), including but not limited to Chinese
hamster ovary (CHO) cells, E5 cells, HeLa cells, baby hamster
kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular
carcinoma cells (e.g., Hep G2), NS0 cells, SP20 cells, Per C6
cells, 293 cells, and a number of other cell lines. In certain
embodiments, cell lines may be selected through determining which
cell lines have high expression levels and produce antibodies with
constitutive antigen binding properties.
[0328] In certain embodiments, the vectors that may be transfected
into a host cell comprise control sequences that are operably
linked to a polynucleotide encoding an anti-TR-2 antibody. In
certain embodiments, control sequences facilitate expression of the
linked polynucleotide, thus resulting in the production of the
polypeptide encoded by the linked polynucleotide. In certain
embodiments, the vector also comprises polynucleotide sequences
that allow chromosome-independent replication in the host cell.
Exemplary vectors include, but are not limited to, plasmids (e.g.,
BlueScript, puc, etc.), cosmids, and YACS.
Certain Antibody Uses
[0329] According to certain embodiments, antibodies are useful for
detecting a particular antigen in a sample. In certain embodiments,
this allows the identification of cells or tissues which produce
the protein. For example, in certain embodiments, anti-TR-2
antibodies may be used to detect the presence of TR-2 in a sample.
In certain embodiments, a method for detecting the presence or
absence of TR-2 in a sample comprises (a) combining an anti-TR-2
antibody and the sample; (b) separating antibodies bound to an
antigen from unbound antibodies; and (c) detecting the presence or
absence of antibodies bound to the antigen.
[0330] Assays in which an antibody may be used to detect the
presence or absence of an antigen include, but are not limited to,
an ELISA and a western blot. In certain embodiments, an anti-TR-2
antibody may be labeled. In certain embodiments, an anti-TR-2
antibody may be detected by a labeled antibody that binds to the
anti-TR-2 antibody. In certain embodiments, a kit for detecting the
presence or absence of TR-2 in a sample is provided. In certain
embodiments, the kit comprises an anti-TR-2 antibody and reagents
for detecting the antibody.
[0331] In certain embodiments, antibodies may be used to
substantially isolate a chemical moiety such as, but not limited
to, a protein. In certain embodiments, the antibody is attached to
a "substrate," which is a supporting material used for immobilizing
the antibody. Substrates include, but are not limited to, tubes,
plates (i.e., multi-well plates), beads such as microbeads,
filters, balls, and membranes. In certain embodiments, a substrate
can be made of water-insoluble materials such as, but not limited
to, polycarbonate resin, silicone resin, or nylon resin. Exemplary
substrates for use in affinity chromatography include, but are not
limited to, cellulose, agarose, polyacrylamide, dextran,
polystyrene, polyvinyl alcohol, and porous silica. There are many
commercially available chromatography substrates that include, but
are not limited to, Sepharose 2B, Sepharose 4B, Sepharose 6B and
other forms of Sepharose (Pharmacia); Bio-Gel (and various forms of
Bio-Gel such as Biogel A, P, or CM), Cellex (and various forms of
Cellex such as Cellex AE or Cellex-CM), Chromagel A, Chromagel P
and Enzafix (Wako Chemical Indus.). The use of antibody affinity
columns is known to a person of ordinary skill in the art. In
certain embodiments, a method for isolating TR-2 comprises (a)
attaching a TR-2 antibody to a substrate; (b) exposing a sample
containing TR-2 to the antibody of part (a); and (c) isolating
TR-2. In certain embodiments, a kit for isolating TR-2 is provided.
In certain embodiments, the kit comprises an anti-TR-2 antibody
attached to a substrate and reagents for isolating TR-2.
[0332] The term "affinity chromatography" as used herein means a
method of separating or purifying the materials of interest in a
sample by utilizing the interaction (e.g., the affinity) between a
pair of materials, such as an antigen and an antibody, an enzyme
and a substrate, or a receptor and a ligand.
[0333] In certain embodiments, antibodies which bind to a
particular protein and block interaction with other binding
compounds may have therapeutic use. In this application, when
discussing the use of anti-TR-2 antibodies to treat diseases or
conditions, such use may include use of the anti-TR-2 antibodies
themselves; compositions comprising anti-TR-2 antibodies; and/or
combination therapies comprising anti-TR-2 antibodies and one or
more additional active ingredients. When anti-TR-2 antibodies are
used to "treat" a disease or condition, such treatment may or may
not include prevention of the disease or condition. In certain
embodiments, anti-TR-2 antibodies can block the interaction of the
TR-2 receptor with its ligand, TRAIL. In certain embodiments,
anti-TR-2 antibodies can activate the TR-2 receptor. In certain
embodiments, anti-TR-2 antibodies can constitutively activate the
TR-2 receptor. Because TR-2 is associated with apoptosis, in
certain embodiments, anti-TR-2 antibodies may have therapeutic use
in treating diseases in which cell death or prevention of cell
death is desired. Such diseases include, but are not limited to,
cancer associated with any tissue expressing TR-2, inflammation,
and viral infections.
[0334] In certain embodiments, an anti-TR-2 antibody is
administered alone. In certain embodiments, an anti-TR-2 antibody
is administered prior to the administration of at least one other
therapeutic agent. In certain embodiments, an anti-TR-2 antibody is
administered concurrent with the administration of at least one
other therapeutic agent. In certain embodiments, an anti-TR-2
antibody is administered subsequent to the administration of at
least one other therapeutic agent. Exemplary therapeutic agents,
include, but are not limited to, at least one other cancer therapy
agent. Exemplary cancer therapy agents include, but are not limited
to, radiation therapy and chemotherapy.
[0335] In certain embodiments, anti-TR-2 antibody pharmaceutical
compositions can be administered in combination therapy, i.e.,
combined with other agents. In certain embodiments, the combination
therapy comprises an anti-TR-2 antibody, in combination with at
least one anti-angiogenic agent. Exemplary agents include, but are
not limited to, in vitro synthetically prepared chemical
compositions, antibodies, antigen binding regions, radionuclides,
and combinations and conjugates thereof. In certain embodiments, an
agent may act as an agonist, antagonist, alllosteric modulator, or
toxin. In certain embodiments, an agent may act to inhibit or
stimulate its target (e.g., receptor or enzyme activation or
inhibition), and thereby promote cell death or arrest cell
growth.
[0336] Exemplary chemotherapy treatments include, but are not
limited to anti-neoplastic agents including, but not limited to,
alkylating agents including, but not limited to: nitrogen mustards,
including, but not limited to, mechlorethamine, cyclophosphamide,
ifosfamide, melphalan and chlorambucil; nitrosoureas, including,
but not limited to, carmustine BCNU, lomustine, CCNU, and
semustine, methyl-CCNU; Temodal.TM., temozolamide;
ethylenimines/methylmelamine, including, but not limited to,
thriethylenemelamine (TEM), triethylene, thiophosphoramide,
thiotepa, hexamethylmelamine (HMM), and altretamine; alkyl
sulfonates, including, but not limited to, busulfan; triazines,
including, but not limited to, dacarbazine (DTIC); antimetabolites,
including, but not limited to, folic acid analogs such as
methotrexate and trimetrexate; pyrimidine analogs, including, but
not limited to, 5-fluorouracil (5FU), fluorodeoxyuridine,
gemcitabine, cytosine arabinoside (AraC, cytarabine),
5-azacytidine, and 2,2''-difluorodeoxycytidine, purine analogs,
including, but not limited to, 6-mercaptopurine, 6-thioguanine,
azathioprine, 2'-deoxycoformycin (pentostatin),
erythrohydroxynonyladenine (EHNA), fludarabine phosphate,
cladribine, and 2-chlorodeoxyadenosine (2-CdA); natural products,
including, but not limited to, antimitotic drugs such as
paclitaxel; vinca alkaloids, including, but not limited to,
vinblastine (VLB), vincristine, and vinorelbine; taxotere;
estramustine and estramustine phosphate; ppipodophylotoxins,
including, but not limited to, etoposide and teniposide;
antibiotics, including, but not limited to, actinomycin D,
daunomycin, rubidomycin, doxorubicin, mitoxantrone, idarubicin,
bleomycins, plicamycin, mithramycin, mitomycin C, and actinomycin;
enzymes, including, but not limited to, L-asparaginase; biological
response modifiers, including, but not limited to,
interferon-alpha, IL-2, G-CSF, and GM-CSF; doxycyckine; irinotecan
hydrochloride; miscellaneous agents, including, but not limited to,
platinium coordination complexes such as cisplatin and carboplatin;
anthracenediones, including, but not limited to, mitoxantrone;
substituted urea, including, but not limited to, hydroxyurea;
methylhydrazine derivatives, including, but not limited to,
N-methylhydrazine (MIH) and procarbazine; adrenocortical
suppressants, including, but not limited to, mitotane (o,p'-DDD)
and aminoglutethimide; hormones and antagonists, including, but not
limited to, adrenocorticosteroid antagonists such as prednisone and
equivalents, dexamethasone and aminoglutethimide; Gemzar.TM.,
gemcitabine; progestin, including, but not limited to,
hydroxyprogesterone caproate, medroxyprogesterone acetate and
megestrol acetate; estrogen, including, but not limited to,
diethylstilbestrol and ethinyl estradiol equivalents; antiestrogen,
including, but not limited to, tamoxifen; androgens, including, but
not limited to, testosterone propionate and
fluoxymesterone/equivalents; antiandrogens, including, but not
limited to, flutamide, gonadotropin-releasing hormone analogs and
leuprolide; and non-steroidal antiandrogens, including, but not
limited to, flutamide.
[0337] Exemplary cancer therapies, which may be administered with
an anti-TR-2 antibody, include, but are not limited to, targeted
therapies. Examples of targeted therapies include, but are not
limited to, use of therapeutic antibodies. Exemplary therapeutic
antibodies, include, but are not limited to, mouse, mouse-human
chimeric, CDR-grafted, humanized, and human antibodies, and
synthetic antibodies, including, but not limited to, those selected
by screening antibody libraries. Exemplary antibodies include, but
are not limited to, those which bind to cell surface proteins Her2,
CDC20, CDC33, mucin-like glycoprotein, and epidermal growth factor
receptor (EGFr) present on tumor cells, and optionally induce a
cytostatic and/or cytotoxic effect on tumor cells displaying these
proteins. Exemplary antibodies also include, but are not limited
to, HERCEPTIN.TM., trastuzumab, which may be used to treat breast
cancer and other forms of cancer; RITUXAN.TM., rituximab,
ZEVALIN.TM., ibritumomab tiuxetan, and LYMPHOCIDE.TM., epratuzumab,
which may be used to treat non-Hodgkin's lymphoma and other forms
of cancer; GLEEVEC.TM., imatinib mesylate, which may be used to
treat chronic myeloid leukemia and gastrointestinal stromal tumors;
and BEXXAR.TM., iodine 131 tositumomab, which may be used for
treatment of non-Hodgkin's lymphoma. Certain exemplary antibodies
also include ERBITUX.TM.; IMC-C.sup.225; Iressa.TM.; gefitinib;
TARCEVA.TM., ertinolib; KDR (kinase domain receptor) inhibitors;
anti VEGF antibodies and antagonists (e.g., Avastin.TM. and
VEGAF-TRAP); anti VEGF receptor antibodies and antigen binding
regions; anti-Ang-1 and Ang-2 antibodies and antigen binding
regions; antibodies to Tie-2 and other Ang-1 and Ang-2 receptors;
Tie-2 ligands; antibodies against Tie-2 kinase inhibitors; and
Campath.RTM., alemtuzumab. In certain embodiments, cancer therapy
agents are other polypeptides which selectively induce apoptosis in
tumor cells, including, but not limited to, TNF-related
polypeptides such as TRAIL.
[0338] In certain embodiments, specific binding agents (including,
but not limited to, anti-IGF-R1 antibodies) that antagonize the
binding of the ligands IGF-1 and/or IGF-2 to insulin-like growth
factor-1 receptor ("IGF-1R") and promote apoptosis of cells
expressing IGF-1R are formulated or administered in combination
with specific binding agents (including, but not limited to, TRAIL
and anti-TR2 antibodies) that agonize and thereby promote apoptosis
of cells expressing TRAIL-R2. Exemplary anti-IGF-1R antibodies are
known in the art and are disclosed, for example, in WO 2006/069202,
filed Dec. 20, 2005, which is incorporated by reference herein for
any purpose.
[0339] In certain embodiments, cancer therapy agents are
anti-angiogenic agents which decrease angiogenesis. Certain such
agents include, but are not limited to, ERBITUX.TM., IMC-C225; KDR
(kinase domain receptor) inhibitory agents (e.g., antibodies and
antigen binding regions that specifically bind to the kinase domain
receptor); anti-VEGF agents (e.g., antibodies or antigen binding
regions that specifically bind VEGF, or soluble VEGF receptors or a
ligand binding region thereof) such as AVASTIN.TM. or
VEGF-TRAP.TM.; anti-VEGF receptor agents (e.g., antibodies or
antigen binding regions that specifically bind thereto); EGFR
inhibitory agents (e.g., antibodies or antigen binding regions that
specifically bind thereto) such as ABX-EGF, panitumumab,
IRESSA.TM., gefitinib, TARCEVA.TM., erlotinib, anti-Ang1 and
anti-Ang2 agents (e.g., antibodies or antigen binding regions
specifically binding thereto or to their receptors, e.g.,
Tie2/Tek); and anti-Tie-2 kinase inhibitory agents (e.g.,
antibodies or antigen binding regions that specifically bind
thereto). In certain embodiments, the pharmaceutical compositions
may also include one or more agents (e.g., antibodies, antigen
binding regions, or soluble receptors) that specifically bind and
inhibit the activity of growth factors, such as antagonists of
hepatocyte growth factor (HGF, also known as Scatter Factor), and
antibodies or antigen binding regions that specifically bind its
receptor "c-met."
[0340] Exemplary anti-angiogenic agents include, but are not
limited to, Campath, IL-8, B-FGF, Tek antagonists (Ceretti et al.,
U.S. Patent Application Publication No. 2003/0162712; U.S. Pat. No.
6,413,932); anti-TWEAK agents (e.g., specifically binding
antibodies or antigen binding regions, or soluble TWEAK receptor
antagonists; see, e.g., Wiley, U.S. Pat. No. 6,727,225); ADAM
disintegrin domain to antagonize the binding of integrin to its
ligands (Fanslow et al., U.S. Patent Application Publication No.
2002/0042368); specifically binding anti-eph receptor and/or
anti-ephrin antibodies or antigen binding regions (U.S. Pat. Nos.
5,981,245; 5,728,813; 5,969,110; 6,596,852; 6,232,447; 6,057,124;
and patent family members thereof); anti-PDGF-BB antagonists (e.g.,
specifically binding antibodies or antigen binding regions) as well
as antibodies or antigen binding regions specifically binding to
PDGF-BB ligands, and PDGFR kinase inhibitory agents (e.g.,
antibodies or antigen binding regions that specifically bind
thereto).
[0341] Exemplary anti-angiogenic/anti-tumor agents include, but are
not limited to, SF-7784 (Pfizer, USA); cilengitide (Merck KgaA,
Germany, EPO 770622); pegaptanib octasodium (Gilead Sciences, USA);
Alphastatin (BioActa, UK); M-PGA (Celgene, USA, U.S. Pat. No.
5,712,291); ilomastat (Arriva, USA, U.S. Pat. No. 5,892,112);
emaxanib (Pfizer, USA, U.S. Pat. No. 5,792,783); vatalanib
(Novartis, Switzerland); 2-methoxyestradiol (EntreMed, USA); TLC
ELL-12 (Elan, Ireland); anecortave acetate (Alcon, USA); alpha-D148
Mab (Amgen, USA); CEP-7055 (Cephalon, USA); anti-Vn Mab (Crucell,
Netherlands); DAC:antiangiogenic (ConjuChem, Canada); Angiocidin
(InKine Pharmaceutical, USA); KM-2550 (Kyowa Hakko, Japan); SU-0879
(Pfizer, USA); CGP-79787 (Novartis, Switzerland, EP 970070); ARGENT
technology (Ariad, USA); YIGSR-Strealth (Johnson & Johnson,
USA); fibrinogen-E fragment (BioActa, UK); angiogenesis inhibitor
(Trigen, UK); TBC-1635 (Encysive Pharmaceuticals, USA); SC-236
(Pfizer, USA); ABT-567 (Abbott, USA); Metastatin (EntreMed, USA);
angiogenesis inhibitor (Tripep, Sweden); maspin (Sosei, Japan);
2-methoxyestradiol (Oncology Sciences Corporation, USA);
ER-68203-00 (IVAX, USA); Benefin (Lane Labs, USA); Tz-93 (Tsumura,
Japan); TAN-1120 (Takeda, Japan); FR-111142 (Fujisawa, Japan, JP
02233610); platelet factor 4 (RepliGen, USA, EP 407122); vascular
endothelial growth factor antagonist (Borean, Denmark);
temsirolimus (CCI-779) (University of South Carolina, USA);
bevacizumab (pINN) (Genentech, USA); angiogenesis inhibitors
(SUGEN, USA); XL 784 (Exelixis, USA); XL 647 (Exelixis, USA); Mab,
alpha5beta3 integrin, Vitaxin and second generation Vitaxin
(Applied Molecular Evolution, USA and MedImmune USA);
Retinostat.RTM. gene therapy (Oxford BioMedica, UK); enzastaurin
hydrochloride (USAN) (Lilly, USA); CEP 7055 (Cephalon, USA and
Sanofi-Synthelabo, France); BC 1 (Genoa Institute of Cancer
Research, Italy); angiogenesis inhibitor (Alchemia, Australia);
VEGF antagonist (Regeneron, USA); rBPI 21 and BPI-derived
antiangiogenic (XOMA, USA); PI 88 (Progen, Australia); cilengitide
(pINN) (Merck KgaA, Germany; Munich Technical University, Germany;
Scripps Clinic and Research Foundation, USA); cetuximab (INN)
(Aventis, France); AVE 8062 (Ajinomoto, Japan); AS 1404 (Cancer
Research Laboratory, New Zealand); SG 292 (Telios, USA); Endostatin
(Boston Children's Hospital, USA); 2-methoxyestradiol (Boston
Childrens Hospital, USA); ZD 6474 (AstraZeneca, UK); ZD 6126
(Angiogene Pharmaceuticals, UK); PPI 2458 (Praecis, USA); AZD 9935
(AstraZeneca, UK); AZD 2171 (AstraZeneca, UK); vatalanib (pINN)
(Novartis, Switzerland and Schering AG, Germany); tissue factor
pathway inhibitors (EntraMed, USA); pegaptanib (Pinn) (Gilead
Sciences, USA); xanthorrhizol (Yonsei University, South Korea);
vaccine, gene-based, VEGF-2 (Scripps Clinic and Research
Foundation, USA); SPV5.2 (Supratek, Canada); SDX 103 (University of
California at San Diego, USA); PX 478 (Pro1X, USA); Metastatin
(EntreMed, USA); troponin I (Harvard University, USA); SU 6668
(SUGEN, USA); OXI 4503 (OXiGENE, USA); o-guanidines (Dimensional
Pharmaceuticals, USA); motuporamine C (British Columbia University,
Canada); CDP 791 (Celltech Group, UK); atiprimod (pINN)
(GlaxoSmithKline, UK); E 7820 (Eisai, Japan); CYC 381 (Harvard
University, USA); AE 941 (Aeterna, Canada); FGF2 cancer vaccine
(EntreMed, USA); urokinase plasminogen activator inhibitor
(Dendreon, USA); oglufanide (pINN) (Melmotte, USA); HIF-1alfa
inhibitors (Xenova, UK); CEP 5214 (Cephalon, USA); BAY RES 2622
(Bayer, Germany); Angiocidin (InKine, USA); A6 (Angstrom, USA); KR
31372 (Korean Research Institute of Chemical Technology, South
Korea); GW 2286 (GlaxoSmithKline, UK); EHT 0101 (ExonHit, France);
CP 868596 (Pfizer, USA); CP 564959 (OSI, USA); CP 547632 (Pfizer,
USA); 786034 (GlaxoSmithKline, UK); KRN 633 (Kirin Brewery, Japan);
drug delivery system, intraocular, 2-methoxyestradiol (EntreMed,
USA); anginex (Maastricht University, Netherlands, and Minnesota
University, USA); ABT 510 (Abbott, USA); AAL 993 (Novartis,
Switzerland); VEGI (ProteomTech, USA); tumor necrosis factor-alpha
inhibitors (National Institute on Aging, USA); SU 11248 (Pfizer,
USA and SUGEN USA); ABT 518 (Abbott, USA); YH16 (Yantai Rongchang,
China); S-3APG (Boston Childrens Hospital, USA and EntreMed, USA);
Mab, KDR (ImClone Systems, USA); Mab, alpha5 beta1 (Protein Design,
USA); KDR kinase inhibitor (Celltech Group, UK, and Johnson &
Johnson, USA); GFB 116 (South Florida University, USA and Yale
University, USA); CS 706 (Sankyo, Japan); combretastatin A4 prodrug
(Arizona State University, USA); chondroitinase AC (IBEX, Canada);
BAY RES 2690 (Bayer, Germany); AGM 1470 (Harvard University, USA,
Takeda, Japan, and TAP, USA); AG 13925 (Agouron, USA);
Tetrathiomolybdate (University of Michigan, USA); GCS 100 (Wayne
State University, USA); CV 247 (Ivy Medical, UK); CKD 732 (Chong
Kun Dang, South Korea); Mab, vascular endothelium growth factor
(Xenova, UK); irsogladine (INN) (Nippon Shinyaku, Japan); RG 13577
(Aventis, France); WX 360 (Wilex, Germany); squalamine (pINN)
(Genaera, USA); RPI 4610 (Sirna, USA); galacto fucan sulphate
(Marinova, Australia); heparanase inhibitors (InSight, Israel); KL
3106 (Kolon, South Korea); Honokiol (Emory University, USA); ZK CDK
(Shering AG, Germany); ZK Angio (Schering AG, Germany); ZK 229561
(Novartis, Switzerland, and Schering AG, Germany); XMP 300 (XOMA,
USA); VGA 1102 (Taisho, Japan); VEGF receptor modulators
(Pharmacopeia, USA); VE-cadherin-2 antagonists (ImClone Systems,
USA); Vasostatin (National Institutes of Health, USA); vaccine,
Flk-1 (ImClone Systems, USA); TZ 93 (Tsumura, Japan); TumStatin
(Beth Israel Hospital, USA); truncated soluble FLT 1 (vascular
endothelial growth factor receptor 1) (Merck & Co, USA); Tie-2
ligands (Regeneron, USA); and thrombospondin 1 inhibitor (Allegheny
Health, Education and Research Foundation, USA).
[0342] Certain cancer therapy agents include, but are not limited
to: thalidomide and thalidomide analogues
(N-(2,6-dioxo-3-piperidyl)phthalimide); tecogalan sodium (sulfated
polysaccharide peptidoglycan); Velcade; bortezomib; rapamycin; TAN
1120
(8-acetyl-7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-methoxy-10-[[octahydro--
5-hydroxy-2-(2-hydroxypropyl)-4,10-dimethylpyrano[3,4-d]-1,3,6-dioxazocin--
8-yl]oxy]-5,12-naphthacenedione); suradista
(7,7'-[carbonylbis[imino(1-methyl-1H-pyrrole-4,2-diyl)carbonylimino(1-met-
hyl-1H-pyrrole-4,2-diyl)carbonylimino]]bis-1,3-naphthalenedisulfonic
acid tetrasodium salt); SU 302; SU 301; SU 1498
((E)-2-cyano-3-[4-hydroxy-3,5-bis(1-methylethyl)phenyl]-N-(3-phenylpropyl-
)-2-pro penamide); SU 1433
(4-(6,7-dimethyl-2-quinoxalinyl)-1,2-benzenediol); ST 1514; SR
25989; soluble Tie-2; SERM derivatives; Pharmos; semaxanib
(pINN)(3-[(3,5-dimethyl-1H-pyrrol-2-yl)methylene]-1,3-dihydro-2H-indol-2--
one); S 836; RG 8803; RESTIN; R 440
(3-(1-methyl-1H-indol-3-yl)-4-(1-methyl-6-nitro-1H-indol-3-yl)-1H-pyrrole-
-2,5-dione); R 123942
(1-[6-(1,2,4-thiadiazol-5-yl)-3-pyridazinyl]-N-[3-(trifluoromethyl)phenyl-
]-4-piperidinamine); prolyl hydroxylase inhibitor; progression
elevated genes; prinomastat (INN)
((S)-2,2-dimethyl-4-[[p-(4-pyridyloxy)phenyl]sulphonyl]-3-thiomorpholinec-
arbohydroxamic acid); NV 1030; NM 3
(8-hydroxy-6-methoxy-alpha-methyl-1-oxo-1H-2-benzopyran-3-acetic
acid); NF 681; NF 050; MIG; METH 2; METH 1; manassantin B
(alpha-[1-[4-[5-[4-[2-(3,4-dimethoxyphenyl)-2-hydroxy-1-methylethoxy]-3-m-
ethoxyphenyl]tetrahydro-3,4-dimethyl-2-furanyl]-2-methoxyphenoxy]ethyl]-1,-
3-benzodioxole-5-methanol); KDR monoclonal antibody; alpha5beta3
integrin monoclonal antibody; LY 290293
(2-amino-4-(3-pyridinyl)-4H-naphtho[1,2-b]pyran-3-carbonitrile); KP
0201448; KM 2550; integrin-specific peptides; INGN 401; GYKI 66475;
GYKI 66462; greenstatin (101-354-plasminogen (human)); gene therapy
for rheumatoid arthritis, prostate cancer, ovarian cancer, glioma,
endostatin, colorectal cancer, ATF BTPI, antiangiogenesis genes,
angiogenesis inhibitor, or angiogenesis; gelatinase inhibitor, FR
111142 (4,5-dihydroxy-2-hexenoic acid
5-methoxy-4-[2-methyl-3-(3-methyl-2-butenyl)oxiranyl]-1-oxaspiro[2.5]oct--
6-yl ester); forfenimex (pINN)
(S)-alpha-amino-3-hydroxy-4-(hydroxymethyl)benzeneacetic acid);
fibronectin antagonist
(1-acetyl-L-prolyl-L-histidyl-L-seryl-L-cysteinyl-L-aspartamide);
fibroblast growth factor receptor inhibitor; fibroblast growth
factor antagonist; FCE 27164
(7,7'-[carbonylbis[imino(1-methyl-1H-pyrrole-4,2-diyl)carbonylimino(1-met-
hyl-1H-pyrrole-4,2-diyl)carbonylimino]]bis-1,3,5-naphthalenetrisulfonic
acid hexasodium salt); FCE 26752
(8,8'-[carbonylbis[imino(1-methyl-1H-pyrrole-4,2-diyl)carbonylimino(1-met-
hyl-1H-pyrrole-4,2-diyl)carbonylimino]]bis-1,3,6-naphthalenetrisulfonic
acid); endothelial monocyte activating polypeptide II; VEGFR
antisense oligonucleotide; anti-angiogenic and trophic factors;
ANCHOR angiostatic agent; endostatin; Del-1 angiogenic protein; CT
3577; contortrostatin; CM 101; chondroitinase AC; CDP 845;
CanStatin; BST 2002; BST 2001; BLS 0597; BIBF 1000; ARRESTIN;
apomigren (1304-1388-type XV collagen (human gene COL15A1
alpha1-chain precursor)); angioinhibin; aaATIII; A 36;
9alpha-fluoromedroxyprogesterone acetate
((6-alpha)-17-(acetyloxy)-9-fluoro-6-methyl-pregn-4-ene-3,20-dione);
2-methyl-2-phthalimidino-glutaric acid
(2-(1,3-dihydro-1-oxo-2H-isoindol-2-yl)-2-methylpentanedioic acid);
Yttrium 90 labelled monoclonal antibody BC-1; Semaxanib
(3-(4,5-Dimethylpyrrol-2-ylmethylene)indolin-2-one)(C15 H14 N2 O);
PI 88 (phosphomannopentaose sulfate); Alvocidib
(4H-1-Benzopyran-4-one,
2-(2-chlorophenyl)-5,7-dihydroxy-8-(3-hydroxy-1-methyl-4-piperidinyl)-cis-
-(-)-) (C21 H20 Cl N O5); E 7820; SU 11248
(5-[3-Fluoro-2-oxo-1,2-dihydroindol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-p-
yrrole-3-carboxylic acid (2-diethylaminoethyl)amide) (C22 H27 F N4
O2); Squalamine (Cholestane-7,24-diol,
3-[[3-[(4-aminobutyl)aminopropyl]amino]-, 24-(hydrogen sulfate),
(3.beta., 5.alpha., 7.alpha.)-) (C34 H65 N3 O5 S); Eriochrome Black
T; AGM 1470 (Carbamic acid, (chloroacetyl)-,
5-methoxy-4-[2-methyl-3-(3-methyl-2-butenyl)oxiranyl]-1-oxaspiro[2,5]
oct-6-yl ester, [3R-[3alpha, 4alpha(2R,3R), 5beta, 6beta]]) (C19
H28 Cl N O6); AZD 9935; BIBF 1000; AZD 2171; ABT 828;
KS-interleukin-2; Uteroglobin; A 6; NSC 639366
(1-[3-(Diethylamino)-2-hydroxypropylamino]-4-(oxyran-2-ylmethylamino)anth-
raquinone fumerate) (C24 H29 N3 O4. C4 H 4 O 4); ISV 616; anti-ED-B
fusion proteins; HUI 77; Troponin I; BC-1 monoclonal antibody; SPV
5.2; ER 68203; CKD 731 (3-(3,4,5-Trimethoxyphenyl)-2(E)-propenoic
acid
(3R,4S,5S,6R)-4-[2(R)-methyl-3(R)-3(R)-(3-methyl-2-butenyl)oxiran-2-yl]-5-
-methoxy-1-oxaspiro[2.5]oct-6-yl ester) (C28 H38 O8); IMC-1C11;
aaATIII; SC 7; CM 101; Angiocol; Kringle 5; CKD 732
(3-[4-[2-(Dimethylamino)ethoxy]phenyl]-2(E)-propenoic acid) (C29
H41 N O6); U 995; Canstatin; SQ 885; CT 2584
(1-[11-(Dodecylamino)-10-hydroxyundecyl]-3,7-dimethylxanthine)(C30
H55 N5 O3); Salmosin; EMAP II; TX 1920
(1-(4-Methylpiperazino)-2-(2-nitro-1H-1-imidazoyl)-1-ethanone) (C10
H15 N5 O3); Alpha-v Beta-x inhibitor; CHIR 11509
(N-(1-Propynyl)glycyl-[N-(2-naphthyl)]glycyl-[N-(carbamoylmethyl)]glycine
bis(4-methoxyphenyl)methylamide)(C36 H37 N5 O6); BST 2002; BST
2001; B 0829; FR 111142; 4,5-Dihydroxy-2(E)-hexenoic acid
(3R,4S,5S,6R)-4-[1(R),2(R)-epoxy-1,5-dimethyl-4-hexenyl]-5-methoxy-1-oxas-
piro[2.5]octan-6-ylester (C22 H34 O7); and kinase inhibitors
including, but not limited to,
N-(4-chlorophenyl)-4-(4-pyridinylmethyl)-1-phthalazinamine;
4-[4-[[[[4-chloro-3-(trifluoromethyl)phenyl]amino]carbonyl]amino]phenoxy]-
-N-methyl-2-pyridinecarboxamide;
N-[2-(diethylamino)ethyl]-5-[(5-fluoro-1,2-dihydro-2-oxo-3H-indol-3-ylide-
ne)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide;
3-[(4-bromo-2,6-difluorophenyl)methoxy]-5-[[[[4-(1-pyrrolidinyl)butyl]ami-
no]carbonyl]amino]-4-isothiazolecarboxamide;
N-(4-bromo-2-fluorophenyl)-6-methoxy-7-[(1-methyl-4-piperidinyl)methoxy]--
4-quinazolinamine;
3-[5,6,7,13-tetrahydro-9-[(1-methylethoxy)methyl]-5-oxo-12H-indeno[2,1-a]-
pyrrolo[3,4-c]carbazol-12-yl]propyl ester N,N-dimethyl-glycine;
N-[5-[[[5-(1,1-dimethylethyl)-2-oxazolyl]methyl]thio]-2-thiazolyl]-4-pipe-
ridinecarboxamide;
N-[3-chloro-4-[(3-fluorophenyl)methoxy]phenyl]-6-[5-[[[2-(methylsulfonyl)-
ethyl]amino]methyl]-2-furanyl]-4-quinazolinamine;
4-[(4-Methyl-1-piperazinyl)methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)-2-pyri-
midinyl]amino]-phenyl]benzamide;
N-(3-chloro-4-fluorophenyl)-7-methoxy-6-[3-(4-morpholinyl)propoxy]-4-quin-
azolinamine;
N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine;
N-(3-((((2R)-1-methyl-2-pyrrolidinyl)methyl)oxy)-5-(trifluoromethyl)pheny-
l)-2-((3-(1,3-oxazol-5-yl)phenyl)amino)-3-pyridinecarboxamide;
2-(((4-fluorophenyl)methyl)amino)-N-(3-((((2R)-1-methyl-2-pyrrolidinyl)me-
thyl)oxy)-5-(trifluoromethyl)phenyl)-3-pyridinecarboxamide;
N-[3-(Azetidin-3-ylmethoxy)-5-trifluoromethyl-phenyl]-2-(4-fluoro-benzyla-
mino)-nicotinamide;
6-fluoro-N-(4-(1-methylethyl)phenyl)-2-((4-pyridinylmethyl)amino)-3-pyrid-
inecarboxamide;
2-((4-pyridinylmethyl)amino)-N-(3-(((2S)-2-pyrrolidinylmethyl)oxy)-5-(tri-
fluoromethyl)phenyl)-3-pyridinecarboxamide;
N-(3-(1,1-dimethylethyl)-1H-pyrazol-5-yl)-2-((4-pyridinylmethyl)amino)-3--
pyridinecarboxamide;
N-(3,3-dimethyl-2,3-dihydro-1-benzofuran-6-yl)-2-((4-pyridinylmethyl)amin-
o)-3-pyridinecarboxamide;
N-(3-((((2S)-1-methyl-2-pyrrolidinyl)methyl)oxy)-5-(trifluoromethyl)pheny-
l)-2-((4-pyridinylmethyl)amino)-3-pyridinecarboxamide;
2-((4-pyridinylmethyl)amino)-N-(3-((2-(1-pyrrolidinyl)ethyl)oxy)-4-(trifl-
uoromethyl)phenyl)-3-pyridinecarboxamide;
N-(3,3-dimethyl-2,3-dihydro-1H-indol-6-yl)-2-((4-pyridinylmethyl)amino)-3-
-pyridinecarboxamide;
N-(4-(pentafluoroethyl)-3-(((2S)-2-pyrrolidinylmethyl)oxy)phenyl)-2-((4-p-
yridinylmethyl)amino)-3-pyridinecarboxamide;
N-(3-((3-azetidinylmethyl)oxy)-5-(trifluoromethyl)phenyl)-2-((4-pyridinyl-
methyl)amino)-3-pyridinecarboxamide;
N-(3-(4-piperidinyloxy)-5-(trifluoromethyl)phenyl)-2-((2-(3-pyridinyl)eth-
yl)amino)-3-pyridinecarboxamide;
N-(4,4-dimethyl-1,2,3,4-tetrahydro-isoquinolin-7-yl)-2-(1H-indazol-6-ylam-
ino)-nicotinamide;
2-(1H-indazol-6-ylamino)-N-[3-(1-methylpyrrolidin-2-ylmethoxy)-5-trifluor-
omethyl-phenyl]-nicotinamide;
N-[1-(2-dimethylamino-acetyl)-3,3-dimethyl-2,3-dihydro-1H-indol-6-yl]-2-(-
1H-indazol-6-ylamino)-nicotinamide;
2-(1H-indazol-6-ylamino)-N-[3-(pyrrolidin-2-ylmethoxy)-5-trifluoromethyl--
phenyl]-nicotinamide;
N-(1-acetyl-3,3-dimethyl-2,3-dihydro-1H-indol-6-yl)-2-(1H-indazol-6-ylami-
no)-nicotinamide;
N-(4,4-dimethyl-1-oxo-1,2,3,4-tetrahydro-isoquinolin-7-yl)-2-(1H-indazol--
6-ylamino)-nicotinamide;
N-[4-(tert-butyl)-3-(3-piperidylpropyl)phenyl][2-(1H-indazol-6-ylamino)(3-
-pyridyl)]carboxamide;
N-[5-(tert-butyl)isoxazol-3-yl][2-(1H-indazol-6-ylamino)(3-pyridyl)]carbo-
xamide; and
N-[4-(tert-butyl)phenyl][2-(1H-indazol-6-ylamino)(3-pyridyl)]carboxamide,
and kinase inhibitors disclosed in U.S. Pat. Nos. 6,258,812;
6,235,764; 6,630,500; 6,515,004; 6,713,485; 5,521,184; 5,770,599;
5,747,498; 5,990,141; U.S. Patent Application Publication No.
US2003/0105091; and Patent Cooperation Treaty publication nos.
WO01/37820; WO01/32651; WO02/68406; WO02/66470; WO02/55501;
WO04/05279; WO04/07481; WO04/07458; WO04/09784; WO02/59110;
WO99/45009; WO98/35958; WO00/59509; WO99/61422; WO00/12089; and
WO00/02871, each of which publications are hereby incorporated by
reference for any purpose.
[0343] TR-2 is expressed in a variety of cells, including liver,
brain, kidney, colon, breast, lung, spleen, thymus, peripheral
blood lymphocytes, pancreas, prostate, testis, ovary, uterus, and
various tissues along the gastro-intestinal tract. Exemplary TR-2
related cancers include, but are not limited to, liver cancer,
brain cancer, renal cancer, breast cancer, pancreatic cancer,
colorectal cancer, lung cancer (small cell lung cancer and
non-small-cell lung cancer), spleen cancer, cancer of the thymus or
blood cells (i.e., leukemia), prostate cancer, testicular cancer,
ovarian cancer, uterine cancer, gastric carcinoma, head and neck
squamous cell carcinoma, melanoma, and lymphoma.
[0344] In certain embodiments, an anti-TR-2 antibody may be used
alone or with at least one additional therapeutic agent for the
treatment of cancer. In certain embodiments, an anti-TR-2 antibody
is used in conjunction with a therapeutically effective amount of
an additional therapeutic agent. Exemplary therapeutic agents that
may be administered with an anti-TR-2 antibody include, but are not
limited to, a member of the geldanamycin family of anisamycin
antibiotics; a Pro-HGF; NK2; a c-Met peptide inhibitor; an
antagonist of Grb2 Src homology 2; a Gab1 modulator;
dominant-negative Src; a von-Hippel-Landau inhibitor, including,
but not limited to, wortmannin; P13 kinase inhibitors, other
anti-receptor therapies, anti EGFr, a COX-2 inhibitor,
Celebrex.TM., celecoxib, Vioxx.TM., rofecoxib; a vascular
endothelial growth factor (VEGF), a VEGF modulator, a fibroblast
growth factor (FGF), an FGF modulator, an epidermal growth factor
(EGF); an EGF modulator; a keratinocyte growth factor (KGF), a
KGF-related molecule, a KGF modulator; and a matrix
metalloproteinase (MMP) modulator.
[0345] In certain embodiments, anti-TR-2 antibody is used with
particular therapeutic agents to treat various cancers. In certain
embodiments, in view of the condition and the desired level of
treatment, two, three, or more agents may be administered. Where
the compounds are used together with one or more other components,
the compound and the one or more other components may be
administered together, separately, or sequentially (e.g., in a
pharmaceutical format). In certain embodiments, such agents may be
provided together by inclusion in the same formulation. In certain
embodiments, such agents and an anti-TR-2 antibody may be provided
together by inclusion in the same formulation. In certain
embodiments, such agents may be formulated separately and provided
together by inclusion in a treatment kit. In certain embodiments,
such agents and an anti-TR-2 antibody may be formulated separately
and provided together by inclusion in a treatment kit. In certain
embodiments, such agents may be provided separately.
[0346] In certain embodiments, when administered by gene therapy,
the genes encoding protein agents and/or an anti-TR-2 antibody may
be included in the same vector. In certain embodiments, the genes
encoding protein agents and/or an anti-TR-2 antibody may be under
the control of the same promoter region. In certain embodiments,
the genes encoding protein agents and/or an anti-TR-2 antibody may
be in separate vectors.
[0347] In certain embodiments, anti-TR-2 antibodies may be used to
treat non-human animals, such as pets (dogs, cats, birds, primates,
etc.), and domestic farm animals (horses cattle, sheep, pigs,
birds, etc.). In certain such instances, an appropriate dose may be
determined according to the animal's body weight. For example, in
certain embodiments, a dose of 0.2-1 mg/kg may be used. In certain
embodiments, the dose may be determined according to the animal's
surface area, an exemplary dose ranging from 0.1 to 20 mg/in.sup.2,
or from 5 to 12 mg/m.sup.2. For small animals, such as dogs or
cats, in certain embodiments, a suitable dose is 0.4 mg/kg. In
certain embodiments, anti-TR-2 antibodies are administered by
injection or other suitable route one or more times per week until
the animal's condition is improved, or it may be administered
indefinitely.
[0348] It is understood that the response by individual patients to
the aforementioned medications or combination therapies may vary,
and an appropriate efficacious combination of drugs for each
patient may be determined by his or her physician.
[0349] The cynomolgus monkey provides a useful model for certain
diseases. Exemplary diseases include, but are not limited to,
transplantation rejection syndrome and inflammatory bowel
disease-like disease. When testing the efficacy of a human MAb in
cynomolgus monkey human disease model, in certain embodiments, it
is useful to determine whether the anti-TR-2 MAb binds to TR-2 in
humans and cynomolgus monkeys at a comparable level.
[0350] In certain embodiments, an anti-TR-2 antibody may be part of
a conjugate molecule comprising all or part of the anti-TR-2
antibody and a cytotoxic agent. The term "cytotoxic agent" refers
to a substance that inhibits or prevents the function of cells
and/or causes the death or destruction of cells. The term includes,
but is not limited to, radioactive isotopes (e.g., I.sup.131,
I.sup.125, Y.sup.90 and Re.sup.186), chemotherapeutic agents, and
toxins such as enzymatically active toxins of bacterial, fungal,
plant or animal origin, or fragments thereof. Exemplary cytotoxic
agents include, but are not limited to, Adriamycin, Doxorubicin,
5-Fluorouracil, Cytosine arabinoside ("Ara-C"), Cyclophosphamide,
Thiotepa, Taxotere (docetaxel), Busulfan, Cytoxin, Taxol,
Methotrexate, Cisplatin, Melphalan, Vinblastine, Bleomycin,
Etoposide, Ifosfamide, Mitomycin C, Mitoxantrone, Vincreistine,
Vinorelbine, Carboplatin, Teniposide, Daunomycin, Caminomycin,
Aminopterin, Dactinomycin, Mitomycins, Esperamicins, Melphalan and
other related nitrogen mustards.
[0351] In certain embodiments, an anti-TR-2 antibody may be part of
a conjugate molecule comprising all or part of the anti-TR-2
antibody and a prodrug. In certain embodiments, the term "prodrug"
refers to a precursor or derivative form of a pharmaceutically
active substance. In certain embodiments, a prodrug is less
cytotoxic to cells compared to the parent drug and is capable of
being enzymatically activated or converted into the more active
cytotoxic parent form. Exemplary prodrugs include, but are not
limited to, phosphate-containing prodrugs, thiophosphate-containing
prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs,
ID-amino acid-modified prodrugs, glycosylated prodrugs,
beta-lactam-containing prodrugs, optionally substituted
phenoxyacetamide-containing prodrugs and optionally substituted
phenylacetamide-containing prodrugs, 5-fluorocytosine and other
5-fluorouridine prodrugs which can be converted into a more active
cytotoxic free drug. Examples of cytotoxic drugs that can be
derivatized into a prodrug form include, but are not limited to,
those cytotoxic agents described above. See, e.g., U.S. Pat. No.
6,702,705.
[0352] In certain embodiments, antibody conjugates function by
having the antibody portion of the molecule target the cytotoxic
portion or prodrug portion of the molecule to a specific population
of cells in the patient. In the case of anti-TR-2 antibodies, such
conjugate molecules may be used, for example, in certain
embodiments, to destroy abnormally proliferating cells, such as
cancer cells.
[0353] In certain embodiments, methods of treating a patient
comprising administering a therapeutically effective amount of an
anti-TR-2 antibody are provided. In certain embodiments, methods of
treating a patient comprising administering a therapeutically
effective amount of an antibody conjugate are provided. In certain
embodiments, an antibody is used in conjunction with a
therapeutically effective amount of at least one additional
therapeutic agent, as discussed above.
[0354] As discussed above, in certain embodiments, anti-TR-2
antibodies may be administered concurrently with one or more other
drugs that are administered to the same patient, each drug being
administered according to a regimen suitable for that medicament.
Such treatment encompasses pre-treatment, simultaneous treatment,
sequential treatment, and alternating regimens. Additional examples
of such drugs include, but are not limited to, antivirals,
antibiotics, analgesics, corticosteroids, antagonists of
inflammatory cytokines, DMARDs, nonsteroidal anti-inflammatories,
chemotherapeutics, inhibitors of angiogenesis, and stimulators of
angiogenesis.
[0355] In certain embodiments, various medical disorders are
treated with anti-TR-2 antibodies in combination with another
stimulator of apoptosis. For example, in certain embodiments,
anti-TR-2 antibodies may be administered in a composition that also
contains a compound that stimulates apoptosis of one or more cells.
In certain embodiments, the anti-TR-2 antibody and stimulators of
apoptosis may be administered as separate compositions, and these
may be administered by the same or different routes.
[0356] In certain embodiments, pharmaceutical compositions are
provided comprising a therapeutically effective amount of an
antibody together with a pharmaceutically acceptable diluent,
carrier, solubilizer, emulsifier, preservative and/or adjuvant.
[0357] In certain embodiments, pharmaceutical compositions are
provided comprising a therapeutically effective amount of an
antibody and a therapeutically effective amount of at least one
additional therapeutic agent, together with a pharmaceutically
acceptable diluent, carrier, solubilizer, emulsifier, preservative
and/or adjuvant.
[0358] In certain embodiments, acceptable formulation materials
preferably are nontoxic to recipients at the dosages and
concentrations employed. In certain embodiments, antibodies of the
present invention are provided in a bufferless formulation as
disclosed in PCT/US06/22599 filed Jun. 8, 2006, which is
incorporated by reference herein for any purpose.
[0359] In certain embodiments, the pharmaceutical composition may
contain formulation materials for modifying, maintaining or
preserving, for example, the pH, osmolarity, viscosity, clarity,
color, isotonicity, odor, sterility, stability, rate of dissolution
or release, adsorption or penetration of the composition. In
certain embodiments, suitable formulation materials include, but
are not limited to, amino acids (such as glycine, glutamine,
asparagine, arginine or lysine); antimicrobials; antioxidants (such
as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite);
buffers (such as borate, bicarbonate, Tris-HCl, citrates,
phosphates or other organic acids); bulking agents (such as
mannitol or glycine); chelating agents (such as ethylenediamine
tetraacetic acid (EDTA)); complexing agents (such as caffeine,
polyvinylpyrrolidone, beta-cyclodextrin or
hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides;
disaccharides; and other carbohydrates (such as glucose, mannose or
dextrins); proteins (such as serum albumin, gelatin or
immunoglobulins); coloring, flavoring and diluting agents;
emulsifying agents; hydrophilic polymers (such as
polyvinylpyrrolidone); low molecular weight polypeptides;
salt-forming counterions (such as sodium); preservatives (such as
benzalkonium chloride, benzoic acid, salicylic acid, thimerosal,
phenethyl alcohol, methylparaben, propylparaben, chlorhexidine,
sorbic acid or hydrogen peroxide); solvents (such as glycerin,
propylene glycol or polyethylene glycol); sugar alcohols (such as
mannitol or sorbitol); suspending agents; surfactants or wetting
agents (such as pluronics, PEG, sorbitan esters, polysorbates such
as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin,
cholesterol, tyloxapal); stability enhancing agents (such as
sucrose or sorbitol); tonicity enhancing agents (such as alkali
metal halides, preferably sodium or potassium chloride, mannitol
sorbitol); delivery vehicles; diluents; excipients and/or
pharmaceutical adjuvants. (Remington's Pharmaceutical Sciences,
18.sup.th Edition, A. R. Gennaro, ed., Mack Publishing Company
(1990).
[0360] In certain embodiments, an antibody and/or an additional
therapeutic molecule is linked to a half-life extending vehicle
known in the art. Such vehicles include, but are not limited to,
the Fc domain, polyethylene glycol, and dextran. Such vehicles are
described, e.g., in U.S. Pat. No. 6,660,843 and published PCT
Application No. WO 99/25044.
[0361] In certain embodiments, the optimal pharmaceutical
composition will be determined by one skilled in the art depending
upon, for example, the intended route of administration, delivery
format and desired dosage. See, for example, Remington's
Pharmaceutical Sciences, supra. In certain embodiments, such
compositions may influence the physical state, stability, rate of
in vivo release and rate of in vivo clearance of the
antibodies.
[0362] In certain embodiments, the primary vehicle or carrier in a
pharmaceutical composition may be either aqueous or non-aqueous in
nature. For example, in certain embodiments, a suitable vehicle or
carrier may be water for injection, physiological saline solution
or artificial cerebrospinal fluid, possibly supplemented with other
materials common in compositions for parenteral administration. In
certain embodiments, neutral buffered saline or saline mixed with
serum albumin are further exemplary vehicles. In certain
embodiments, pharmaceutical compositions comprise Tris buffer of
about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which may
further include sorbitol or a suitable substitute therefor. In
certain embodiments, a pharmaceutical composition is an aqueous or
liquid formulation comprising an acetate buffer of about pH
4.0-5.5, a polyol (polyalcohol), and optionally, a surfactant,
wherein the composition does not comprise a salt, e.g., sodium
chloride, and wherein the composition is isotonic for the patient.
Exemplary polyols include, but are not limited to, sucrose,
glucose, sorbitol, and mannitol. An exemplary surfactant includes,
but is not limited to, polysorbate. In certain embodiments, a
pharmaceutical composition is an aqueous or liquid formulation
comprising an acetate buffer of about pH 5.0, sorbitol, and a
polysorbate, wherein the composition does not comprise a salt,
e.g., sodium chloride, and wherein the composition is isotonic for
the patient. Certain exemplary compositions are found, for example,
in U.S. Pat. No. 6,171,586. Additional pharmaceutical carriers
include, but are not limited to, oils, including petroleum oil,
animal oil, vegetable oil, peanut oil, soybean oil, mineral oil,
sesame oil, and the like. In certain embodiments, aqueous dextrose
and glycerol solutions can also be employed as liquid carriers,
particularly for injectable solutions. In certain embodiments, a
composition comprising an antibody, with or without at least one
additional therapeutic agent, may be prepared for storage by mixing
the selected composition having the desired degree of purity with
optional formulation agents (Remington's Pharmaceutical Sciences,
supra) in the form of a lyophilized cake or an aqueous solution.
Further, in certain embodiments, a composition comprising an
antibody, with or without at least one additional therapeutic
agent, may be formulated as a lyophilizate using appropriate
excipient solutions (e.g., sucrose) as diluents.
[0363] In certain embodiments, anti-TR-2 antibodies are
administered in the form of a physiologically acceptable
composition comprising purified recombinant protein in conjunction
with physiologically acceptable carriers, excipients or diluents.
In certain embodiments, such carriers are nontoxic to recipients at
the dosages and concentrations employed. In certain embodiments,
preparing such compositions may involve combining the anti-TR-2
antibodies with buffers, antioxidants such as ascorbic acid, low
molecular weight polypeptides (such as those having fewer than 10
amino acids), proteins, amino acids, carbohydrates such as glucose,
sucrose or dextrins, chelating agents such as EDTA, glutathione
and/or other stabilizers, and excipients. In certain embodiments,
appropriate dosages are determined in standard dosing trials, and
may vary according to the chosen route of administration. In
certain embodiments, in accordance with appropriate industry
standards, preservatives may also be added, which include, but are
not limited to, benzyl alcohol. In certain embodiments, the amount
and frequency of administration may be determined based on such
factors as the nature and severity of the disease being treated,
the desired response, the age and condition of the patient, and so
forth.
[0364] In certain embodiments, pharmaceutical compositions can be
selected for parenteral delivery. The preparation of certain such
pharmaceutically acceptable compositions is within the skill of the
art.
[0365] In certain embodiments, the formulation components are
present in concentrations that are acceptable to the site of
administration. In certain embodiments, buffers are used to
maintain the composition at physiological pH or at a slightly lower
pH, typically within a pH range of from about 5 to about 8.
[0366] In certain embodiments, when parenteral administration is
contemplated, a therapeutic composition may be in the form of a
pyrogen-free, parenterally acceptable aqueous solution comprising
the desired antibody, with or without additional therapeutic
agents, in a pharmaceutically acceptable vehicle. In certain
embodiments, a vehicle for parenteral injection is sterile
distilled water in which the antibody, with or without at least one
additional therapeutic agent, is formulated as a sterile, isotonic
solution, properly preserved. In certain embodiments, the
preparation can involve the formulation of the desired molecule
with an agent, such as injectable microspheres, bio-erodible
particles, polymeric compounds (such as polylactic acid or
polyglycolic acid), beads, or liposomes, that may provide for the
controlled or sustained release of the product which may then be
delivered via a depot injection. In certain embodiments, hyaluronic
acid may also be used, and may have the effect of promoting
sustained duration in the circulation. In certain embodiments,
implantable drug delivery devices may be used to introduce the
desired molecule.
[0367] In certain embodiments, a pharmaceutical composition may be
formulated for inhalation. In certain embodiments, an antibody,
with or without at least one additional therapeutic agent, may be
formulated as a dry powder for inhalation. In certain embodiments,
an inhalation solution comprising an antibody, with or without at
least one additional therapeutic agent, may be formulated with a
propellant for aerosol delivery. In certain embodiments, solutions
may be nebulized. Pulmonary administration is further described in
PCT publication no. WO94/20069, which describes pulmonary delivery
of chemically modified proteins.
[0368] In certain embodiments, it is contemplated that formulations
may be administered orally. In certain embodiments, an antibody,
with or without at least one additional therapeutic agent, that is
administered in this fashion may be formulated with or without
those carriers customarily used in the compounding of solid dosage
forms such as tablets and capsules. In certain embodiments, a
capsule may be designed to release the active portion of the
formulation at the point in the gastrointestinal tract when
bioavailability is maximized and pre-systemic degradation is
minimized. In certain embodiments, at least one additional agent
can be included to facilitate absorption of the antibody and/or any
additional therapeutic agents. In certain embodiments, diluents,
flavorings, low melting point waxes, vegetable oils, lubricants,
suspending agents, tablet disintegrating agents, and/or binders may
also be employed.
[0369] In certain embodiments, a pharmaceutical composition may
involve an effective quantity of antibodies, with or without at
least one additional therapeutic agent, in a mixture with non-toxic
excipients which are suitable for the manufacture of tablets. In
certain embodiments, by dissolving the tablets in sterile water, or
another appropriate vehicle, solutions may be prepared in unit-dose
form. Suitable excipients include, but are not limited to, inert
diluents, such as calcium carbonate, sodium carbonate or
bicarbonate, lactose, or calcium phosphate; and binding agents,
such as starch, gelatin, and acacia; and lubricating agents such as
magnesium stearate, stearic acid, and talc.
[0370] Additional pharmaceutical compositions will be evident to
those skilled in the art, including formulations involving
antibodies, with or without at least one additional therapeutic
agent, in sustained- or controlled-delivery formulations. In
certain exemplary sustained- or controlled-delivery formulations
include, but are not limited to, liposome carriers, bio-erodible
microparticles, porous beads, and depot injections. Certain
exemplary techniques for preparing certain formulations are known
to those skilled in the art. See for example, PCT publication no.
WO93/15722, which describes the controlled release of porous
polymeric microparticles for the delivery of pharmaceutical
compositions. In certain embodiments, sustained-release
preparations may include semipermeable polymer matrices in the form
of shaped articles, e.g. films, or microcapsules. Sustained release
matrices include, but are not limited to, polyesters, hydrogels,
polylactides (U.S. Pat. No. 3,773,919 and EP 058,481), copolymers
of L-glutamic acid and gamma ethyl-L-glutamate (Sidman at al.,
Biopolymers, 22:547-556 (1983)), poly (2-hydroxyethyl-methacrylate)
(Langer et al., J. Biomed. Mater. Res., 15:167-277 (1981) and
Langer, Chem. Tech., 12:98-105 (1982)), ethylene vinyl acetate
(Langer et al., supra), and poly-D(-)-3-hydroxybutyric acid (EP
133,988). In certain embodiments, sustained release compositions
may also include liposomes, which can be prepared, in certain
embodiments, by any of several methods known in the art. See e.g.,
Eppstein et al., Proc. Natl. Acad. Sci. USA, 82:3688-3692 (1985);
EP 036,676; EP 088,046 and EP 143,949.
[0371] In certain embodiments, the pharmaceutical composition to be
used for in vivo administration is sterile. In certain embodiments,
the pharmaceutical composition to be used for in vivo
administration is made sterile by filtration through sterile
filtration membranes. In certain embodiments, where the composition
is lyophilized, sterilization using sterile filtration membranes
may be conducted either prior to or following lyophilization and
reconstitution. In certain embodiments, the composition for
parenteral administration may be stored in lyophilized form or in a
solution. In certain embodiments, parenteral compositions 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.
[0372] In certain embodiments, after the pharmaceutical composition
has been formulated, it may be stored in sterile vials as a
solution, suspension, gel, emulsion, solid, or as a dehydrated or
lyophilized powder. In certain embodiments, such formulations may
be stored either in a ready-to-use form or in a form (e.g., a
lyophilized form) that is reconstituted prior to
administration.
[0373] In certain embodiments, kits for producing a single-dose
administration unit are provided. In certain embodiments, the kits
may each contain both a first container having a dried protein and
a second container having an aqueous formulation. In certain
embodiments, kits containing single and/or multi-chambered
pre-filled syringes (e.g., liquid syringes and lyosyringes) are
included.
[0374] In certain embodiments, the effective amount of a
pharmaceutical composition comprising an antibody, with or without
at least one additional therapeutic agent, to be employed
therapeutically will depend, for example, upon the therapeutic
context and objectives. One skilled in the art will appreciate that
the appropriate dosage levels for treatment, according to certain
embodiments, will thus vary depending, in part, upon the molecule
delivered, the indication for which the antibody, with or without
at least one additional therapeutic agent, is being used, the route
of administration, and the size (body weight, body surface or organ
size) and/or condition (the age and general health) of the patient.
In certain embodiments, the clinician may titer the dosage and
modify the route of administration to obtain the optimal
therapeutic effect. In certain embodiments, a typical dosage may
range from about 0.1 .mu.g/kg to up to about 100 mg/kg or more,
depending on the factors mentioned above. In certain embodiments,
the dosage may range from 0.1 .mu.g/kg up to about 100 mg/kg; or 1
.mu.g/kg up to about 100 mg/kg; or 5 .mu.g/kg up to about 100
mg/kg; or 0.1 mg/kg up to about 100 mg/kg.
[0375] In certain embodiments, the frequency of dosing will take
into account the pharmacokinetic parameters of the antibody and/or
any additional therapeutic agents in the formulation used. In
certain embodiments, a clinician will administer the composition
until a dosage is reached that achieves the desired effect. In
certain embodiments, the composition may therefore be administered
as a single dose, or as two or more doses (which may or may not
contain the same amount of the desired molecule) over time, or as a
continuous infusion via an implantation device or catheter. Certain
methods of further refining the appropriate dosage are within the
skill in the art. In certain embodiments, appropriate dosages may
be ascertained through use of appropriate dose-response data.
[0376] In certain embodiments, the route of administration of the
pharmaceutical composition is in accord with known methods, e.g.
orally, through injection by intravenous, intraperitoneal,
intracerebral (intra-parenchymal), intracerebroventricular,
intramuscular, intra-ocular, intraarterial, intraportal, or
intralesional routes; by sustained release systems or by
implantation devices. In certain embodiments, the compositions may
be administered by bolus injection or continuously by infusion, or
by implantation device.
[0377] As discussed above, in various embodiments, any efficacious
route of administration may be used to administer anti-TR-2
antibodies. If injected, in certain embodiments, anti-TR-2
antibodies may be administered, for example, via intra-articular,
intravenous, intramuscular, intralesional, intraperitoneal,
intracranial, intranasal, inhalation or subcutaneous routes by
bolus injection or by continuous infusion. Exemplary methods of
administration include, but are not limited to, sustained release
from implants, aerosol inhalation, eyedrops, oral preparations,
including pills, syrups, lozenges, and chewing gum, and topical
preparations such as lotions, gels, sprays, ointments, and other
suitable techniques.
[0378] In certain embodiments, administration by inhalation is
beneficial when treating diseases associated with pulmonary
disorders. In certain embodiments, anti-TR-2 antibodies may be
administered by implanting cultured cells that express the
antibodies. In certain embodiments, the patient's own cells are
induced to produce by transfection in vivo or ex vivo with one or
more vectors that encode an anti-TR-2 antibody. In certain
embodiments, this vector can be introduced into the patient's
cells, for example, by injecting naked DNA or liposome-encapsulated
DNA that encodes an anti-TR-2 antibody, or by other methods of
transfection. When anti-TR-2 antibodies are administered in
combination with one or more other biologically active compounds,
in certain embodiments, these may be administered by the same or by
different routes, and may be administered together, separately, or
sequentially.
[0379] In certain embodiments, the composition may be administered
locally via implantation of a membrane, sponge or another
appropriate material onto which the desired molecule has been
absorbed or encapsulated. In certain embodiments, where an
implantation device is used, the device may be implanted into any
suitable tissue or organ, and delivery of the desired molecule may
be via diffusion, timed-release bolus, or continuous
administration.
[0380] In certain embodiments, it may be desirable to use a
pharmaceutical composition comprising an antibody, with or without
at least one additional therapeutic agent, in an ex vivo manner. In
such embodiments, cells, tissues and/or organs that have been
removed from the patient are exposed to a pharmaceutical
composition comprising an antibody, with or without at least one
additional therapeutic agent, after which the cells, tissues and/or
organs are subsequently implanted back into the patient.
[0381] In certain embodiments, an antibody and any additional
therapeutic agents can be delivered by implanting certain cells
that have been genetically engineered, using methods such as those
described herein, to express and secrete the polypeptides. In
certain embodiments, such cells may be animal or human cells, and
may be autologous, heterologous, or xenogeneic. In certain
embodiments, the cells may be immortalized. In certain embodiments,
in order to decrease the chance of an immunological response, the
cells may be encapsulated to avoid infiltration of surrounding
tissues. In certain embodiments, the encapsulation materials are
typically biocompatible, semi-permeable polymeric enclosures or
membranes that allow the release of the protein product(s) but
prevent the destruction of the cells by the patient's immune system
or by other detrimental factors from the surrounding tissues.
EXAMPLES
Example 1
Production of Certain Human Monoclonal Antibodies
[0382] Human anti-TR-2 antibodies were produced in one of two ways.
Transgenic mice expressing human immunoglobulin genes
(Xenomouse.RTM.) were exposed to human TR-2. Certain human
anti-TR-2 monoclonal antibodies were produced from those mice using
hybridoma techniques. Certain other human anti-TR-2 monoclonal
antibodies were produced from those mice using XenoMax technology,
which incorporates the selected lymphocyte antibody method ("SLAM")
technique (see, e.g., U.S. Pat. No. 5,627,052; and Babcook et al.,
Proc. Natl. Acad. Sci. USA 93:7843-7848 (1996)).
[0383] The methodology used to produce human anti-TR-2 monoclonal
antibodies in transgenic mice expressing human immunoglobulin genes
was as follows. Five groups of mice were immunized with recombinant
human TR-2 with a C-terminal hexahistidine tag (TR-2-His) (mature
amino acid sequence ALITQQDLAPQQRAAPQQKRSSPSEGLCPPGHHISEDGRDCISCKY
GQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVCQCEEGTFREEDS
PEMCRKCRTGCPRGMVKVGDCTPWSDIECVHKESGTKHSGEAPAVEETVT
SSPGTPASRSGSSHHHHHH (SEQ ID NO: 140)) (Genbank Reference Number
NM-003842), as shown in FIG. 1. The mice in group one, group three,
group four, and group five were engineered to produce antibodies of
the IgG2 isotype (FIG. 2). The mice in group two were engineered to
produce antibodies of the IgG4 isotype (FIG. 2). Group one included
7 mice, group two included 8 mice, group three included 8 mice,
group four included 10 mice, and group five included 5 mice. The
mice in group one, group two, and group three were immunized by
injection of TR-2-His into the footpad (10 .mu.g per injection),
while the mice in group four and group five were immunized
intraperitoneally (10 .mu.g per injection) with TR-2-His. On day 0,
10 .mu.g antigen was administered by the described route. At
specified intervals, booster injections were administered to the
mice. Group one mice had nine booster injections, at days 5, 11,
14, 18, 24, 28, 34, 42, and 46. Group two and group three mice had
7 booster injections; those for group 2 were at days 3, 7, 10, 14,
17, 24, and 27, and those for group three were at days 5, 8, 15,
21, 26, 30, and 33. Group four and group five mice had 5 booster
injections, at days 14, 28, 42, 56, and 72. Each first injection
and each booster injection contained 10 .mu.g TR-2-His with an
adjuvant, either Titermax Gold (Groups one, two, and three), alum
gel (groups one, two, and three), Complete Freund's Adjuvant (CFA)
(groups four and five), Incomplete Freund's Adjuvant (IFA) (groups
four and five), or Dulbecco's phosphate-buffered saline (D-PBS)
(groups one, two, three, four, and five) (see FIG. 1). Mice were
bled after three injections (groups four and five), after four
injections (groups one, two, and three), after six injections
(groups one and two), and after ten injections (group one). The
reactivity of each bleed to TR-2-His was assessed by ELISA, as
shown in FIG. 2.
[0384] The ELISA assay was performed as follows. Multiwell plates
were coated with soluble TR-2-His (0.5 .mu.g/mL) by passive
adsorption overnight at 4.degree. C. The coated wells were washed
and blocked for 30 minutes with milk. Ten .mu.L of each mouse serum
was combined with 40 .mu.L milk and incubated in the wells of
different plates for 1 hour, 1.25 hours, or 2 hours. The plates
were washed five times with water. The plates were then incubated
with a goat anti-human IgG Fc-specific horseradish
peroxidase-conjugated antibody (Pierce) at a final concentration of
1 .mu.g/mL for 1 hour at room temperature. The plates were washed
five times with water. The plates were incubated with K blue
substrate (Neogen) for 30 minutes. Negative controls included blank
wells lacking TR-2-His and wells including TR-2-His but incubated
with naive G2 sera expected to lack anti-TR-2 antibodies.
[0385] The methodologies used to produce human anti-TR-2 monoclonal
antibodies were as follows. For XenoMax technology, CD19+ B cells
were isolated from the hyperimmune transgenic mice that were
harvested on day 37 (mouse M712-7 from group three), or day 76
(mouse M564-1 from group four, and mice M564-3, M564-5, and M563-5
from group five after the initiation of immunization. The B cells
were cultured for 1 week to allow their expansion and consequent
differentiation into plasma cells. The supernatants containing the
secreted antibody was saved for further analysis and the plasma
cells in each well were frozen at -80 degrees celcius in media
containing 10% DMSO and 90% FCS. For hybridoma technology, the
cells from the remaining hyperimmune transgenic mice were harvested
on day 31, 37 or 46 for further analysis as shown in FIG. 1.
[0386] For XenoMax technology, supernates from the B cell cultures
were screened by ELISA for the presence of antibodies to TR-2.
Anti-TR-2 antibodies were detected by assessing binding to
immobilized TR-2-His using an anti-human IgG antibody detection
reagent as follows. Plates were coated with soluble TR-2-His (0.5
.mu.g/ml) by passive adsorption overnight at 4.degree. C. After
washing the plates five times with water and blocking the wells in
the plates with milk for 30 minutes, 10 .mu.L cell culture
supernate from each individual hybridoma was combined with 40 .mu.L
milk and incubated in the wells of different plates for 1 hour,
1.25 hours, or 2 hours. The plates were washed five times with
water, and incubated with a goat anti-human IgG Fc-specific
horseradish peroxidase (Pierce)-conjugated antibody at a final
concentration of 1 .mu.g/mL for 1 hour at room temperature. After
washing the plates five times with water, the plates were incubated
with K blue substrate (Neogen) for 30 minutes. Negative controls
included blank wells lacking TR-2-His and wells using naive G2 sera
expected to lack anti-TR-2 antibodies. Positive samples were
screened by ELISA a second time against TR-2-His to confirm the
identity of cells producing antibodies specific for TR-2.
[0387] The antibodies reactive with TR-2, identified above, were
screened for their ability to induce apoptosis of WM-266 melanoma
cells (ATCC Cat. No. CRL-1676) using an apoptosis assay. WM-266
cells were cultured in a microtiter plate at a density of 4500
cells/well in normal culture medium as recommended by ATCC
overnight. For B cell cultures, 20 .mu.L of antigen-specific B cell
culture supernatant or control B cell culture supernatant was added
to 180 .mu.L of apoptosis medium mixture (cell culture medium
containing 1 .mu.g/mL cycloheximide (CHX) and 0.5% fetal calf serum
("FCS")). The culture media from the WM-266 cells was removed and
the antibody-apoptosis medium mixture was added to the cells one
row at a time. The cells were incubated with the antibody-apoptosis
medium for 20 hours to allow apoptosis to occur. The DNA-binding
fluorescent dyes propidium iodide (Sigma) and Hoechst 33342
(Molecular Probes) were added to each well at a final concentration
of 0.5 .mu.g/mL and 2.5 .mu.g/mL, respectively. Hoechst 33342 is
membrane-permeable, and thus labels both live and dead cells;
propidium iodide is not membrane-permeable, and thus labels only
dead cells. After one hour at 37.degree. C., images of each well
were captured and analyzed for total number of cells (by assessing
the amount of Hoechst label) and total number of dead cells (by
assessing the amount of propidium iodide label). The percent
apoptosis was determined as (propidium iodide-positive
cells/Hoechst-positive cells).times.100.
[0388] For XenoMax technology, the antibodies from several wells
that displayed the best induction of apoptosis were selected for
rescue using the haemolytic plaque assay. TR-2-His was biotinylated
and coated onto streptavidin-coated sheep red blood cells. Plasma
cells corresponding to antigen-specific wells were thawed and
incubated with the antigen-coated red blood cells in the presence
of complement and guinea pig anti-human IgG enhancing serum. Plasma
cells producing antibodies against TR-2-His caused the sheep red
blood cells around them to lyse and thus allowed the identification
of antigen-specific plasma cells in the mixture. Those plasma cells
were isolated by micromanipulation of single cells from the
mixture.
[0389] After isolation of the desired single plasma cells, mRNA was
extracted from those cells. The mRNAs encoding the heavy and light
chain variable sequences were converted to cDNA and amplified by
reverse transcriptase PCR using degenerate antisense primers
specific for the leader sequences and the constant regions of human
IgG2 and human kappa mRNA The primer sequences are provided in
Table 2 below:
TABLE-US-00002 TABLE 2 Primer Name Primer seq AS-Ck RT 5' GTA GGT
GCT GTC CT 3' (SEQ ID NO: 97) AS-.gamma.CH1 RT 5' TGA GTT CCA CGA
CA 3' (SEQ ID NO: 98) AS-C Lambda RT 5' CTT CCA AGC CAC T 3' (SEQ
ID NO: 99) AS-C Lambda RT 5' CA (GA) GC ACT GTC A 3' (SEQ ID NO:
100) AS-Ck outer 5' GTA GGT GCT GTC CTT GCT 3' (SEQ ID NO: 101)
AS-Ck middle 5' CTC TGT GAC ACT CTC CTG GGA 3' (SEQ ID NO: 102)
AS-Ck inner with Xba I 5' GCT CTA GAT TGG AGG GCG TTA TCC ACC TTC
CAC T 3' (SEQ ID NO: 103) AS-Ck inner with Nhe I 5' AAC TAG CTA GCA
GTT CCA GAT TTC AAC TGC TCA TCA GAT 3' (SEQ ID NO: 104) AS-CL outer
5' GCT CCC GGG TAG AAG TCA 3' (SEQ ID NO: 105) AS-CL middle 5'
AC(CT) AGT GTG GCC TTG TTG GCT T 3' (SEQ ID NO: 106) AS-CL inner 5'
GCT CTA GAG GG(CT) GGG AAC AGA GTG AC 3' (SEQ ID NO: 107)
AS.gamma.-CH1 outer 5' ACG ACA CCG TCA CCG GTT 3' (SEQ ID NO: 108)
AS.gamma.-CH1 middle 5' AAG TAG TCC TTG ACC AGG CAG CCC A 3' (SEQ
ID NO: 109) AS.gamma.-CH1 inner with Xba I 5' GCT CTA GAG GGT GCC
AGG GGG AAG ACC GAT 3' (SEQ ID NO: 110) (G1 specific) AS.gamma.-CH1
inner with Xba I 5' GCT CTA GAG CAG GGC GCC AGG GGG AAG A 3' (SEQ
ID NO: 111) (G2, G3 & G4 specific) S-Vk1&2 Leader outer 5'
ATG AGG (CG)TC CC(CT) GCT CAG CT 3' (SEQ ID NO: 112) S-Vk3 Leader
outer 5' ATG GAA (AG)CC CCA GC(GT) CAG CTT 3'(SEQ ID NO: 113) S-Vk4
Leader outer 5' ATG GTG TTG CAG ACC CAG GTC T 3' (SEQ ID NO: 114)
S-Vk1&2 Leader inner with Bgl II 5' GAA GAT CTC ACC ATG AGG
(CG)TC CC(CT) GCT CAG CT(CT) CT 3' (SEQ ID NO: 115) S-Vk3 Leader
inner with Bgl II 5' GAA GAT CTC ACC ATG GAA (AG)CC CCA GC(GT) CAG
CTT CTC TT 3' (SEQ ID NO: 116) S-Vk4 Leader inner with Bgl II 5'
GAA GAT CTC ACC ATG GTG TTG CAG ACC CAG GTC TTC AT 3' (SEQ ID NO:
117) S-VL1-4 Leader outer 5' C(GA)T C(AT)C CAC CAT G(GA)C (CA)(TA)G
3' (SEQ ID NO: 118) S-VL1 Leader outer 5' CAC CAT G(GA)C C(TA)G
(GC)T(CT) CCC T 3' (SEQ ID NO: 119) S-VL2 Leader outer 5' ACC ATG
GCC TGG (GA)CT C(TC)(GT) CT 3' (SEQ ID NO: 120) S-VL3 Leader outer
5' CAC CAT GGC (CA)TG G(GA)(TC) C(CGA)(CT) T 3' (SEQ ID NO: 121)
S-VL4 Leader outer 5' CAC CAT GGC (CT)TG G(GA)(TC) CC(CA) A(CT)T 3'
(SEQ ID NO: 122) S-VL1 Leader inner with Bgl II 5' GAA GAT CTC ACC
ATG (GA)CC (TA)G(GC) T(CT)C CCT CT 3' (SEQ ID NO: 123) S-VL2 Leader
inner with Bgl II 5' GAA GAT CTC ACC ATG GCC TGG (GA)CT C(TC) (GT)
CT(CG) (TC)T 3' (SEQ ID NO: 124) (Also amplifies VL5-7,9,10) S-VL3
Leader inner with Bgl II 5' GAA GAT CTC ACC ATG GC(CA) TGG
(GA)(TC)C (CGA)(CT)T CTC 3' (SEQ ID NO: 125) S-VL4 Leader inner
with Bgl II 5' GAA GAT CTC ACC ATG GC(CT) TGG (GA)(TC)C C(CA)A
(CT)TC 3' (SEQ ID NO: 126) S-VH1 Leader outer 5' CAC CAT GGA (GC)TG
GAC CTG GAG (GCA)(AGTC)T C 3' (SEQ ID NO: 127) S-VH2 Leader outer
5' CAC CAT GGA CAT ACT TTG (CT)TC CAC GCT C 3' (SEQ ID NO: 128)
S-VH3 Leader outer 5' CAC CAT GGA [AG]TT [TG]GG [AG]CT [GCT][ACT]G
CT 3' (SEQ ID NO: 129) S-VH4 Leader outer 5' CAC CAT GAA [AG]CA
[TC]CT GTG GTT CTT CCT [TC]CT 3' (SEQ ID NO: 130) S-VH5 Leader
outer 5' CAC CAT GGG GTC AAC CG[CT[ CAT CCT 3' (SEQ ID NO: 131)
S-VH6 Leader outer 5' CAC CAT GTC TGT CTC CTT CCT CAT CTT C 3' (SEQ
ID NO: 132) S-VH1 Leader inner with Bgl II 5' GAA GAT CTC ACC ATG
GA[GC] TGG ACC TGG AG[GCA] [AGTC]TC C 3' (SEQ ID NO: 133) S-VH2
Leader inner with Bgl II 5' GAA GAT CTC ACC ATG GAC ATA CTT TG[CT]
TCC ACG CTC C 3' (SEQ ID NO: 134) S-VH3 Leader inner with Bgl II 5'
GAA GAT CTC ACC ATG GA[AG] TTGG[AG] CT[GCT] [ACT]GC TGG (GAC)TT
TT(TC) CT 3' (SEQ ID NO: 135) S-VH4 Leader inner with Bgl II 5' GAA
GAT CT C ACC ATG AA[AG] CA[TC] CTG TGG TTC TTC CT[TC] CTC 3' (SEQ
ID NO: 136) S-VH5 Leader inner with Bgl II 5' GAA GAT CTC ACC ATG
GGG TCA ACC G[CT]C ATC CT 3' (SEQ ID NO: 137) S-VH6 Leader inner
with Bgl II 5' GAA GAT CTC ACC ATG TCT GTC TCC TTC CTC ATC TTC T 3'
(SEQ ID NO: 138) S-VH7 Leader inner with Bgl II 5' GAA GAT CTC ACC
ATG GAC TGG ACC TGG AGG ATC CTC TTC TTG GT 3' (SEQ ID NO: 139)
[0390] The primers introduced the following restriction sites at
the 5' end (BgIII) and the 3' end (Xba1) of the heavy chain cDNA.
Similarly, the primers introduced the following restriction sites
at the 5' end (BgIII) and the 3' end (NheI) of the kappa chain
cDNA.
[0391] The variable heavy chain cDNA amplicon was digested with
appropriate restriction enzymes for the restriction enzyme sites
that were added during the PCR reaction. The products of that
digestion were cloned into each of an IgG1, IgG2, and IgG4
expression vector with compatible overhangs for cloning. The IgG2
and IgG4 expression vectors were digested with BamHI and XbaI to
generate compatible overhangs for sub-cloning. The IgG1 expression
construct was digested with BamHI and NheI to generate compatible
overhangs for sub-cloning. These vectors were generated by cloning
the constant domain of human IgG1, IgG2 or IgG4 into the multiple
cloning site of the vector pcDNA3.1+/Hygro (Invitrogen).
[0392] The variable light chain cDNA amplicon was also digested
with appropriate restriction enzymes for the restriction enzyme
sites that were added during the PCR reaction. The products of that
digestion were cloned into an IgK expression vector that had been
digested with BamHI and NheI to provide compatible overhangs for
sub-cloning. That vector was generated by cloning the constant
domain of the human Igk gene into the multiple cloning site of the
vector pcDNA4.1+/Neo (Invitrogen).
[0393] The heavy chain and the light chain expression vectors were
then co-lipofected into a 60 mm dish of 70% confluent human
embryonal kidney 293 cells (ATCC, Cat. No. CRL-1573). For 24 hours,
the transfected cells were allowed to secrete a recombinant
antibody with the identical specificity as the original plasma
cell. The supernatant (3 mL) was harvested from the HEK 293 cells
and the secretion of an intact antibody was demonstrated with a
sandwich ELISA to specifically detect human IgG. Control plates
were coated with 2 mg/mL Goat anti-human IgG H+ L O/N as for
binding plates. The plates were washed five times with water.
Recombinant antibodies were titrated 1:2 for 7 wells from the
undiluted lipofection supernatant. The plates were washed five
times with dH2O. A goat anti-human IgG Fc-specific HRP-conjugated
antibody was added at a final concentration of 1 .mu.g/mL for 1
hour at room temperature for the secretion and the two binding
assays. The plates were washed five times with dH2O. The plates
were developed with the addition of tetramethylbenzidine (TMB) for
30 minutes and the ELISA was stopped by the addition of 1 M
phosphoric acid.
[0394] In addition to the XenoMax methodology described above,
certain antibodies were obtained using hybridoma technology.
Immunized mice were sacrificed by cervical dislocation, and the
draining lymph nodes were harvested and pooled from each cohort.
The lymphoid cells were dissociated by grinding in Dulbecco's
Modified Eagle's Medium ("DMEM") to release the cells from the
tissues. Recovered cells were suspended in DMEM. The cells were
counted, and 0.9 mL DMEM per 100 million lymphocytes was added to
the cell pellet to resuspend the cells gently but completely. The
resuspended cells were incubated with 100 .mu.L of CD90.sup.+
magnetic beads per 100 million cells at 4.degree. C. for 15
minutes. The magnetically-labeled cell suspension (containing up to
10.sup.8 positive cells (or up to 2.times.10.sup.9 total cells))
was loaded onto an LS.sup.+ column. The column was washed with
DMEM. The total effluent was collected as the CD90.sup.-negative
fraction, which was expected to contain mostly B cells.
[0395] The fusion was performed by mixing washed enriched B cells
from above and nonsecretory myeloma P3X63Ag*.653 cells (ATCC (CRL
1580, see, e.g., Kearney et al., J. Immunol. 123, 1979, 1548-1550))
at a ratio of 1:1. The cell mixture was gently pelleted by
centrifugation at 800.times.g. After complete removal of the
supernatant from the cells, the cells were treated with 2 to 4 ml
of Pronase solution (CalBiochem; 0.5 mg/ml in phosphate-buffered
saline ("PBS")) for no more than 2 minutes. Three to five mL of
fetal bovine serum ("FBS") was added to stop the enzyme activity
and the suspension was adjusted to a 40 mL total volume using
electro cell fusion solution ("ECFS") (0.3M sucrose, 0.1 mM
magnesium acetate, 0.1 mM calcium acetate). The supernatant was
removed after centrifugation and the cells were resuspended in 40
mL ECFS. This wash step was repeated and the cells again were
resuspended in 40 mL ECFS to a concentration of 2.times.10.sup.6
cells/mL.
[0396] Electro-cell fusion was performed using a fusion generator
(model ECM2001, Genetronic, Inc.). The fusion chamber size used was
2.0 mL, using the following instrument settings: alignment
condition: 50 V, 50 seconds; membrane breaking at 3000 V, 30
microseconds; post-fusion holding time: 3 seconds.
[0397] After electro-cell fusion took place, the cell suspensions
were carefully removed from the fusion chamber under sterile
conditions and transferred into a sterile tube containing the same
volume of hybridoma culture medium, containing DMEM, (JRH
Biosciences), 15% FBS (Hyclone), supplemented with L-glutamine,
penicillin/streptomycin, OPI (oxaloacetate, pyruvate, bovine
insulin), and IL-6 (Boehringer Mannheim). The cells were incubated
for 15 to 30 minutes at 37.degree. C., and then centrifuged at
400.times.g (1000 rpm) for 5 minutes. The cells were gently
resuspended in a small volume of hybridoma selection medium
(hybridoma culture medium supplemented with 0.5.times. hyaluronic
acid (Sigma)). The total volume was adjusted appropriately with
more hybridoma selection medium based on a final plating volume of
5.times.10.sup.6 B cells total per 96-well plate and 200 .mu.L per
well. The cells were mixed gently, pipetted into 96-well plates,
and allowed to grow. On day 7 or 10, one-half the medium was
removed, and the cells were re-fed with fresh hybridoma selection
medium.
[0398] After 14 days of culture, hybridoma supernatants were
screened for TR2-specific monoclonal antibodies by ELISA. In the
Primary screen, the ELISA plates (Fisher, Cat. No. 12-565-136) were
coated with 50 .mu.L/well of TR2 protein (2 .mu.g/mL) in Coating
Buffer (0.1 M Carbonate Buffer, pH 9.6, NaHCO.sub.3 8.4 g/L), then
incubated at 4.degree. C. overnight. After incubation, the plates
were washed with Washing Buffer (0.05% Tween 20 in PBS) one time.
200 .mu.L/well Blocking Buffer (0.5% BSA, 0.1% Tween 20, 0.01%
Thimerosal in 1.times.PBS) were added and the plates were incubated
at room temperature for 1 hour. After incubation, the plates were
washed with Washing Buffer one time. Aliquots (50 .mu.L/well) of
hybridoma supernatantsand positive and negative controls were
added, and the plates were incubated at room temperature for 2
hours. The positive control used throughout was serum from a
hyperimmune XenoMouse animal and the negative control was serum
from the KLH-immunized XenoMouse animal. After incubation, the
plates were washed three times with Washing Buffer. 100 .mu.L/well
of detection antibody goat anti-huIgGFc-HRP (Caltag Inc., Cat. No.
H10507, using concentration was 1:2000 dilution) was added and the
plates were incubated at room temperature for 1 hour. After
incubation, the plates were washed three times with Washing Buffer.
100 .mu.l/well of TMB (BioFX Lab. Cat. No. TMSK-0100-01) was added,
and the plates were allowed to develop for about 10 minutes (until
negative control wells barely started to show color). 50 .mu.l/well
stop solution (TMB Stop Solution (BioFX Lab. Cat. No. STPR-0100-01)
was then added and the plates were read on an ELISA plate reader at
a wavelength of 450 nm.
[0399] The antibodies produced by the hybridomas were analyzed
using the same apoptosis assay described above. WM-266 cells were
cultured at a density of 4500 cells/well in normal culture medium
overnight in a microtiter plate. A 2.times. apoptosis medium
mixture was prepared using cell culture medium without FCS and
additionally including 1.8 .mu.g/mL cycloheximide and 0.9% FCS.
Separate microtiter plates were used to titrate hybridoma
supernatant 1:2 (in the 2.times. apoptosis medium mixture) in
parallel with an isotype-matched negative control anti-KLH
antibody. The culture media was removed from the WM-266 cells and
100 .mu.L of the antibody-apoptosis medium mixture was added to
each cell-containing well, one row at a time. The microtiter plates
were incubated for 20 hours to allow apoptosis to occur. The
DNA-binding fluorescent dyes propidium iodide (Sigma) and Hoechst
33342 (Molecular Probes) were added to each well at a final
concentration of 0.5 .mu.g/mL and 2.5 .mu.g/mL respectively. After
1 hour at 37.degree. C., fluorescent images of each well were
captured and analyzed for total number of dead cells (PI) and total
number of cells (Hoechst). The percent apoptosis was determined as
(PI-positive cells/Hoechst-positive cells).times.100.
[0400] Seventeen different anti-TR-2 antibodies were obtained
(Antibodies A-Q) using either the XenoMax or hybridoma
methodologies. All of the antibodies were sequenced, and the
sequences of the heavy and light chain variable regions identified
(see FIGS. 3-19). Alignments of the heavy chains and the light
chains of the seventeen antibodies are shown in FIGS. 20 and
21.
[0401] Certain antibodies were examined for their ability to induce
apoptosis in cells, using a similar apoptosis assay to the one
described above. WM-266 melanoma cells were cultured in a
microtiter plate at a density of 4500 cells/well in normal culture
medium overnight. In a separate microtiter plate, the recombinant
antibodies to be tested, an appropriate positive control (M413, a
mouse IgG1 anti-TR-2 antibody having a heavy chain variable
sequence: MEVQLVESGGGLVQPGGSLKLSCAASGFTFSTYGMSWVRQTPDKRLELVA
LINSQGGSTYNSDSVKGRFTISRDNARNTLYLQMSSLKSEDTAMYYCARRD
YESLDSWGQGTSVTVSSG (SEQ ID NO: 141) and a light chain variable
sequence: DIVLTQSPASLPVSLGQRATISCRASESVEYSGTSLIQWYRQKPGQPPKLLIY
AASNVDSEVPARFSGSGSGTDFSLYIHPVEEDDIAMYFCQQSRKVPWTFGG
GTKLEIKRTDAAPGLEAA (SEQ ID NO: 142)), and isotype-matched negative
control antibodies (a potential anti-TR2 antibody that failed to
show activity) were titrated such that the final concentration of
antibody would cover a range of 0.0001 .mu.g/mL to 5 .mu.g/mL. The
antibodies were mixed in apoptosis medium containing a final
concentration of 0.9 .mu.g/mL CHX and 0.45% FCS. The culture media
was removed from the WM-266 cells and the antibody-apoptosis medium
mixture was added to the cells. After 20 hours of culture, the
cells were stained with propidium iodide (Sigma) and Hoechst 33342
(Molecular Probes). After 1 hour at 37.degree. C., an image of each
well was captured and analyzed for total number of dead cells (PI)
and total number of cells (Hoechst). The percent apoptosis was
determined as (PI-positive cells/Hoechst-positive cells).times.100.
Significant cell death was observed in cells treated with M413 or
with certain anti-TR-2 antibodies described above.
Example 2
Kinetic Analyses of Anti-TR-2 Antibody Binding to TR-2
[0402] The kinetics of the binding of anti-TR-2 antibodies A to Q
to TR-2 was analyzed using a Biacore.RTM. 2000 instrument.
High-density goat anti-human antibody surfaces were prepared on
CM-5 Biacore.RTM. chips using routine amine coupling. Each purified
anti-TR-2 antibody was diluted to approximately 1 .mu.g/ml in HBS-P
running buffer containing 100 .mu.g/ml BSA. Each anti-TR-2 antibody
was captured on a separate surface using a two minute contact time
and a five minute wash to stabilize the anti-TR-2 antibody surface
on the chip.
[0403] To analyze the kinetics of TR-2 binding to each individual
anti-TR-2 antibody, 226 nM recombinant human TR-2-His (described in
Example 1) was kinetically injected over each anti-TR-2 surface for
one minute (using kinject) at 25.degree. C., followed by a five
minute dissociation period. The baseline drift resulting from a
buffer injection lacking TR-2 over the anti-TR-2 antibody surface
was subtracted from the observed binding on each of the other
surfaces. Additionally, the data for TR-2 binding to anti-TR-2
antibody were normalized for the amount of monoclonal antibody
captured on each surface. Each data set was fit globally to a 1:1
interaction model to determine binding kinetics. The k.sub.a,
k.sub.d, and K.sub.d values obtained for each antibody are shown in
Table 3.
TABLE-US-00003 TABLE 3 Kinetics of TR-2 binding to anti-TR-2
antibody at 25.degree. C. Antibody k.sub.a (M.sup.-1s.sup.-1)
k.sub.d (s.sup.-1) K.sub.d (nM) A 5.3 .times. 10.sup.5 3.7 .times.
10.sup.-3 6.9 B 5.7 .times. 10.sup.5 1.1 .times. 10.sup.-2 19 C 6.8
.times. 10.sup.5 2.6 .times. 10.sup.-3 3.9 D 6.2 .times. 10.sup.5
2.7 .times. 10.sup.-3 4.5 E 8.7 .times. 10.sup.5 1.8 .times.
10.sup.-3 2.1 F 3.8 .times. 10.sup.5 5.0 .times. 10.sup.-3 13 G 6.0
.times. 10.sup.5 1.9 .times. 10.sup.-2 31 H 8.6 .times. 10.sup.5
8.4 .times. 10.sup.-3 9.8 I 2.9 .times. 10.sup.5 1.3 .times.
10.sup.-3 4.4 J 5.7 .times. 10.sup.5 7.1 .times. 10.sup.-3 12 K 6.8
.times. 10.sup.5 1.2 .times. 10.sup.-2 18 L 6.0 .times. 10.sup.5
1.1 .times. 10.sup.-2 18 M 3.4 .times. 10.sup.5 1.2 .times.
10.sup.-2 37 N 8.1 .times. 10.sup.5 5.5 .times. 10.sup.-2 68* O 4.4
.times. 10.sup.5 8.4 .times. 10.sup.-3 19 P 8.1 .times. 10.sup.5
2.7 .times. 10.sup.-2 33* Q 1.2 .times. 10.sup.6 1.6 .times.
10.sup.-2 13* *Data for that sample exhibited heterogeneity and fit
poorly to a 1:1 model.
Example 3
Cell Killing Assays
[0404] Cell killing assays were performed with certain human
anti-TR-2 antibodies described in Example 2 to determine the degree
to which each antibody triggered apoptosis and cell death. Certain
human anti-TR-2 antibodies, as well as mouse anti-TR-2 antibodies
M412 and M413, were immobilized in separate wells of 96-well
Protein G-coated plates (reactin-bind Protein G coated plates,
Pierce Cat. No. 15131). M412 is a mouse IgG1 anti-TR-2 antibody
having a heavy chain variable sequence:
KVQLQQSGTELVKPGASVKLSCKASGYTFTEYIIHWVKQRSGQGLEWIGWF
YPGSGYIKYNEKFKDKATMTADKSSSTVYMELSRLTSEDSAVYFCTRHEED
GYYAAYWGQGTLVTVSA (SEQ ID NO: 143) and a light chain variable
sequence: DIVMTQSH KFMSTSVGDRVSITCKASQDVSSAVAWYQQKPGQSPKLLIYWA
STRHTGVPDRFTGSGSGTDYTLTISSVQAEDLALYYCQQHYSTPYTFGGGT KLEIKR (SEQ ID
NO: 144). M413 is a mouse IgG1 anti-TR-2 antibody as described
above in Example 1. Each antibody was added at a concentration of
50 .mu.g/ml to a first well, and serially diluted 1:3.times. in
each of seven additional wells. Each antibody dilution was
performed in triplicate. Plates were incubated for 24 hours at
4.degree. C. prior to use. Following the washing of each well with
culture media (RPMI plus 10% FBS), one of four different cell lines
was plated onto each immobilized antibody, at a density of 50,000
cells per well in a total volume of 200 .mu.L. The cell lines
tested were COLO 205 cells (human colon adenocarcinoma), MDA-231
cells (human breast cancer), WM35 cells (human melanoma), and WM793
cells (human melanoma). Cells were incubated at 37.degree. C./6%
CO.sub.2 for 24 hours, followed by a 6 hour incubation with
.sup.3H-thymidine. The percentage of viable cells was assessed by
determining the level of .sup.3H-thymidine incorporation in the
treated cells relative to the level of .sup.3H-thymidine
incorporation into the untreated cells. The ED.sub.50 of each
antibody was derived from the cell viability titration curve by
determining the concentration of antibody that reduced the
viability of treated cells by 50% relative to untreated cells. The
ED.sub.50 of the human antibodies for COLO 205 cells ranged from 0
.mu.g/ml to 3.25 .mu.g/ml. The mouse antibodies M412 and M413 had
ED.sub.50s of 1.85 .mu.g/ml and 0.07 .mu.g/ml, respectively, for
those cells. The ED.sub.50 of the human antibodies for MDA-231
cells ranged from 0.05 .mu.g/ml to 0.5 .mu.g/ml. The mouse
antibodies M412 and M413 had ED.sub.50s of 0.6 .mu.g/ml and 0.07
.mu.g/ml, respectively, for those cells. The ED.sub.50 of the human
antibodies for WM35 cells ranged from 0.1 .mu.g/ml to 0.6 .mu.g/ml.
The mouse antibodies M412 and M413 had ED.sub.50s of 1.85 .mu.g/ml
and 0.07 .mu.g/ml, respectively, for those cells. The ED.sub.50 of
the human antibodies for WM793 cells ranged from 0.02 .mu.g/ml to
0.2 .mu.g/ml. The mouse antibodies M412 and M413 had ED.sub.50s of
1.85 .mu.g/ml and 0.05 .mu.g/ml, respectively, for those cells.
Example 4
Human TR-2 Expression in Tumor Cell Lines
[0405] Human tumor cell lines were screened for expression of TR-2.
Cell lines used included those from breast, central nervous system,
colon, liver, lung, cervix, uterine, ovarian, pancreatic, prostate,
and renal cancers, as well as leukemia and melanoma.
[0406] The expression of TR-2 on human tumor cells was determined
using a cell-based array. Briefly, 4.times.10.sup.5 cells in 100 MI
CBA buffer (PBS, 3% FBS, 0.02% Azide) were distributed into each of
the wells of 20 V-bottom 96-well plates. CBA buffer (150 .mu.L) was
added to each well and the plates were centrifuged to spin down the
cells. The medium was discarded and 100 .mu.L of antibody solution
(one of the antibodies A to Q) at 10 .mu.g/ml was added to the cell
pellet resuspended in PBS containing 2% PBS ("assay buffer"). After
a 25 minute incubation on ice, the cells were washed once in assay
buffer. 100 .mu.L of a secondary goat anti-human IgG Fc-specific
horseradish peroxidase (HRP, Pierce) was added to the wells, and
the plates were incubated on ice for 20 minutes. The plates were
washed twice with assay buffer, and 100 .mu.L of the TMB substrate
(ZYMED) was added for 10 minutes at room temperature. The plates
were centrifuged and 50 .mu.L of each supernate was transferred
into a clean plate containing 50 .mu.L stop solution (BioFX
Laboratories). Optical density readings were performed at 450 nm
using the SpectraMax/plus reader (Molecular Devices). The data were
normalized by subtracting the optical density values obtained from
an isotype control antibody.
[0407] Several cell lines had an OD.sub.450 greater than 0.1 in the
assay, including breast cancer cell lines HS 578.T (OD of 0.122)
and T-47D (OD of 0.112), colon cancer cell lines TE 671(u) (OD of
0.109), HT-29 (OD of 0.193), SW-948 (OD of 0.122), KM-12 (OD of
0.354), and HCC-2998 (OD of 0.133), liver cancer cell lines NCI-N87
(OD of 0.154) and NCI-SNU-5 (OD of 0.137), leukemia cell lines
HL-60 (OD of 0.233) and hPBMC(OD of 0.131), non-small-cell lung
cancer cell line JY (OD of 0.118), CCRF-CEM (OD of 0.106),
NCI-H2126 (OD of 0.108) and NCI-H460 (OD of 0.122), melanoma cell
lines SK-mel-5 (OD of 0.131), LOX IMVI (OD of 0.102), RPMI 7951 (OD
of 0.101), and UACC-62 (OD of 0.127), pancreas cancer cell lines
HPAF II (OD of 0.117) and CAPAN-1 (OD of 0.101), prostate cancer
cell line LNCaP (OD of 0.174), and renal carcinoma cell lines
Caki-1 (OD of 0.148) and UO-31 (OD of 0.104). The greatest
expression of TR-2 among the tumor cell lines studied was found in
colon cancer cell lines KM-12 and HT-29, and in leukemia cell line
HL-60. None of the central nervous system, small-cell liver,
cervical, uterine, or ovarian cancer cell lines studied had an
OD450 greater than background.
[0408] To determine TR-2 expression profile on human tumor cell
lines, the above human tumor cell lines were assayed with the mouse
anti-TR-2 antibody M412. The expression of TR-2 on human tumor
cells was determined using a cell-based array. Briefly,
4.times.10.sup.5 cells in 100 MI CBA buffer (PBS, 3% FBS, 0.02%
Azide) were distributed into each of the wells of 20 V-bottom
96-well plates. CBA buffer (150 .mu.L) was added to each well and
the plates were centrifuged to spin down the cells. The medium was
discarded and 100 .mu.L of mouse anti-TR-2 monoclonal antibody M412
at 10 .mu.g/ml was added to the cell pellet resuspended in PBS
containing 2% PBS ("assay buffer"). After a 25 minute incubation on
ice, the cells were washed once in assay buffer. 100 .mu.L of a
secondary goat anti-mouse IgG Fc-specific horseradish peroxidase
(HRP, Pierce) was added to the wells, and the plates were incubated
on ice for 20 minutes. The plates were washed twice with assay
buffer, and 100 .mu.L of the TMB substrate (ZYMED) was added for 10
minutes at room temperature. The plates were centrifuged and 50
.mu.L of each supernate was transferred into a clean plate
containing 50 .mu.L stop solution (BioFX Laboratories). Optical
density readings were performed at 450 nm using the SpectraMax/plus
reader (Molecular Devices). The data were normalized by subtracting
the optical density values obtained from an isotype control
antibody.
[0409] Many of the cell lines had TR-2 expression. The highest
expressors (those with an OD.sub.450 nm greater than 0.3) included
breast cancer cell lines HS 578.T (OD of 0.403), MDA-MB-231 (OD of
0.408), and T-47D (OD of 0.366), CNS cancer cell lines SF-295 (OD
of 0.354) and U251 (OD of 0.323), colon cancer cell lines HCT-116
(OD of 0.41), HT-29 (OD of 0.869), SW-707 (OD of 0.323), SW-948 (OD
of 0.423), KM-12 (OD of 0.77), and HCC-2998 (OD of 0.635), liver
cancer cell line NCI-SNU-1 (OD of 0.354), leukemia cell line A 673
(OD of 0.347), non-small-cell lung cancer cell lines HOP-62 (OD of
0.313), HOP-62 (OD of 0.47), NCI-H2126 (OD of 0.501), NCI-H460 (OD
of 0.326), small cell lung cancer line A549 (OD of 0.381), melanoma
cell lines LOX IMVI (OD of 0.573), RPMI 7951 (OD of 0.322), and
UACC-62 (OD of 0.319), ovarian cancer cell line IGROV1 (OD of
0.312), prostate cancer cell lines DU 145 (OD of 0.372), 22Rv1 (OD
of 0.301), and LNCaP(OD of 0.63), and renal carcinoma cell lines
Caki-1 (OD of 0.93), Caki-2 (OD of 0.443), SN12C(OD of 0.313), and
UO-31 (OD of 0.331). The greatest expression of TR-2 among the
tumor cell lines treated with mouse anti-TR-2 antibody was found in
renal carcinoma cell line Caki-1, and in colon cancer cell lines
HT-29 and KM-12.
Example 5
Antibody Cross-Reactivity
[0410] The ability of certain of the human anti-TR-2 antibodies to
block the binding of the others to TR-2 was assessed, as described
in Jia et al., J. Immunol. Methods 288: 91-98 (2004). The beads
were conjugated with anti-human IgG antibodies using the coupling
procedure taken directly from the Luminex 100 User's Manual,
Version 1.7. After the beads were activated, they were coupled to a
Pharmingen mouse anti-hIgG mAb, following the manufacturer's
instructions. Two experiments were performed. In a first
experiment, the coated beads were incubated for two hours at room
temperature. In a second experiment, the coated beads were
incubated overnight at 4.degree. C. At the end of the incubation,
the coated beads were blocked and then counted using a Coulter cell
counter. Conjugated beads were either used immediately or were
stored at 4.degree. C. in the dark for future use.
[0411] The categorization of the anti-TR-2 antibodies based on
epitope cross-reactivity was performed by the following steps.
First, each set of bead-mouse anti-hIgG complexes from above were
separately incubated with a reference antibody ("reference
antibody") on a rotator overnight at 4.degree. C. The reference
antibody was selected from anti-TR-2 antibodies A-Q, described
above. After antibody capture, 2000 of each bead-mouse
anti-hIgG-reference Ab complexes were pooled together in one tube,
and then immediately added to each well of a 96-well plate and
aspirated. TR-2 (50 ng) was added to each well and incubated for 1
hour at room temperature. After washing the wells, 100-500 ng/mL of
another of the human anti-TR-2 antibodies (the "probe antibody")
was added to each well and incubated for 2 hours at room
temperature. After washing the wells, bound probe antibody was
detected using 1 .mu.g/ml of a biotinylated version of the same
monoclonal mouse anti-hIgG used for capturing the reference
antibody. Following incubation and washing of the wells, 0.5
.mu.g/ml streptavidin-phycoerythrin was added. The mixture was
incubated for 30 minutes at room temperature and then the
phycoerythrin signal was detected using the Luminex 100. An
additional set of wells lacking antigen was used as a negative
control to aid in data analysis.
[0412] The data was analyzed in a two-step process. First, the data
was normalized using the negative control values. Second, the
anti-TR-2 antibodies were clustered according to their ability to
impede binding of one or more other anti-TR-2 antibodies. For the
clustering analysis, a dissimilarity matrix was generated from the
normalized intensity matrix. Antibodies were clustered based on the
values in the average dissimilarity matrix using the SPLUS 2000
agglomerative nesting hierarchical clustering subroutine with the
Manhattan metric, using an input dissimilarity matrix of the actual
average dissimilarity matrix.
[0413] Based on the findings, the antibodies were placed into four
different epitope groups. Within any one group, the binding of one
of the group members to TR-2 blocks the binding of another member
of the same group to TR-2. However, the binding of one of the
members of group 1 to TR-2, for example, does not block the binding
of one of the members of groups 2, 3, or 4 to TR-2. Those groups
are shown in FIG. 22.
Example 6
Epitope Mapping
[0414] To identify the specific region of TR-2 important for
binding to certain described anti-TR-2 antibodies, an epitope
mapping study was performed. An N-avidin-TR-2 construct was made by
PCR-amplifying the coding sequence for mature TR-2 (MacFarlane,
1997) from a template source and cloning it into a pCEP4 vector
(Invitrogen) containing the chicken avidin sequence in an
orientation such that upon insertion at a HindIII site, the TR-2
sequence was joined at the C-terminus of the avidin sequence. The
forward primer for the mature TR-2 coding sequence was
GTAAGCAAGCTTGGCTC TGATCACCCAACAAGA (SEQ ID NO: 145), and the
reverse primer was GATTAGGGATCCAGAGGCAGGAGTCCCTGG (SEQ ID NO: 146).
The amino acid sequence of the resulting avidin-TR-2 fusion protein
was MVHATSPLLLLLL
LSLALVAPGLSARKCSLTGKWTNDLGSNMTIGAVNSKGEFTGTYTTAVTATS
NEIKESPLHGTQNTINKRTQPTFGFTVNWKFSESTTVFTGQCFIDRNGKEVL
KTMWLLRSSVNDIGDDWKATRVGINIFTRLRTQKEQLLASLALITQQDLAPQ
QRAAPQQKRSSPSEGLCPPGHHISEDGRDCISCKYGQDYSTHWNDLLFCL
RCTRCDSGEVELSPCTTTRNTVCQCEEGTFREEDSPEMCRKCRTGCPRG
MVKVGDCTPWSDIECVHKESGTKHSGEAPAVEETVTSSPGTPAS (SEQ ID NO: 69).
[0415] Twelve molecules comprising N-avidin and truncations of
human TR-2 were synthesized as described below. Three molecules had
only C-terminal truncations of human TR-2 (TR-2-1 through TR-2-3),
and nine molecules had truncations at both the N- and the
C-terminus of human TR-2 (TR-2-4 through TR-2-13) (shown
schematically in FIG. 23). Polynucleotides encoding human TR-2
truncations were prepared by PCR amplification using the primers
described below. To form each of the twelve molecules, the
truncated human TR-2 resulting from the amplification was inserted
into the pCEP4 vector (Invitrogen) containing the chicken avidin
sequence that is described above. The polynucleotide encoding amino
acids 1-43 of mature TR-2 was amplified using the forward primer
GTAAGCAAGCTTGGCTCTGATCACCCAACAAGA (SEQ ID NO: 145) and the reverse
primer TAGTTGGGATCCTCAGGAGATGCAATCTCT ACCGT (SEQ ID NO: 147). The
amino acid sequence of TR-2-1 was
MVHATSPLLLLLLLSLALVAPGLSARKCSLTGKWTNDLGSNMTIGAVNS
KGEFTGTYTTAVTATSNEIKESPLHGTQNTINKRTQPTFGFTVNWKFSESTT
VFTGQCFIDRNGKEVLKTMWLLRSSVNDIGDDWKATRVGINIFTRLRTQKEQ
LLASLALITQQDLAPQQRAAPQQKRSSPSEGLCPPGHHISEDGRDCIS (SEQ ID NO:
70).
[0416] The polynucleotide encoding amino acids 1-85 of mature TR-2
was amplified using the forward primer
GTAAGCAAGCTTGGCTCTGATCACCCAACAAGA (SEQ ID NO: 145) and the reverse
primer GGTAGTGGATCCTCACTGACACACTGTGTTTCTGG (SEQ ID NO: 148). The
amino acid sequence of TR-2-2 was
MVHATSPLLLLLLLSLALVAPGLSARKCSLTGKWTNDLGSNMTIGAVNSKG
EFTGTYTTAVTATSNEIKESPLHGTQNTINKRTQPTFGFTVNWKFSESTTVFT
GQCFIDRNGKEVLKTMWLLRSSVNDIGDDWKATRVGINIFTRLRTQKEQLLA
SLALITQQDLAPQQRAAPQQKRSSPSEGLCPPGHHISEDGRDCISCKYGQD
YSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVCQ (SEQ ID NO: 71).
[0417] The polynucleotide encoding amino acids 1-126 of mature TR-2
was amplified using the forward primer GTAAGCAAGCTTGGCTCTGATC
ACCCAACAAGA (SEQ ID NO: 145) and the reverse primer GTAATGGGATCCTC
AGACACATTCGATGTCACTCC (SEQ ID NO: 149). The amino acid sequence of
TR-2-3 was MVHATSPLLLLLLLSLALVAPGLSARKCSLTGKWTNDLGSNMTIGA
VNSKGEFTGTYTTAVTATSNEIKESPLHGTQNTI NKRTQPTFGFTVNWKFSE
STTVFTGQCFIDRNGKEVLKTMWLLRSSVNDIGDDWKATRVGINIFTRLRTQ
KEQLLASLALITQQDLAPQQRAAPQQKRSSPSEGLCPPGHHISEDGRDCISC
KYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVCQCEEGTFREE
DSPEMCRKCRTGCPRGMVKVGDCTPWSDIECV (SEQ ID NO: 72).
[0418] The polynucleotide encoding amino acids 16-43 of mature TR-2
was amplified using the forward primer GTAATGAAGCTTGCCACAACA
AAAGAGGTCCAG (SEQ ID NO: 150) and the reverse primer TAGTTGGGAT
CCTCAGGAGATGCAATCTCTACCGT (SEQ ID NO: 147). The amino acid sequence
of TR-2-4 was MVHATSPLLLLLLLSLALVAPGLSARKCSLTGKWTNDLGSNMTIGAVNSKGE
FTGTYTTAVTATSNEIKESPLHGTQNTINKRTQPTFGFTVNWKFSESTTVFT
GQCFIDRNGKEVLKTMWLLRSSVNDIGDDWKATRVGINIFTRLRTQKEQLLA
SLPQQKRSSPSEGLCPPGHHISEDGRDCIS (SEQ ID NO: 73).
[0419] The polynucleotide encoding amino acids 16-85 of mature TR-2
was amplified using the forward primer GTAATGAAGCTTGCCACAACAAA
AGAGGTCCAG (SEQ ID NO: 150) and the reverse primer GGTAGTGGA
TCCTCACTGACACACTGTGTTTCTGG (SEQ ID NO: 148). The amino acid
sequence of TR-2-5 was MVHATSPLLLLLLLSLALVAPGLSARKCSLTGKWTNDL
GSNMTIGAVNSKGEFTGTYTTAVTATSNEIKESPLHGTQNTINKRTQPTFGF
TVNWKFSESTTVFTGQCFIDRNGKEVLKTMWLLRSSVNDIGDDWKATRVGI
NIFTRLRTQKEQLLASLPQQKRSSPSEGLCPPGHHISEDGRDCISCKYGQDY
STHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVCQ (SEQ ID NO: 74).
[0420] The polynucleotide encoding amino acids 16-126 of mature
TR-2 was amplified using the forward primer GTAATGAAGCTTGCCACAACAAA
AGAGGTCCAG (SEQ ID NO: 150) and the reverse primer GTAATGGGATCCTCA
GACACATTCGATGTCACTCC (SEQ ID NO: 149). The amino acid sequence of
TR-2-6 was MVHATSPLLLLLLLSLALVAPGLSARKCSLTGKWTNDLGSNMTIGA
VNSKGEFTGTYTTAVTATSNEIKESPLHGTQNTINKRTQPTFGFTVNWKFSE
STTVFTGQCFIDRNGKEVLKTMWLLRSSVNDIGDDWKATRVGINIFTRLRTQ
KEQLLASLPQQKRSSPSEGLCPPGHHISEDGRDCISCKYGQDYSTHWNDLL
FCLRCTRCDSGEVELSPCTTTRNTVCQCEEGTFREEDSPEMCRKCRTGCP
RGMVKVGDCTPWSDIECV (SEQ ID NO: 75).
[0421] The polynucleotide encoding amino acids 42-85 of mature TR-2
was amplified using the forward primer
GATTGAAAGCTTGATCTCCTGCAAATATGGACAG (SEQ ID NO: 151) and the reverse
primer GGTAGTGGATCCTCACTGACACACTGTGTTTCTGG (SEQ ID NO: 148). The
amino acid sequence of TR-2-7 was
MVHATSPLLLLLLLSLALVAPGLSARKCSLTGKWTNDLGSNMTIGAVNS
KGEFTGTYTTAVTATSNEIKESPLHGTQNTINKRTQPTFGFTVNWKFSESTT
VFTGQCFIDRNGKEVLKTMWLLRSSVNDIGDDWKATRVGINIFTRLRTQKEQ
LLASLISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVCQ (SEQ ID NO:
76).
[0422] The polynucleotide encoding amino acids 42-126 of mature
TR-2 was amplified using the forward primer
GATTGAAAGCTTGATCTCCTGCAAATATGGACAG (SEQ ID NO: 151) and the reverse
primer GTAATGGGATCCTCAGACACATTCGATGTCACTCC (SEQ ID NO: 149). The
amino acid sequence of TR-2-9 was
MVHATSPLLLLLLLSLALVAPGLSARKCSLTGKWTNDLGSNMTIGA
VNSKGEFTGTYTTAVTATSNEIKESPLHGTQNTINKRTQPTFGFTVNWKFSE
STTVFTGQCFIDRNGKEVLKTMWLLRSSVNDIGDDWKATRVGINIFTRLRTQ
KEQLLASLISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVC
QCEEGTFREEDSPEMCRKCRTGCPRGMVKVGDCTPWSDIECV (SEQ ID NO: 77).
[0423] The polynucleotide encoding amino acids 85-154 of mature
TR-2 was amplified using the forward primer
GTAATGAAGCTTGCAGTGCGAAGAAGGCACCT (SEQ ID NO: 152) and the reverse
primer GATTAGGGATCCAGAGGCAGGAGTCCCTGG (SEQ ID NO: 146). The amino
acid sequence of TR-2-10 was
MVHATSPLLLLLLLSLALVAPGLSARKCSLTGKWTNDLGSNMTIGAVNSKG
EFTGTYTTAVTATSNEIKESPLHGTQNTINKRTQPTFGFTVNWKFSESTTVFT
GQCFIDRNGKEVLKTMWLLRSSVNDIGDDWKATRVGINIFTRLRTQKEQLLA
SLQCEEGTFREEDSPEMCRKCRTGCPRGMVKVGDCTPWSDIECVHKESGT
KHSGEAPAVEETVTSSPGTPAS (SEQ ID NO: 78).
[0424] The polynucleotide encoding amino acids 42-154 of mature
TR-2 was amplified using the forward primer GATTGAAAGCTTGATCTCCTGC
AAATATGGACAG (SEQ ID NO: 151) and the reverse primer GATTAGGGATCCA
GAGGCAGGAGTCCCTGG (SEQ ID NO: 146). The amino acid sequence of
TR-2-11 was MVHATSPLLLLLLLSLALVAPGLSARKCSLTGKWTNDLGSNMTIGAVN
SKGEFTGTYTTAVTATSNEIKESPLHGTQNTINKRTQPTFGFTVNWKFSEST
TVFTGQCFIDRNGKEVLKTMWLLRSSVNDIGDDWKATRVGINIFTRLRTQKE
QLLASLISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVCQC
EEGTFREEDSPEMCRKCRTGCPRGMVKVGDCTPWSDIECVHKESGTKHS
GEAPAVEETVTSSPGTPAS (SEQ ID NO: 79).
[0425] The polynucleotide encoding amino acids 16-66 of mature TR-2
was amplified using the forward primer TGATTGAAGCTTGCCACAACAA
AAGAGGTCCAG (SEQ ID NO: 150) and the reverse primer GATGGAGGATCCT
CAACACCTGGTGCAGCGCAAG (SEQ ID NO: 153). The amino acid sequence of
TR-2-12 was MVHATSPLLLLLLLSLALVAPGLSARKCSLTGKWTNDLGSNMTI
GAVNSKGEFTGTYTTAVTATSNEIKESPLHGTQNTINKRTQPTFGFTVNWKF
SESTTVFTGQCFIDRNGKEVLKTMWLLRSSVNDIGDDWKATRVGINIFTRLR
TQKEQLLASLPQQKRSSPSEGLCPPGHHISEDGRDCISYKYGQDYSTHWND LLFCLRCTRC (SEQ
ID NO: 80).
[0426] The polynucleotide encoding amino acids 16-74 of mature TR-2
was amplified using the forward primer TGATTGAAGCTTGCCACAACA
AAAGAGGTCCAG (SEQ ID NO: 150) and the reverse primer GTAAGTGGATCC
TCAGCAGGGACTTAGCTCCACT (SEQ ID NO: 154). The amino acid sequence of
TR-2-13 was MVHATSPLLLLLLLSLALVAPGLSARKCSLTGKWTNDLGSNMT
IGAVNSKGEFTGTYTTAVTATSNEIKESPLHGTQNTINKRTQPTFGFTVNWK
FSESTTVFTGQCFIDRNGKEVLKTMWLLRSSVNDIGDDWKATRVGINIFTRL
RTQKEQLLASLPQQKRSSPSEGLCPPGHHISEDGRDCISCKYGQDYSTHW
NDLLFCLRCTRCDSGEVELS (SEQ ID NO: 81). Four molecules comprising
N-avidin and truncations of TR-2 from cynomolgus monkey were
synthesized as described below. The polynucleotide encoding amino
acids 1 to 132 of mature cyno TR-2 was amplified using the forward
primer GTTAGTAAGCTTGGCTCCAATCACCCGAC (SEQ ID NO: 155) and the
reverse primer GTTGATGGATCCTTCTTTGTGGACACTCGAT (SEQ ID NO: 156).
The amino acid sequence of cyno TR-2 (short) was MVHATS
PLLLLLLLSLALVAPGLSARKCSLTGKWTNDLGSNMTIGAVNSKGEFTGTYT
TAVTATSNEIKESPLHGTQNTINKRTQPTFGFTVNWKFSESTTVFTGQCFIDR
NGKEVLKTMWLLRSSVNDIGDDWKATRVGINIFTRLRTQKEQLLASLAPITR
QSLDPQRRAAPQQKRSSPTEGLCPPGHHISEDSRDCISCKYGQDYSTHWN
DFLFCLRCTKCDSGEVEVSSCTTTRNTVCQCEEGTFREEDSPEICRKCRTG
CPRGMVKVKDCTPWSDIECPQRRIQT (SEQ ID NO: 82).
[0427] The polynucleotide encoding amino acids 1 to 154 of mature
cyno TR-2 was amplified using the forward primer
GTTAGTAAGCTTGGCTCCA ATCACCCGAC (SEQ ID NO: 155) and the reverse
primer GTAGTTGGATCCTC AAGAAGCAGGAGTCCCAGGG (SEQ ID NO: 157). The
amino acid sequence of cyno TR-2 (long) was
MVHATSPLLLLLLLSLALVAPGLSARKCSLTGKWTNDLG
SNMTIGAVNSKGEFTGTYTTAVTATSNEIKESPLHGTQNTINKRTQPTFGFTV
NWKFSESTTVFTGQCFIDRNGKEVLKTMWLLRSSVNDIGDDWKATRVGINIF
TRLRTQKEQLLASLAPITRQSLDPQRRAAPQQKRSSPTEGLCPPGHHISEDS
RDCISCKYGQDYSTHWNDFLFCLRCTKCDSGEVEVSSCTTTRNTVCQCEE
GTFREEDSPEICRKCRTGCPRGMVKVKDCTPWSDIECVHKESGTKHTGEV PAVEKTVTTSPGTPAS
(SEQ ID NO: 83).
[0428] The polynucleotide encoding amino acids 1 to 85 of mature
cyno TR-2 was amplified using the forward primer
GTTAGTAAGCTTGGCTCCA ATCACCCGAC (SEQ ID NO: 155) and the reverse
primer GTATGAGGGATCCTC ACTGACACACCGTGTTTCTGG (SEQ ID NO: 158). The
amino acid sequence of cyno 1-85 was
MVHATSPLLLLLLLSLALVAPGLSARKCSLTGKWTNDLGSNMT
IGAVNSKGEFTGTYTTAVTATSNEIKESPLHGTQNTINKRTQPTFGFTVNWK
FSESTTVFTGQCFIDRNGKEVLKTMWLLRSSVNDIGDDWKATRVGINIFTRL
RTQKEQLLASLAPITRQSLDPQRRAAPQQKRSSPTEGLCPPGHHISEDSRD
CISCKYGQDYSTHWNDFLFCLRCTKCDSGEVEVSSCTTTRNTVCQ (SEQ ID NO: 84).
[0429] The polynucleotide encoding amino acids 16 to 85 of mature
cyno TR-2 was amplified using the forward primer
GTATGGAAGCTTGCCACAA CAAAAGAGATCCAGC (SEQ ID NO: 159) and the
reverse primer GTATGAGGG ATCCTCACTGACACACCGTGTTTCTGG (SEQ ID NO:
158). The amino acid sequence of cyno 16-85 was
MVHATSPLLLLLLLSLALVAPGLSARKCSLTGKW
TNDLGSNMTIGAVNSKGEFTGTYTTAVTATSNEIKESPLHGTQNTINKRTQP
TFGFTVNWKFSESTTVFTGQCFIDRNGKEVLKTMWLLRSSVNDIGDDWKAT
RVGINIFTRLRTQKEQLLASLPQQKRSSPIEGLCPPGHHISEDSRDCISCKYG
QDYSTHWNDFLFCLRCTKCDSGEVEVSSCTTTRNTVCQ (SEQ ID NO: 85).
[0430] Four N-avidin-fused chimeras were also made using different
portions of human TR-2 and cyno TR-2, as shown in FIG. 25. Each
chimera was constructed by preparing two PCR products with
overlapping ends that were then amplified together using the same
5' and 3' primers. To form each of the chimeras, the amplified
polynucleotide was then subcloned into the pCEP4 vector
(Invitrogen) containing the chicken avidin sequence that is
described above. An alignment of the human, cyno (short), and mouse
TR-2 sequences is shown in FIG. 26.
[0431] Cyno/human chimera #1 was prepared by amplifying a region of
mature cyno TR-2 corresponding to amino acids 1-16 using the
forward primer GTTAGTAAGCTTGGCTCCAATCACCCGAC (SEQ ID NO: 155) and
the reverse primer GGACCTCTTTTGTTGTGGAGCCGCTCTTCGCTGG (SEQ ID NO:
159) and amplifying a region of mature human TR-2 corresponding to
amino acids 17-85 using the forward primer
CAGCGAAGAGCGGCTCCACAACAAAAG AGGTCCAG (SEQ ID NO: 160) and the
reverse primer GGTAGTGGATCCTCACT GACACACTGTGTTTCTGG (SEQ ID NO:
148). Overlapping PCR of the cyno and human TR-2 fragments was
performed using the forward primer for the cyno TR-2 amino acids
1-16 fragment, above (SEQ ID NO: 155) and the reverse primer for
the human TR-2 amino acids 17-85 fragment, above (SEQ ID NO: 148).
The amino acid sequence for cyno/human chimera #1 was
MVHATSPLLLLLLLSLALVAPGLSARKCSLTGKWT
NDLGSNMTIGAVNSKGEFTGTYTTAVTATSNEIKESPLHGTQNTINKRTQPT
FGFTVNWKFSESTTVFTGQCFIDRNGKEVLKTMWLLRSSVNDIGDDWKATR
VGINIFTRLRTQKEQLLASLAPITRQSLDPQRRAAPQQKRSSPSEGLCPPGH
HISEDGRDCISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTV CQ (SEQ ID NO:
86).
[0432] Cyno/human chimera #2 was prepared by amplifying a region of
mature cyno TR-2 corresponding to amino acids 1-16 using the
forward primer GTTAGTAAGCTTGGCTCCAATCACCCGAC (SEQ ID NO: 155) and
the reverse primer GGACCTCTTTTGTTGTGGAGCCGCTCTTCGCTGG (SEQ ID NO:
159) and amplifying a region of mature human TR-2 corresponding to
amino acids 17-154 using the forward primer
CAGCGAAGAGCGGCTCCACAACAAAA GAGGTCCAG (SEQ ID NO: 160) and the
reverse primer GATTAGGGATCCTCAA GAGGCAGGAGTCCCTGG (SEQ ID NO: 146).
Overlapping PCR of the cyno and human TR-2 fragments was performed
using the forward primer for the cyno TR-2 amino acids 1-16
fragment, above (SEQ ID NO: 155) and the reverse primer for the
human TR-2 amino acids 17-154 fragment, above (SEQ ID NO: 146). The
amino acid sequence for cyno/human chimera #2 was
MVHATSPLLLLLLLSLALVAPGLSARKCSLTGKW
TNDLGSNMTIGAVNSKGEFTGTYTTAVTATSNEIKESPLHGTQNTINKRTQP
TFGFTVNWKFSESTTVFTGQCFIDRNGKEVLKTMWLLRSSVNDIGDDWKAT
RVGINIFTRLRTQKEQLLASLAPITRQSLDPQRRAAPQQKRSSPSEGLCPPG
HHISEDGRDYISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNT
VCQCEEGTFREEDSPEMCRKCRTGCPRGMVKVGDCTPWSDIECVHKESG
TKHSGEAPAVEETVTSSPGTPAS (SEQ ID NO: 87).
[0433] Cyno/human chimera #3 was prepared by amplifying a region of
mature human TR-2 corresponding to amino acids 1-16 using the
forward primer GTAAGCAAGCTTGGCTCTGATCACCCAACAAGA (SEQ ID NO: 145)
and the reverse primer GGATCTCTTTTGTTGTGGGGCCGCTCTCTGCTGG G (SEQ ID
NO: 161) and amplifying a region of mature cynoTR-2 corresponding
to amino acids 17-85 using the forward primer CAGCAGAGAGCGGCCCCACA
ACAAAAGAGATCCAGC (SEQ ID NO: 162) and the reverse primer GTATGAGG
GATCCTCACTGACACACCGTGTTTCTGG (SEQ ID NO: 158). Overlapping PCR of
the cyno and human TR-2 fragments was performed using the forward
primer for the human TR-2 amino acids 1-16 fragment, above (SEQ ID
NO: 145) and the reverse primer for the cyno TR-2 amino acids 17-85
fragment, above (SEQ ID NO: 158). The amino acid sequence for
cyno/human chimera #3 was MVHATSPLLLLLLLSLALVAPGLSAR
KCSLTGKWTNDLGSNMTIGAVNSKGEFTGTYTTAVTATSNEIKESPLHGTQN
TINKRTQPTFGFTVNWKFSESTTVFTGQCFIDRNGKEVLKTMWLLRSSVNDI
GDDWKATRVGINIFTRLRTQKEQLLASLALITQQDLAPQQRAAPQQKRSSPT
EGLCPPGHHISEDSRDCISCKYGQDYSTHWNDFLFCLRCTKCDSGEVEVSS CTTTRNTVCQ (SEQ
ID NO: 88).
[0434] Cyno/human chimera #4 was prepared by amplifying a region of
mature human TR-2 corresponding to amino acids 1-16 using the
forward primer GTAAGCAAGCTTGGCTCTGATCACCCAACAAGA (SEQ ID NO: 145)
and the reverse primer GGATCTCTTTTGTTGTGGGGCCGCTCTCTGCTGG G (SEQ ID
NO: 161) and amplifying a region of mature cyno TR-2 corresponding
to amino acids 17-154 using the forward primer CAGCAGAGAGCGGCCCCACA
ACAAAAGAGATCCAGC (SEQ ID NO: 162) and the reverse primer GTAGTTGGA
TCCTCAAGAAGCAGGAGTCCCAGGG (SEQ ID NO: 157). Overlapping PCR of the
cyno and human TR-2 fragments was performed using the forward
primer for the human TR-2 amino acids 1-16 fragment, above (SEQ ID
NO: 145) and the reverse primer for the cyno TR-2 amino acids
17-154 fragment, above (SEQ ID NO: 157). The amino acid sequence
for cyno/human chimera #4 was MVHATSPLLLLLLLSLALVAPGLSAR
KCSLTGKWTNDLGSNMTIGAVNSKGEFTGTYTTAVTATSNEIKESPLHGTQN
TINKRTQPTFGFTVNWKFSESTTVFTGQCFIDRNGKEVLKTMWLLRSSVNDI
GDDWKATRVGINIFTRLRTQKEQLLASLALITQQDLAPQQRAAPQQKRSSPT
EGLCPPGHHISEDSRDCISCKYGQDYSTHWNDFLFCLRCTKCDSGEVEVSS
CTTTRNTVCQCEEGTFREEDSPEICRKCRTGCPRGMVKVKDCTPWSDIECV
HKESGTKHTGEVPAVEKTVTTSPGTPAS (SEQ ID NO: 89). Four additional
modified TR-2 proteins were constructed by replacing short regions
of human TR-2 with the corresponding mouse TR-2 sequence, in the
context of an N-avidin fusion. Human/mouse TR-2 #1 comprised the
mouse TR-2 sequence from amino acids 1-22 and the human TR-2
sequence from amino acids 23-150. Human/mouse TR-2 #2 comprised the
human TR-2 sequence from amino acids 1-28 and 35-150 and the mouse
TR-2 sequence from amino acids 29-34. Human/mouse TR-2 #3 comprised
the human TR-2 sequence from amino acids 1-53 and 60-150 and the
mouse TR-2 sequence from amino acids 54-59. Human/mouse TR-2 #4
comprised the human TR-2 sequence from amino acids 1-66 and 76-150
and the mouse TR-2 sequence from amino acids 67-75. To form each of
the modified proteins, the amplified polynucleotide was then
subcloned into the pCEP4 vector (Invitrogen) containing the chicken
avidin sequence that is described above.
[0435] Human/mouse TR-2 #1 was prepared by amplifying a region of
mature human TR-2 corresponding to amino acids 23-150 using the
forward primer CAGCGGCCGGAGGAGAGCCCCTCAGAGGGATTGT (SEQ ID NO: 163)
and the reverse primer GATTGAGGATCCCTAAGAGGCAGGAGTCCCTGG (SEQ ID
NO: 164) and amplifying a region of mature mouse TR-2 corresponding
to amino acids 1-22 using the forward primer TGAATGAAGCTTGGTTCCAGTA
ACAGCTAACCCA (SEQ ID NO: 165) and the reverse primer TCCCTCTGAGGG
GCTCTCCTCCGGCCGCTGTAG (SEQ ID NO: 166). Overlapping PCR of the
human and mouse TR-2 fragments was performed using the forward
primer for the mouse TR-2 amino acids 1-22 fragment, above (SEQ ID
NO: 165) and the reverse primer for the human TR-2 amino acids
23-150 fragment, above (SEQ ID NO: 164). The amino acid sequence
for human/mouse TR-2 #1 was MVHATSPLLLLLLLSLALV
APGLSARKCSLTGKWTNDLGSNMTIGAVNSKGEFTGTYTTAVTATSNEIKES
PLHGTQNTINKRTQPTFGFTVNWKFSESTTVFTGQCFIDRNGKEVLKTMWL
LRSSVNDIGDDWKATRVGINIFTRLRTQKEQLLASLVPVTANPAHNRPAGLQ
RPEESPSEGLCPPGHHISEDGRDCISCKYGQDYSTHWNDLLFCLRCTRCDS
GEVELSPCTTTRNTVCQCEEGTFREEDSPEMCRKCRTGCPRGMVKVGDCT
PWSDIECVHKESGTKHSGEAPAVEETVTSSPGTPAS (SEQ ID NO: 90).
[0436] Human/mouse TR-2 #2 was prepared by amplifying a region of
mature human TR-2 corresponding to amino acids 1-28 using the
forward primer GTAAGCAAGCTTGGCTCTGATCACCCAACAAGA (SEQ ID NO: 145)
and the reverse primer CAGGTACTGGCCTGCTAGACACAATCCCTCTGAGGGG (SEQ
ID NO: 167), amplifying a region of mature human TR-2 corresponding
to amino acids 35-150 using the forward primer
CTAGCAGGCCAGTACCTGTCAG AAGACGGTAGAGATTGC (SEQ ID NO: 168), and the
reverse primer GATTGAG GATCCCTAAGAGGCAGGAGTCCCTGG (SEQ ID NO: 164)
and amplifying a region of mature mouse TR-2 corresponding to amino
acids 29-34 using the forward primer
CAGGTACTGGCCTGCTAGACACAATCCCTCTGAGGGG (SEQ ID NO: 169) and the
reverse primer CTAGCAGGCCAGTACCTGTCAGAAGACGG TAGAGATTGC (SEQ ID NO:
170). Overlapping PCR of the human and mouse TR-2 fragments was
performed using the forward primer for the human TR-2 amino acids
1-28 fragment, above (SEQ ID NO: 145) and the reverse primer for
the human TR-2 amino acids 35-150 fragment, above (SEQ ID NO: 170).
The amino acid sequence for human/mouse TR-2 #2 was
MVHATSPLLLLLLLSLALVAPGLSARKCSLTGKW
TNDLGSNMTIGAVNSKGEFTGTYTTAVTATSNEIKESPLHGTQNTINKRTQP TF
GFTVNWKFSESTTVFTGQCFIDRNGKEVLKTMWLLRSSVNDIGDDWKATRV
GINIFTRLRTQKEQLLASLALITQQDLAPQQRAAPQQKRSSPSEGLCLAGQY
LSEDGRDCISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVC
QCEEGTFREEDSPEMCRKCRTGCPRGMVKVGDCTPWSDIECVHKESGTK
HSGEAPAVEETVTSSPGTPAS (SEQ ID NO: 91).
[0437] Human/mouse TR-2 #3 was prepared by amplifying a region of
mature human TR-2 corresponding to amino acids 1-53 using the
forward primer GTAAGCAAGCTTGGCTCTGATCACCCAACAAGA (SEQ ID NO: 145)
and the reverse primer TGAATCCAGAGAATGGTTGGAGTGAGTGCTATAGTCCTG TC
(SEQ ID NO: 171), and amplifying a region of mature human TR-2
corresponding to amino acids 60-154 using the forward primer
TCCAACCATTCTCTGGATTCA TGCTTGCGCTGCACCAGG (SEQ ID NO: 172) and the
reverse primer GATTG AGGATCCCTAAGAGGCAGGAGTCCCTGG (SEQ ID NO: 173)
The above primers include nucleotides encoding mouse TR-2
corresponding to amino acids 54-59. Overlapping PCR of the human
and mouse TR-2 fragments was performed using the forward primer for
the human TR-2 amino acids 1-53 fragment, above (SEQ ID NO: 145)
and the reverse primer for the human TR-2 amino acids 60-154
fragment, above (SEQ ID NO: 173). The amino acid sequence for
human/mouse TR-2 #3 was MVHATSPLLLLLLLSLALVAPGLSARKCSLTGKWT
NDLGSNMTIGAVNSKGEFTGTYTTAVTATSNEIKESPLHGTQNTINKRTQPT
FGFTVNWKFSESTTVFTGQCFIDRNGKEVLKTMWLLRSSVNDIGDDWKATR
VGINIFTRLRTQKEQLLASLALITQQDLAPQQRAAPQQKRSSPSEGLCPPGH
HISEDGRDCISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTV
CQCEEGTFREEDSPEMCRKCRTGCPRGMVKVGDCTPWSDIECVHKESGT
KHSGEAPAVEETVTSSPGTPAS (SEQ ID NO: 92).
[0438] Human/mouse chimera #4 was prepared by amplifying a region
of mature human TR-2 corresponding to amino acids 1-66 using the
forward primer GTAAGCAAGCTTGGCTCTGATCACCCAACAAGA (SEQ ID NO: 145)
and the reverse primer TCGGGTTTCTACGACTTTATCTTCCTTACACCTGG
TGCAGCGCAAG (SEQ ID NO: 174), and amplifying a region of mature
human TR-2 corresponding to amino acids 76-154 using the forward
primer AAGGAAG ATAAAGTCGTAGAAACCCGATGCACCACGACCAGAAAC AC (SEQ ID
NO: 175) and the reverse primer GATTGAGGATCCCTAAGAGGCA GGAGTCCCTGG
(SEQ ID NO: 176). The above primers include nucleotides encoding
mouse TR-2 corresponding to amino acids 67-75. Overlapping PCR of
the human and mouse TR-2 fragments was performed using the forward
primer for the human TR-2 amino acids 1-66 fragment, above (SEQ ID
NO: 145) and the reverse primer for the human TR-2 amino acids
76-154 fragment, above (SEQ ID NO: 176). The amino acid sequence
for human/mouse TR-2 #4 was MVHATSPLLLLLLLSLALVAPGL
SARKCSLTGKWTNDLGSNMTIGAVNSKGEFTGTYTTAVTATSNEIKESPLHG
TQNTINKRTQPTFGFTVNWKFSESTTVFTGQCFIDRNGKEVLKTMWLLRSS
VNDIGDDWKATRVGINIFTRLRTQKEQLLASLALITQQDLAPQQRAAPQQKR
SSPSEGLCPPGHHISEDGRDCISCKYGQDYSTHSNHSLDSCLRCTRCDSGE
VELSPCTTTRNTVCQCEEGTFREEDSPEMCRKCRTGCPRGMVKVGDCTP
WSDIECVHKESGTKHSGEAPAVEETVTSSPGTPAS (SEQ ID NO: 93).
[0439] Expression of avidin fusion proteins was performed by
transient transfection of human 293T adherent cells in vented T75
tissue culture flasks. Cells were grown and maintained in DMEM with
10% dialyzed FBS and 1.times. pen-step-glutamine at 37.degree. C.
with 5% CO.sub.2. To prepare for transfection, approximately
3.times.10.sup.6 293T cells were inoculated into each of a series
of clean 175 flasks containing 15 ml growth medium, and all of the
flasks were grown overnight for approximately 20 hours. Each of the
pCEP4-Avidin(N)-TR-2 constructs were transfected into different
cells as follows. 15 .mu.g DNA was mixed with 75 .mu.L
Lipofectamine 2000 (Invitrogen) in the presence of Opti-MEM medium
(Invitrogen) to form a DNA-Lipofectamine complex. The complex was
incubated for 20 minutes. During that incubation period, the growth
medium was aspirated from the T75 flasks and replaced with 15 mL
Opti-MEM. Following incubation, each transfection complex was
inoculated into a different flask and incubated at 37.degree. C.
for 4 to 5 hours. At the end of the incubation period, the Opti-MEM
medium in each flask was replaced with fresh growth medium.
Approximately 48 hours post-transfection, the conditioned media was
harvested and transferred to 50 ml tubes (Falcon). The tubes were
centrifuged at 2000.times.g for 10 minutes at 4.degree. C. to
remove cells and debris, and subsequently transferred to a clean 50
mL tube. A control flask lacking transfected DNA was also made
following the same protocol, yielding negative control conditioned
media for binding experiments.
[0440] The concentration of each N-avidin-TR-2 fusion protein was
determined using a quantitative FACS-based assay. The avidin fusion
proteins were captured on 6.7 .mu.m biotin polystyrene beads
(Spherotech, Inc.). Two samples were prepared for each fusion
protein: 5 .mu.L (approximately 3.5.times.10.sup.5) bead suspension
plus 20 .mu.L of 1.times. conditioned media, and 5 .mu.L bead
suspension plus 200 .mu.L of 1.times. conditioned media. All
samples were incubated for 1 hour at room temperature with
rotation. Conditioned media was removed from each sample by
centrifugation and washing with PBS containing 0.5% BSA (BPBS). The
avidin beads were stained with 200 .mu.L of a 0.5 .mu.g/mL solution
of a goat FITC-labeled anti-avidin antibody (Vector Labs,
Burlingame, Calif.) in BPBS. The reaction was allowed to proceed at
room temperature for 45 minutes with the reaction tubes covered by
foil. Following incubation, the beads were collected again by
centrifugation and washing with BPBS, and resuspended for analysis
in 0.5 ml BPBS. The FITC fluorescence was detected using a FACScan
(Becton Dickinson Bioscience). The signal was converted to protein
mass using a standard curve derived with recombinant avidin.
[0441] The binding of two human anti-TR-2 antibodies to each of the
human TR-2 truncations, to human TR-2, and to TR-2 from cynomolgus
monkey was assessed. The binding assay was performed as follows.
Biotin beads, described above, were loaded with approximately 100
ng of one of the N-Avidin TR-2 fusion proteins per
3.5.times.10.sup.5 beads and brought to volume with growth medium.
The beads were mixed with 1 .mu.g of FITC-conjugated human
anti-TR-2 monoclonal antibody in 0.2 mL BPBS. After incubation for
1 hour at room temperature, 3 mL BPBS was added and the
antibody-bead complexes were collected by centrifugation for 5
minutes at 750.times.g. The pellet was washed in 3 mL BPBS. The
antibody bound to the avidin-bead complexes was detected by FACS
analysis. The mean fluorescent intensity was recorded for each
sample. Binding of those antibodies to conditioned media lacking
TR-2 was used as a negative control ("Neg CM"). The results are
shown in FIG. 24.
[0442] The observed binding patterns of the two antibodies were
similar. The strongest observed binding was to the positive
control, human TR-2, with an average fluorescent intensity of 7349.
Observed binding of the antibodies (as measured in fluorescent
intensity) to truncation TR-2-2 was 6561-6693, to truncations
TR-2-3 and TR-2-5 was 3158-3866, to truncation TR-2-6 was
1959-2202, and to truncation TR-2-1 was 662-759. Binding of the
antibodies to full-length TR-2 from cynomolgus monkey (as measured
in fluorescent intensity) was 666-764. The antibodies did not bind
to mouse or rat TR-2, or to truncations TR-2-4, TR-2-7, TR-2-9,
TR-2-10, TR-2-11, TR-2-12, or TR-2-13, as determined by the fact
that the binding was similar to the background for the
experiment.
[0443] TR-2-1 is a C-terminal truncation of TR-2 after amino acid
43, and TR-2-2, -3, -5, and -6 all include at least amino acids 16
to 85. Binding occurred when the entire region from amino acids 1
to 85 was present (see results for TR-2-2). The addition of amino
acids 86 to 126 decreased binding by approximately two-fold
(compare results for TR-2-2 to TR-2-3). The absence of amino acids
1 to 15 from the N-terminus of TR-2 in TR-2-2 decreased binding by
approximately two-fold (compare results for TR-2-2 to TR-2-5). The
simultaneous absence of amino acids 1 to 15 and the addition of
amino acids 86 to 126 decreased binding by approximately three-fold
(compare results for TR-2-2 to TR-2-6). Elimination of residues 44
to 85 (TR-2-1) reduced binding to about 11% of that observed to
TR-2-2. Those results indicate that one or more residues in the
regions of amino acids 1 to 15 (SEQ ID NO: 94; ALITQQDLAPQQRAA) and
44 to 85 (SEQ ID NO: 95; CKYGQDYSTHWNDLL FCLRCTRCDSGEVE
LSPCTTTRNTVCQ) are important for binding of those two human
anti-TR-2 antibodies and human TR-2.
[0444] The binding of a human anti-TR-2 antibody to each of the
cyno TR-2 truncations, to human/cyno chimeras, and to human TR-2
comprising certain mouse TR-2 domains was also assessed. The
anti-TR-2 antibody bound strongly to full-length human TR-2
(fluorescent intensity ("FI") of 5681). The binding of the
anti-TR-2 antibody to the full-length long version of cyno TR-2 was
about five-fold reduced (FI of 1573) from that to full-length human
TR-2. Only background levels of binding were observed to the
full-length short version of cyno TR-2 (FI of 209) and to cyno TR-2
truncations 17-154 (FI of 51), cyno 1-85 (FI of 11), and cyno 17-85
(FI of 8).
[0445] The binding of certain human anti-TR-2 antibodies to
cyno/human TR-2 chimeras was also assessed (see FIG. 27). Observed
binding (FI) of the antibodies to the four chimeras was as follows:
cyno/human chimera #1: FI of 5977; cyno/human chimera #2: FI of 47;
cyno/human chimera #3: FI of 12; cyno/human chimera #4: FI of 1507.
As above, observed binding of the antibodies to full-length human
TR-2 was 5681, while binding of the antibodies to full-length cyno
TR-2 was 1573 (long form) and 209 (short form).
[0446] Because the antibody binding to cyno/human chimera #1 was
similar to that to the truncation TR-2-5, replacement of amino
acids 1-16 with the corresponding cyno sequence apparently did not
affect antibody binding in the context of human amino acids 17-85.
However, replacement of amino acids 1-16 with the corresponding
cyno sequence in the context of the full-length human TR-2
(cyno/human #2) significantly abrogated binding, confirming that at
least one amino acid in the region from 1-16 forms part of the
epitope. Binding to cyno/human chimeras #3, and #4 was
significantly attenuated from that to full-length human TR-2,
suggesting that amino acids 17-85 of the human sequence are
important for binding. Overall, one or more of the amino acids in
the region of 1-85 of the human sequence (SEQ ID NO: 96;
ALITQQDLAPQQRAAPQQ
KRSSPSEGLCPPGHHISEDGRDCISCKYGQDYSTHWNDLLFCLRCTRCDSG
EVELSPCTTTRNTVCQ) are involved in epitope binding. Similarly,
replacement of various human sequences in the region of amino acids
1-85 with the corresponding mouse sequence significantly attenuates
antibody binding (see FIG. 27), further confirming that one or more
amino acids in that region are involved in epitope binding.
Sequence CWU 1
1
2041363DNAHomo sapiens 1caggtgcagc tggtgcagtc tggggctgag gtgaagaagc
ctggggcctc agtgaaggtc 60tcctgcaagg cttctggata caccttcacc agttatgata
tcaactgggt gcgacaggcc 120actggacaag ggcttgagtg gatgggatgg
atgaacccta acagtgataa cacaggctat 180gcacagaagt tccagggcag
agtcaccatg accaggaaca cctccataag cacagcctac 240atggagttga
gcagcctgag atctgaggac acggccgtgt attactgtgc gagatggaat
300cactatggtt cggggagtca ttttgactac tggggccagg gaaccctggt
caccgtctcc 360tca 3632121PRTHomo sapiens 2Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asp Ile
Asn Trp Val Arg Gln Ala Thr Gly Gln Gly Leu Glu Trp Met 35 40 45
Gly Trp Met Asn Pro Asn Ser Asp Asn Thr Gly Tyr Ala Gln Lys Phe 50
55 60 Gln Gly Arg Val Thr Met Thr Arg Asn Thr Ser Ile Ser Thr Ala
Tyr 65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Trp Asn His Tyr Gly Ser Gly Ser His
Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 3360DNAHomo sapiens 3caggtgcagc tgcaggagtc gggcccagga
ctggtgaagc cttcacagac cctgtccctc 60acctgcactg tctctggtgg ctccatcagc
agtggtggtc actactggag ctggatccgc 120cagcacccag ggaagggcct
ggagtggatt gggtacatct attacagtgg gagcacctac 180tacaacccgt
ccctcaagag tcgagttacc atatcagtag acacgtctaa gaaccagttc
240tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtattattg
tgcgagagat 300gacagcagtg gctggggttt tgactactgg ggccagggaa
tcctggtcac cgtctcctca 3604120PRTHomo sapiens 4Gln Val Gln Leu Gln
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser
Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30 Gly
His Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu 35 40
45 Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser
50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn
Gln Phe 65 70 75 80Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr
Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Asp Asp Ser Ser Gly Trp Gly
Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Ile Leu Val Thr Val Ser Ser
115 1205360DNAHomo sapiens 5caggtgcagc tgcaggagtc gggcccagga
ctggtgaagc cttcacagac cctgtccctc 60acctgcactg tctctggtgg ctccatcagc
agtggtggtc actactggag ctggatccgc 120cagcacccag ggaagggcct
ggagtggatt gggtacatct attacagtgg gagcgcctac 180tacaacccgt
ccctcaagag tcgagttacc atatcagtag acacgtctaa gaaccagttc
240tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtattactg
tgcgagagat 300gacagcagtg gctggggttt tgactactgg ggccagggaa
tcctggtcac cgtctcctca 3606120PRTHomo sapiens 6Gln Val Gln Leu Gln
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser
Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30 Gly
His Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu 35 40
45 Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Ala Tyr Tyr Asn Pro Ser
50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn
Gln Phe 65 70 75 80Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr
Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Asp Asp Ser Ser Gly Trp Gly
Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Ile Leu Val Thr Val Ser Ser
115 1207360DNAHomo sapiens 7caggtgcagc tgcaggagtc gggcccagga
ctggtgaagc cttcacagac cctgtccctc 60acctgcactg tctctggtgg ctccatcagc
agtggtggtc actactggag ctggatccgc 120cagcacccag ggaagggcct
ggagtggatt gggtacatct attacagtgg gagcgcctac 180tacaacccgt
ccctcaagag tcgagttacc atatcagtag acacgtctaa gaaccagttc
240tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtattactg
tgcgagagat 300gacagcagtg gctggggttt tgactactgg ggccagggaa
tcctggtcac cgtctcctca 3608120PRTHomo sapiens 8Gln Val Gln Leu Gln
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser
Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30 Gly
His Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu 35 40
45 Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Ala Tyr Tyr Asn Pro Ser
50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn
Gln Phe 65 70 75 80Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr
Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Asp Asp Ser Ser Gly Trp Gly
Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Ile Leu Val Thr Val Ser Ser
115 1209363DNAHomo sapiens 9caggtgcagc tggtggagtc tgggggaggc
ttggtcaagc ctggagggtc cctgagactc 60tcctgtgcag cctctggatt caccttcagt
gactactaca tgaactggat ccgccaggct 120ccagggaagg gactggagtg
ggtttcacac attagtagta gtggtagtat cttagactac 180gcagactctg
tgaagggccg attcaccatc tccagggaca acgccaagaa ctcactgtat
240ctgcaaatga acagcctgag agtcgaggac acggccgtgt attactgtgc
gagagatggg 300gctgcagctg gtacggatgc ttttgatctc tggggccaag
ggacaatggt caccgtctct 360tca 36310121PRTHomo sapiens 10Gln Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25
30 Tyr Met Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ser His Ile Ser Ser Ser Gly Ser Ile Leu Asp Tyr Ala Asp
Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Ser Leu Tyr 65 70 75 80Leu Gln Met Asn Ser Leu Arg Val Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Ala Ala Ala Gly
Thr Asp Ala Phe Asp Leu Trp Gly 100 105 110 Gln Gly Thr Met Val Thr
Val Ser Ser 115 120 11366DNAHomo sapiens 11caggtgcagc tggtggagtc
tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cgtctggatt
caccttcagt tactatggca tacactgggt ccgccaggct 120ccaggcaagg
ggctggagtg ggtggcagtt atatggtatg atggaagtaa taaatactat
180gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa
cacgctgtat 240ctgcaaatga acagcctgag agccgaggac acggctgtgt
attactgtgc gagagggagg 300tatagcagct cgtcctggtg gtacttcgat
ctctggggcc gtggcaccct ggtcactgtc 360tcctca 36612122PRTHomo sapiens
12Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr
Tyr 20 25 30 Gly Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Arg Tyr
Ser Ser Ser Ser Trp Trp Tyr Phe Asp Leu Trp 100 105 110 Gly Arg Gly
Thr Leu Val Thr Val Ser Ser 115 120 13366DNAHomo sapiens
13caggtgcagg ctgagcagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc
60acctgcactg tctctggtgg ctccatcagt aattactact ggagctggat ccggcagccc
120ccagggaagg gactggagtg gattgggtat atctattaca gtgggagcac
caagtacaac 180ccctccctca agagtcgagt caccatatca gtagacacgt
ccaagaacca gttctccctg 240aagctaacct ctgtgaccac tgcggacacg
gccgtgtatt actgtgcgag agactcccct 300cgtggattta gtggctacga
ggcttttgac tcctggggcc agggaaccct ggtcaccgtc 360tcctca
36614122PRTHomo sapiens 14Gln Val Gln Ala Glu Gln Ser Gly Pro Gly
Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val
Ser Gly Gly Ser Ile Ser Asn Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg
Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr
Tyr Ser Gly Ser Thr Lys Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg
Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75
80Lys Leu Thr Ser Val Thr Thr Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95 Arg Asp Ser Pro Arg Gly Phe Ser Gly Tyr Glu Ala Phe Asp
Ser Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
15360DNAHomo sapiens 15caggtgcagc tgcaggagtc gggcccagga ctggtgaagc
cttcacagac cctgtccctc 60acctgcactg tctctggtgg ctccatcagc agtgataatt
actactggag ctggatccgc 120cagcacccag ggaagggcct ggagtggatt
gggtacatct attacagtgg gagcacctac 180tacaacccgt ccctcaagag
tcgagttacc atatcagtag acacgtctaa gaaccagttc 240tccctgaagc
tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagga
300gttaactgga actttctttt tgatatctgg ggccaaggga caatggtcac
cgtctcttca 36016120PRTHomo sapiens 16Gln Val Gln Leu Gln Glu Ser
Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr
Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Asp 20 25 30 Asn Tyr Tyr
Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu 35 40 45 Trp
Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55
60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe
65 70 75 80Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val
Tyr Tyr 85 90 95 Cys Ala Arg Gly Val Asn Trp Asn Phe Leu Phe Asp
Ile Trp Gly Gln 100 105 110 Gly Thr Met Val Thr Val Ser Ser 115
12017348DNAHomo sapiens 17caggtgcagc tggtggagtc tgggggaggc
ttggtcaagc ctggagggtc cctgagactc 60tcctgtgcag cctctggatt caccttcagt
gactactaca tgagctggat ccgccaggct 120ccagggaagg ggctggagtg
ggtttcatac attagtagaa gtggtagtac catatactac 180gcagactctg
tgaagggccg attcaccatc tccagggaca acgccaagaa ctcactgtat
240ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc
gagatcttta 300ggcggtatgg acgtctgggg ccaagggacc acggtcaccg tctcctca
34818116PRTHomo sapiens 18Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Ser Trp Ile Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser
Arg Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Arg Ser Leu Gly Gly Met Asp Val Trp Gly Gln Gly Thr
Thr Val 100 105 110 Thr Val Ser Ser 115 19387DNAHomo sapiens
19caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc
60tcctgtgcag cgtctggatt caccttcaat aactatggca tgcactgggt ccgccaggct
120ccaggcaagg ggctggagtg ggtggcagtt atatggtatg atggaagtaa
taaatactat 180gcagactccg tgaagggccg attcaccatc tccagagaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agccgaggac
acggctgtgt attactgtgc gagagatagg 300accgtatata gcaactcgtc
acccttttac tactactact acggtatgga cgtctggggc 360caagggacca
cggtcaccgt ctcctca 38720129PRTHomo sapiens 20Gln Val Gln Leu Val
Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asn Tyr 20 25 30 Gly
Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr 65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Thr Val Tyr Ser Asn Ser
Ser Pro Phe Tyr Tyr Tyr 100 105 110 Tyr Tyr Gly Met Asp Val Trp Gly
Gln Gly Thr Thr Val Thr Val Ser 115 120 125 Ser 21387DNAHomo
sapiens 21caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc
cctgagactc 60tcctgtgcag cgtctggatt caccttcagt acctatggca tgcactgggt
ccgccaggct 120ccaggcaagg ggctggagtg ggtggcagtt atatggtatg
atggaagtaa taaatactat 180gcagactccg tgaagggccg attcaccatc
tccagagaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgag
agccgaggac acggctgtgt attattgtgc gagagatagg 300accgtatata
gcagctcgtc acccttttac tactactact acggtatgga cgtctggggc
360caagggacca cggtcaccgt ctcctca 38722129PRTHomo sapiens 22Gln Val
Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20
25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala
Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
Lys Asn Thr Leu Tyr 65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Thr Val Tyr
Ser Ser Ser Ser Pro Phe Tyr Tyr Tyr 100 105 110 Tyr Tyr Gly Met Asp
Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser 115 120 125 Ser
23357DNAHomo sapiens 23caggtgcagc tacagcagtg gggcgcacga ctgttgaagc
cttcggagac cctgtccctc 60acctgcgctg tctatggtgg gtccttcagt ggttactact
ggagctggat ccgccagccc 120ccagggaagg ggctggagtg gattggggaa
atcaatcata gtggaagcac caactacaac 180ccgtccctca agagtcgagt
caccatatca gtagacacgt ccaagaacca gttctccctg 240aagctgaggt
ctgtgaccgc cgcggacacg gctgtgtatt actgtgcgag agggggaagc
300agtggctact ggtacttcga tctctggggc cgtggcaccc tggtcactgt ctcctca
35724119PRTHomo sapiens 24Gln Val Gln Leu Gln Gln Trp Gly Ala Arg
Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val
Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg
Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn
His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg
Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75
80Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95 Arg Gly Gly Ser Ser Gly Tyr Trp Tyr Phe Asp Leu Trp Gly
Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 25363DNAHomo
sapiens 25gaggtgcagg tggtggagtc tgggggaggc
ctggtcaagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt caccttcagt
agctatagca tgaactgggt ccgccaggct 120ccagggaagg ggctggagtg
ggtctcatcc attagtagta gtagtagtta catatactac 180gcagactcag
tgaagggccg attcaccatc tccagagaca acgccaagaa ctcactgtat
240ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc
gagggggggc 300agcagctggt acggggactg gttcgacccc tggggccagg
gaaccctggt caccgtctcc 360tca 36326121PRTHomo sapiens 26Glu Val Gln
Val Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25
30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp
Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Ser Leu Tyr 65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Ser Ser Trp Tyr
Gly Asp Trp Phe Asp Pro Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr
Val Ser Ser 115 120 27378DNAHomo sapiens 27cagctggtgg agtctggggg
aggcgtggtc cagcctggga ggtccctgag actctcctgt 60gcagcgtctg gattcacctt
cagtagctat ggcatgcact gggtccgcca ggctccaggc 120aaggggctgg
agtgggtggc agttatatgg tatgatggaa gaaataaata ctatgcagac
180tccgtgaagg gccgattcac catctccaga gacaattcca agaacacgct
gtatctgcaa 240atgaacagcc tgagagccga ggacacggct gtgtattact
gtgcgagaga agtgggatat 300tgtactaatg gtgtatgctc ctactactac
tacggtatgg acgtctgggg ccaagggacc 360acggtcaccg tctcctca
37828126PRTHomo sapiens 28Gln Leu Val Glu Ser Gly Gly Gly Val Val
Gln Pro Gly Arg Ser Leu 1 5 10 15 Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Ser Tyr Gly Met 20 25 30 His Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val Ala Val 35 40 45 Ile Trp Tyr Asp
Gly Arg Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly 50 55 60 Arg Phe
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 65 70 75
80Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg
85 90 95 Glu Val Gly Tyr Cys Thr Asn Gly Val Cys Ser Tyr Tyr Tyr
Tyr Gly 100 105 110 Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val
Ser Ser 115 120 125 29366DNAHomo sapiens 29caggtgcagc tgcaggagtc
gggcccagga ctggtgaagc cttcacagac cctgtccctc 60acctgcactg tctctggtgg
ctccatcagc agtggtgatt acttctggag ctggatccgc 120cagctcccag
ggaagggcct ggagtgcatt gggcacatcc ataacagtgg gaccacctac
180tacaatccgt ccctcaagag tcgagttacc atatcagtag acacgtctaa
gaagcagttc 240tccctgaggc tgagttctgt gactgccgcg gacacggccg
tatattactg tgcgagagat 300cgagggggtg actactacta tggtatggac
gtctggggcc aagggaccac ggtcaccgtc 360tcctca 36630122PRTHomo sapiens
30Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1
5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser
Gly 20 25 30 Asp Tyr Phe Trp Ser Trp Ile Arg Gln Leu Pro Gly Lys
Gly Leu Glu 35 40 45 Cys Ile Gly His Ile His Asn Ser Gly Thr Thr
Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val
Asp Thr Ser Lys Lys Gln Phe 65 70 75 80Ser Leu Arg Leu Ser Ser Val
Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Asp Arg
Gly Gly Asp Tyr Tyr Tyr Gly Met Asp Val Trp 100 105 110 Gly Gln Gly
Thr Thr Val Thr Val Ser Ser 115 120 31366DNAHomo sapiens
31caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc
60acctgcagtg tctctggtgg ctccatcagc agtggtggtt actactggag ctggatccgc
120cagcacccag ggaagggcct ggagtggatt gggtacatct attacagtgg
gagcacctac 180tgcaacccgt ccctcaagag tcgagttacc atatcagtcg
acacgtctaa gaaccagttc 240tccctgaagc tgagctctgt gactgccgcg
gacacggccg tgtattactg tgcgagagac 300aatggttcgg ggagttatga
ctggttcgac ccctggggcc agggaatcct ggtcaccgtc 360tcctca
36632122PRTHomo sapiens 32Gln Val Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Val
Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30 Gly Tyr Tyr Trp Ser Trp
Ile Arg Gln His Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Tyr
Ile Tyr Tyr Ser Gly Ser Thr Tyr Cys Asn Pro Ser 50 55 60 Leu Lys
Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75
80Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
85 90 95 Cys Ala Arg Asp Asn Gly Ser Gly Ser Tyr Asp Trp Phe Asp
Pro Trp 100 105 110 Gly Gln Gly Ile Leu Val Thr Val Ser Ser 115 120
33366DNAHomo sapiens 33caggtgcaga tgcaggagtc gggcccagga ctggtgaagc
cttcacagac cctgtccctc 60acctgcactg tctctggtgg ctccatcagc agtggtgatt
actactggag ctggatccgc 120cagcacccag ggaagaacct ggagtggatt
gggtacatct attacagtgg gagcacctac 180tacaacccgt ccctcaagag
tcgagttacc atatcagtag acacgtctaa gaaccagttc 240tccctgaagc
tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagac
300aatggttcgg ggagttatga ctggttcgac ccctggggcc agggaaccct
ggtcaccgtc 360tcctca 36634122PRTHomo sapiens 34Gln Val Gln Met Gln
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser
Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30 Asp
Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Asn Leu Glu 35 40
45 Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser
50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn
Gln Phe 65 70 75 80Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr
Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Asp Asn Gly Ser Gly Ser Tyr
Asp Trp Phe Asp Pro Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val
Ser Ser 115 120 35321DNAHomo sapiens 35gacatccaga tgacccagtc
tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggcaagtca
gagcattagc atttatttaa attggtatca gcagaaacca 120gggaaagccc
ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatta
180aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag
tctgcaacct 240gaagatattg caacttacta ctgtcaacag agttacaaaa
ccccgctcac tttcggcgga 300gggaccaagg tggagatcaa a 32136107PRTHomo
sapiens 36Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser
Ile Ser Ile Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser
Gly Val Pro Leu Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80Glu Asp Ile Ala
Thr Tyr Tyr Cys Gln Gln Ser Tyr Lys Thr Pro Leu 85 90 95 Thr Phe
Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 37321DNAHomo sapiens
37gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgttggaga cagagtcacc
60atcacttgcc gggcaagtca gggccttaga aatgatttag gctggtttca gcagaaacca
120gggaaagtca ctaagcgcct gatctatgct gcatccagtt tgcaaagagg
ggtcccatca 180aggttcagcg gcagtggatc tgggacagaa ttcactctca
caatcagcag cctgcagcct 240gaagattttg caacttatta ctgtctacag
cattatagtt tcccgtggac gttcggccaa 300gggaccaagg tggagatcaa a
32138107PRTHomo sapiens 38Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Gly Leu Arg Asn Asp 20 25 30 Leu Gly Trp Phe Gln Gln
Lys Pro Gly Lys Val Thr Lys Arg Leu Ile 35 40 45 Tyr Ala Ala Ser
Ser Leu Gln Arg Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Tyr Ser Phe Pro Trp
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
39321DNAHomo sapiens 39gacatccaga tgacccagtc tccatcctcc ctgtctgcat
ctgttggaga cagagtcacc 60atcacttgcc gggcaagtca gggccttaga aatgatttag
gctggtttca gcagaaacca 120gggaaagccc ctaagcgcct gatctatgct
gcatccagtt tgcaaagagg ggtcccatca 180aggttcagcg gcagtggatc
tgggacagaa ttcactctca caatcagcag cctgcagcct 240gaagatttta
caacttattt ctgtctacag cataatagtt tcccgtggac gttcggccaa
300gggaccaagg tggaaatcaa a 32140107PRTHomo sapiens 40Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Leu Arg Asn Asp 20 25
30 Leu Gly Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile
35 40 45 Tyr Ala Ala Ser Ser Leu Gln Arg Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro 65 70 75 80Glu Asp Phe Thr Thr Tyr Phe Cys Leu Gln
His Asn Ser Phe Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys 100 105 41321DNAHomo sapiens 41gacatccaga tgacccagtc
tccatcctcc ctgtctgcat ctgttggaga cagagtcacc 60atcacttgcc gggcaagtca
gggccttaga aatgatttag gctggtttca gcagaaacca 120gggaaagccc
ctaagcgcct gatctatgct gcatccagtt tgcaaagagg ggtcccatca
180aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag
cctgcagcct 240gaagatttta caacttattt ctgtctacag cataatagtt
tcccgtggac gttcggccaa 300gggaccaagg tggaaatcaa a 32142107PRTHomo
sapiens 42Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly
Leu Arg Asn Asp 20 25 30 Leu Gly Trp Phe Gln Gln Lys Pro Gly Lys
Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Arg
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80Glu Asp Phe Thr
Thr Tyr Phe Cys Leu Gln His Asn Ser Phe Pro Trp 85 90 95 Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 43321DNAHomo sapiens
43gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc
60atcacttgcc ggtcaagtca gagcattagt aactatataa attggtatca acagagacca
120gggaaagccc cgaacctcct gatccatgat gtatccagtt tccaaagtgc
ggtcccatca 180aggttcagtc gcagtggatc tgggacagtt ttcactctca
ccatcagcag tctgcaacct 240gaagattttg caacttactt ctgtcaacag
acttacatta ccccattcac tttcggccct 300gggaccaaag tggatatcaa a
32144107PRTHomo sapiens 44Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg
Ser Ser Gln Ser Ile Ser Asn Tyr 20 25 30 Ile Asn Trp Tyr Gln Gln
Arg Pro Gly Lys Ala Pro Asn Leu Leu Ile 35 40 45 His Asp Val Ser
Ser Phe Gln Ser Ala Val Pro Ser Arg Phe Ser Arg 50 55 60 Ser Gly
Ser Gly Thr Val Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75
80Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Thr Tyr Ile Thr Pro Phe
85 90 95 Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys 100 105
45321DNAHomo sapiens 45gacatccaga tgacccagtc tccatcctcc ctgtctgcat
ctgtaggaga cagagtcacc 60atcacttgcc gggcgagtca gggcattagc aattatttag
cctggtatca gcagaaacca 120gggaaagttc ctaagctcct gatctatgct
gcatccactt tgcaatcagg ggtcccatct 180cggttcagtg gcagtggatc
tgggacagat ttcactctca ccatcagcag cctgcagcct 240gaagatgttg
caacttatta ctgtcaaaag tataacagtg ccccgctcac tttcggcgga
300gggaccaagg tggagatcaa a 32146107PRTHomo sapiens 46Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn Tyr 20 25
30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile
35 40 45 Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro 65 70 75 80Glu Asp Val Ala Thr Tyr Tyr Cys Gln Lys
Tyr Asn Ser Ala Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val
Glu Ile Lys 100 105 47339DNAHomo sapiens 47gacatcgtga tgacccagtc
tccagactcc ctggctgtgt ctctgggcga gagggccacc 60atcaactgca agtccagcca
gagtgtttta tacaggtcca acaataagat ctacttagct 120tggtaccagc
agaaaccagg acagcctcct aagctgctca tttactgggc atcgacccgg
180gaatccgggg tccctgaccg attcagtggc agcgggtctg ggacagattt
cactctcacc 240atcagcagcc tgctggctga agatgtggca gtttattact
gtcagcaata ttatagtact 300ccattcactt tcggccctgg gaccaaagtg gatatcaaa
33948113PRTHomo sapiens 48Asp Ile Val Met Thr Gln Ser Pro Asp Ser
Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys
Ser Ser Gln Ser Val Leu Tyr Arg 20 25 30 Ser Asn Asn Lys Ile Tyr
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu
Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75
80Ile Ser Ser Leu Leu Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln
85 90 95 Tyr Tyr Ser Thr Pro Phe Thr Phe Gly Pro Gly Thr Lys Val
Asp Ile 100 105 110 Lys 49336DNAHomo sapiens 49gatattgtga
tgactcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60atctcctgca
ggtctagtca gagcctcctg cgtcgtaatg gatacaacta tttggattgg
120tacctgcaga agccagggca gtctccacaa ctcctgatct atttgggttc
taatcgggcc 180tccggggtcc cagacaggtt cagtggcagt ggatcaggca
cagattttac actgaaaatc 240agcagagtgg aggctgagga tgttggggtt
tattactgca tgcaagctct acaaactccg 300ctcactttcg gcggagggac
cgaggtggag atcaaa 33650112PRTHomo sapiens 50Asp Ile Val Met Thr Gln
Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser
Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Arg Arg 20 25 30 Asn Gly
Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45
Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50
55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys
Ile 65
70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln
Ala 85 90 95 Leu Gln Thr Pro Leu Thr Phe Gly Gly Gly Thr Glu Val
Glu Ile Lys 100 105 110 51339DNAHomo sapiens 51gacatcgtga
tgacccagtt tccagactcc ctggctgtgt ctctgggcga gagggccacc 60atcaactgca
agtccagcca gagtgtttta cacagctcca acaataagaa ctacttaact
120tggtaccagc tgaaaccagg acagcctcct aagttgctca tttactgggc
atctacccgg 180gaatccgggg tccctgaccg attcagtggc agcgggtctg
ggacagattt cactctcacc 240atcagcagcc tgcaggctga agatgtggca
gtttattact gtcaccaata ttatagtact 300ccgtccagtt ttggccaggg
gaccaagctg gagatcaaa 33952113PRTHomo sapiens 52Asp Ile Val Met Thr
Gln Phe Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala
Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu His Ser 20 25 30 Ser
Asn Asn Lys Asn Tyr Leu Thr Trp Tyr Gln Leu Lys Pro Gly Gln 35 40
45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr 65 70 75 80Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr
Tyr Cys His Gln 85 90 95 Tyr Tyr Ser Thr Pro Ser Ser Phe Gly Gln
Gly Thr Lys Leu Glu Ile 100 105 110 Lys 53321DNAHomo sapiens
53gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgttggaga cagagtcacc
60atcacttgcc ggacaagtca gagcattagc acctatttaa attggtatca gcagaaacca
120gggaaagccc ctaagctcct gatctctgct acatccagtt tgcaaagtgg
ggtcccatca 180aggttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg caacttacta ctgtcaacag
agttacagta ccccgctcac tttcggcgga 300gggaccaagg tggagatcaa a
32154107PRTHomo sapiens 54Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg
Thr Ser Gln Ser Ile Ser Thr Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Ala Thr Ser
Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu
85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
55321DNAHomo sapiens 55gacatccaga tgacccagtc tccatcctcc ctgtctgcat
ctgtaggaga cagagtcacc 60atcacttgcc gggcaagtca gagcattagc agctatttaa
attggtatca gcagaaacca 120gggaaagccc ctaagctcct gatctctgct
acatccagtt ttcaaagtgg ggtcccatca 180aggttcagtg gcagtggatc
tgggacagat ttcactctca ccatcagcag tctgcaacct 240gaagattttg
cagcttacta ctgtcaacag agttacagta ccccgctcac tttcggcgga
300gggaccaagg tggagatcaa a 32156107PRTHomo sapiens 56Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25
30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45 Ser Ala Thr Ser Ser Phe Gln Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro 65 70 75 80Glu Asp Phe Ala Ala Tyr Tyr Cys Gln Gln
Ser Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val
Glu Ile Lys 100 105 57339DNAHomo sapiens 57gacatcgtga tgacccagtc
tccagactcc ctggctgtgt ctctgggcga gagggccacc 60atcaactgca agtccagcca
gagtgtttta cacagctcca acaataagaa ttatttagtt 120tggtaccagc
agaaaccagg acagcctcct aagctgctca tttactgggc atctacccgg
180gaatccgggg tccctgaccg attcagtggc agcgggtctg ggacagattt
cactctcacc 240atcagcagcc tgcaggctga agatgtggca gtttattact
gtcagcaata ttatagtact 300cctctcactt tcggcggagg gaccaaggtg gagatcaaa
33958113PRTHomo sapiens 58Asp Ile Val Met Thr Gln Ser Pro Asp Ser
Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys
Ser Ser Gln Ser Val Leu His Ser 20 25 30 Ser Asn Asn Lys Asn Tyr
Leu Val Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu
Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75
80Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln
85 90 95 Tyr Tyr Ser Thr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val
Glu Ile 100 105 110 Lys 59321DNAHomo sapiens 59gacatccaga
tgacccagtc tccatcttcc gtgtctgcat ctgtaggaga cagagtcacc 60atcacttgtc
gggcgagtca gggtattagc agctggttag tctggtatca gcagaaacca
120gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg
ggtcccatca 180aggttcagcg gcagtggatc tgggacagat ttcactctca
ccatcagcag cctgcagcct 240gaagattttg caacttacta ttgtcagcag
gctaacagtt tccctttcac tttcggcgga 300gggaccaagg tggagatcaa a
32160107PRTHomo sapiens 60Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Val Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser
Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Phe
85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
61321DNAHomo sapiens 61gacatccaga tgacccagtc tccatcctca ctgtctgcat
ctgtaggaga cagagtcacc 60atcacttgtc gggcgagtca gggcattagc aattatttag
cctggtttca gcagaaacca 120gggaaagccc ctaagtccct gatctatgct
gcatccagtt tgcaaagtgg ggtcccatca 180aaattcagcg gcagtggatc
tgggacagat ttcactctca ccatcagcag cctgcagcct 240gaagattttg
caacttatta ctgccaacag tataatagtt accctctcac tttcggcgga
300gggaccaagg tggagatcaa a 32162107PRTHomo sapiens 62Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn Tyr 20 25
30 Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Leu Ile
35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Lys Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro 65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Tyr Asn Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val
Glu Ile Lys 100 105 63324DNAHomo sapiens 63gaaattgtgt tgacgcagtc
tccaggcacc ctgtctttgt ctccagggga aagagccacc 60ctctcctgca gggccagtca
gggtattagt agaagctact tagcctggta ccagcagaaa 120cctggccagg
ctcccagcct cctcatctat ggtgcatcca gcagggccac tggcatccca
180gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag
cagactggag 240cctgaagatt ttgcagtgta ttactgtcaa caatttggta
gttcaccgtg gacgttcggc 300caagggacca aggtggaaat caaa 32464108PRTHomo
sapiens 64Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser
Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Gly
Ile Ser Arg Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Gln Ala Pro Ser Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala
Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80Pro Glu Asp Phe
Ala Val Tyr Tyr Cys Gln Gln Phe Gly Ser Ser Pro 85 90 95 Trp Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 65321DNAHomo
sapiens 65gacattcaga tgacccagtc tccatcctcc gtgtctgcat ctgtaggaga
cagagtcacc 60atcacttgtc gggcgagtca gggtattagc agctggttag cctggtatca
gcagaaacca 120gggaaagccc caaagttcct gatctttgtt gcatccagtt
tccaaagtgg ggtcccatca 180aggttcagcg gcagtggatc tgggacagat
ttcactctca ccatcagcag cctgcagcct 240gaagattttg caacttacta
ttgtcaacag gctaacagtt tccctcggac gttcggccaa 300gggaccaagg
tggaaatcaa a 32166107PRTHomo sapiens 66Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Phe Leu Ile 35 40 45 Phe
Val Ala Ser Ser Phe Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe
Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
105 67321DNAHomo sapiens 67gacatccaga tgacccagtc tccatcttcc
gtgtctgcat ctgttggaga cagagtcacc 60atcacttgtc gggcgagtca gggtattagc
agctggttag cctggtatca gcagaaacca 120gggaaagccc ctaagttcct
gatctttgtt gcatccagtt tgcaaagtgg ggtcccatca 180aggttcagcg
gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct
240gaagattttg caacttacta ttgtcaacag gctaacagtt tccctcggac
gttcggccaa 300gggaccaagg tggaaatcaa a 32168107PRTHomo sapiens 68Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp
20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Phe
Leu Ile 35 40 45 Phe Val Ala Ser Ser Leu Gln Ser Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro 65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Ala Asn Ser Phe Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys 100 105 69312PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide sequence 69Met Val His
Ala Thr Ser Pro Leu Leu Leu Leu Leu Leu Leu Ser Leu 1 5 10 15 Ala
Leu Val Ala Pro Gly Leu Ser Ala Arg Lys Cys Ser Leu Thr Gly 20 25
30 Lys Trp Thr Asn Asp Leu Gly Ser Asn Met Thr Ile Gly Ala Val Asn
35 40 45 Ser Lys Gly Glu Phe Thr Gly Thr Tyr Thr Thr Ala Val Thr
Ala Thr 50 55 60 Ser Asn Glu Ile Lys Glu Ser Pro Leu His Gly Thr
Gln Asn Thr Ile 65 70 75 80Asn Lys Arg Thr Gln Pro Thr Phe Gly Phe
Thr Val Asn Trp Lys Phe 85 90 95 Ser Glu Ser Thr Thr Val Phe Thr
Gly Gln Cys Phe Ile Asp Arg Asn 100 105 110 Gly Lys Glu Val Leu Lys
Thr Met Trp Leu Leu Arg Ser Ser Val Asn 115 120 125 Asp Ile Gly Asp
Asp Trp Lys Ala Thr Arg Val Gly Ile Asn Ile Phe 130 135 140 Thr Arg
Leu Arg Thr Gln Lys Glu Gln Leu Leu Ala Ser Leu Ala Leu 145 150 155
160Ile Thr Gln Gln Asp Leu Ala Pro Gln Gln Arg Ala Ala Pro Gln Gln
165 170 175 Lys Arg Ser Ser Pro Ser Glu Gly Leu Cys Pro Pro Gly His
His Ile 180 185 190 Ser Glu Asp Gly Arg Asp Cys Ile Ser Cys Lys Tyr
Gly Gln Asp Tyr 195 200 205 Ser Thr His Trp Asn Asp Leu Leu Phe Cys
Leu Arg Cys Thr Arg Cys 210 215 220 Asp Ser Gly Glu Val Glu Leu Ser
Pro Cys Thr Thr Thr Arg Asn Thr 225 230 235 240Val Cys Gln Cys Glu
Glu Gly Thr Phe Arg Glu Glu Asp Ser Pro Glu 245 250 255 Met Cys Arg
Lys Cys Arg Thr Gly Cys Pro Arg Gly Met Val Lys Val 260 265 270 Gly
Asp Cys Thr Pro Trp Ser Asp Ile Glu Cys Val His Lys Glu Ser 275 280
285 Gly Thr Lys His Ser Gly Glu Ala Pro Ala Val Glu Glu Thr Val Thr
290 295 300 Ser Ser Pro Gly Thr Pro Ala Ser 305 310
70201PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide sequence 70Met Val His Ala Thr Ser Pro Leu
Leu Leu Leu Leu Leu Leu Ser Leu 1 5 10 15 Ala Leu Val Ala Pro Gly
Leu Ser Ala Arg Lys Cys Ser Leu Thr Gly 20 25 30 Lys Trp Thr Asn
Asp Leu Gly Ser Asn Met Thr Ile Gly Ala Val Asn 35 40 45 Ser Lys
Gly Glu Phe Thr Gly Thr Tyr Thr Thr Ala Val Thr Ala Thr 50 55 60
Ser Asn Glu Ile Lys Glu Ser Pro Leu His Gly Thr Gln Asn Thr Ile 65
70 75 80Asn Lys Arg Thr Gln Pro Thr Phe Gly Phe Thr Val Asn Trp Lys
Phe 85 90 95 Ser Glu Ser Thr Thr Val Phe Thr Gly Gln Cys Phe Ile
Asp Arg Asn 100 105 110 Gly Lys Glu Val Leu Lys Thr Met Trp Leu Leu
Arg Ser Ser Val Asn 115 120 125 Asp Ile Gly Asp Asp Trp Lys Ala Thr
Arg Val Gly Ile Asn Ile Phe 130 135 140 Thr Arg Leu Arg Thr Gln Lys
Glu Gln Leu Leu Ala Ser Leu Ala Leu 145 150 155 160Ile Thr Gln Gln
Asp Leu Ala Pro Gln Gln Arg Ala Ala Pro Gln Gln 165 170 175 Lys Arg
Ser Ser Pro Ser Glu Gly Leu Cys Pro Pro Gly His His Ile 180 185 190
Ser Glu Asp Gly Arg Asp Cys Ile Ser 195 200 71243PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
sequence 71Met Val His Ala Thr Ser Pro Leu Leu Leu Leu Leu Leu Leu
Ser Leu 1 5 10 15 Ala Leu Val Ala Pro Gly Leu Ser Ala Arg Lys Cys
Ser Leu Thr Gly 20 25 30 Lys Trp Thr Asn Asp Leu Gly Ser Asn Met
Thr Ile Gly Ala Val Asn 35 40 45 Ser Lys Gly Glu Phe Thr Gly Thr
Tyr Thr Thr Ala Val Thr Ala Thr 50 55 60 Ser Asn Glu Ile Lys Glu
Ser Pro Leu His Gly Thr Gln Asn Thr Ile 65 70 75 80Asn Lys Arg Thr
Gln Pro Thr Phe Gly Phe Thr Val Asn Trp Lys Phe 85 90 95 Ser Glu
Ser Thr Thr Val Phe Thr Gly Gln Cys Phe Ile Asp Arg Asn 100 105 110
Gly Lys Glu Val Leu Lys Thr Met Trp Leu Leu Arg Ser Ser Val Asn 115
120 125 Asp Ile Gly Asp Asp Trp Lys Ala Thr Arg Val Gly Ile Asn Ile
Phe 130 135 140 Thr Arg Leu
Arg Thr Gln Lys Glu Gln Leu Leu Ala Ser Leu Ala Leu 145 150 155
160Ile Thr Gln Gln Asp Leu Ala Pro Gln Gln Arg Ala Ala Pro Gln Gln
165 170 175 Lys Arg Ser Ser Pro Ser Glu Gly Leu Cys Pro Pro Gly His
His Ile 180 185 190 Ser Glu Asp Gly Arg Asp Cys Ile Ser Cys Lys Tyr
Gly Gln Asp Tyr 195 200 205 Ser Thr His Trp Asn Asp Leu Leu Phe Cys
Leu Arg Cys Thr Arg Cys 210 215 220 Asp Ser Gly Glu Val Glu Leu Ser
Pro Cys Thr Thr Thr Arg Asn Thr 225 230 235 240Val Cys Gln
72284PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide sequence 72Met Val His Ala Thr Ser Pro Leu
Leu Leu Leu Leu Leu Leu Ser Leu 1 5 10 15 Ala Leu Val Ala Pro Gly
Leu Ser Ala Arg Lys Cys Ser Leu Thr Gly 20 25 30 Lys Trp Thr Asn
Asp Leu Gly Ser Asn Met Thr Ile Gly Ala Val Asn 35 40 45 Ser Lys
Gly Glu Phe Thr Gly Thr Tyr Thr Thr Ala Val Thr Ala Thr 50 55 60
Ser Asn Glu Ile Lys Glu Ser Pro Leu His Gly Thr Gln Asn Thr Ile 65
70 75 80Asn Lys Arg Thr Gln Pro Thr Phe Gly Phe Thr Val Asn Trp Lys
Phe 85 90 95 Ser Glu Ser Thr Thr Val Phe Thr Gly Gln Cys Phe Ile
Asp Arg Asn 100 105 110 Gly Lys Glu Val Leu Lys Thr Met Trp Leu Leu
Arg Ser Ser Val Asn 115 120 125 Asp Ile Gly Asp Asp Trp Lys Ala Thr
Arg Val Gly Ile Asn Ile Phe 130 135 140 Thr Arg Leu Arg Thr Gln Lys
Glu Gln Leu Leu Ala Ser Leu Ala Leu 145 150 155 160Ile Thr Gln Gln
Asp Leu Ala Pro Gln Gln Arg Ala Ala Pro Gln Gln 165 170 175 Lys Arg
Ser Ser Pro Ser Glu Gly Leu Cys Pro Pro Gly His His Ile 180 185 190
Ser Glu Asp Gly Arg Asp Cys Ile Ser Cys Lys Tyr Gly Gln Asp Tyr 195
200 205 Ser Thr His Trp Asn Asp Leu Leu Phe Cys Leu Arg Cys Thr Arg
Cys 210 215 220 Asp Ser Gly Glu Val Glu Leu Ser Pro Cys Thr Thr Thr
Arg Asn Thr 225 230 235 240Val Cys Gln Cys Glu Glu Gly Thr Phe Arg
Glu Glu Asp Ser Pro Glu 245 250 255 Met Cys Arg Lys Cys Arg Thr Gly
Cys Pro Arg Gly Met Val Lys Val 260 265 270 Gly Asp Cys Thr Pro Trp
Ser Asp Ile Glu Cys Val 275 280 73186PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
sequence 73Met Val His Ala Thr Ser Pro Leu Leu Leu Leu Leu Leu Leu
Ser Leu 1 5 10 15 Ala Leu Val Ala Pro Gly Leu Ser Ala Arg Lys Cys
Ser Leu Thr Gly 20 25 30 Lys Trp Thr Asn Asp Leu Gly Ser Asn Met
Thr Ile Gly Ala Val Asn 35 40 45 Ser Lys Gly Glu Phe Thr Gly Thr
Tyr Thr Thr Ala Val Thr Ala Thr 50 55 60 Ser Asn Glu Ile Lys Glu
Ser Pro Leu His Gly Thr Gln Asn Thr Ile 65 70 75 80Asn Lys Arg Thr
Gln Pro Thr Phe Gly Phe Thr Val Asn Trp Lys Phe 85 90 95 Ser Glu
Ser Thr Thr Val Phe Thr Gly Gln Cys Phe Ile Asp Arg Asn 100 105 110
Gly Lys Glu Val Leu Lys Thr Met Trp Leu Leu Arg Ser Ser Val Asn 115
120 125 Asp Ile Gly Asp Asp Trp Lys Ala Thr Arg Val Gly Ile Asn Ile
Phe 130 135 140 Thr Arg Leu Arg Thr Gln Lys Glu Gln Leu Leu Ala Ser
Leu Pro Gln 145 150 155 160Gln Lys Arg Ser Ser Pro Ser Glu Gly Leu
Cys Pro Pro Gly His His 165 170 175 Ile Ser Glu Asp Gly Arg Asp Cys
Ile Ser 180 185 74228PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide sequence 74Met Val His
Ala Thr Ser Pro Leu Leu Leu Leu Leu Leu Leu Ser Leu 1 5 10 15 Ala
Leu Val Ala Pro Gly Leu Ser Ala Arg Lys Cys Ser Leu Thr Gly 20 25
30 Lys Trp Thr Asn Asp Leu Gly Ser Asn Met Thr Ile Gly Ala Val Asn
35 40 45 Ser Lys Gly Glu Phe Thr Gly Thr Tyr Thr Thr Ala Val Thr
Ala Thr 50 55 60 Ser Asn Glu Ile Lys Glu Ser Pro Leu His Gly Thr
Gln Asn Thr Ile 65 70 75 80Asn Lys Arg Thr Gln Pro Thr Phe Gly Phe
Thr Val Asn Trp Lys Phe 85 90 95 Ser Glu Ser Thr Thr Val Phe Thr
Gly Gln Cys Phe Ile Asp Arg Asn 100 105 110 Gly Lys Glu Val Leu Lys
Thr Met Trp Leu Leu Arg Ser Ser Val Asn 115 120 125 Asp Ile Gly Asp
Asp Trp Lys Ala Thr Arg Val Gly Ile Asn Ile Phe 130 135 140 Thr Arg
Leu Arg Thr Gln Lys Glu Gln Leu Leu Ala Ser Leu Pro Gln 145 150 155
160Gln Lys Arg Ser Ser Pro Ser Glu Gly Leu Cys Pro Pro Gly His His
165 170 175 Ile Ser Glu Asp Gly Arg Asp Cys Ile Ser Cys Lys Tyr Gly
Gln Asp 180 185 190 Tyr Ser Thr His Trp Asn Asp Leu Leu Phe Cys Leu
Arg Cys Thr Arg 195 200 205 Cys Asp Ser Gly Glu Val Glu Leu Ser Pro
Cys Thr Thr Thr Arg Asn 210 215 220 Thr Val Cys Gln 225
75269PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide sequence 75Met Val His Ala Thr Ser Pro Leu
Leu Leu Leu Leu Leu Leu Ser Leu 1 5 10 15 Ala Leu Val Ala Pro Gly
Leu Ser Ala Arg Lys Cys Ser Leu Thr Gly 20 25 30 Lys Trp Thr Asn
Asp Leu Gly Ser Asn Met Thr Ile Gly Ala Val Asn 35 40 45 Ser Lys
Gly Glu Phe Thr Gly Thr Tyr Thr Thr Ala Val Thr Ala Thr 50 55 60
Ser Asn Glu Ile Lys Glu Ser Pro Leu His Gly Thr Gln Asn Thr Ile 65
70 75 80Asn Lys Arg Thr Gln Pro Thr Phe Gly Phe Thr Val Asn Trp Lys
Phe 85 90 95 Ser Glu Ser Thr Thr Val Phe Thr Gly Gln Cys Phe Ile
Asp Arg Asn 100 105 110 Gly Lys Glu Val Leu Lys Thr Met Trp Leu Leu
Arg Ser Ser Val Asn 115 120 125 Asp Ile Gly Asp Asp Trp Lys Ala Thr
Arg Val Gly Ile Asn Ile Phe 130 135 140 Thr Arg Leu Arg Thr Gln Lys
Glu Gln Leu Leu Ala Ser Leu Pro Gln 145 150 155 160Gln Lys Arg Ser
Ser Pro Ser Glu Gly Leu Cys Pro Pro Gly His His 165 170 175 Ile Ser
Glu Asp Gly Arg Asp Cys Ile Ser Cys Lys Tyr Gly Gln Asp 180 185 190
Tyr Ser Thr His Trp Asn Asp Leu Leu Phe Cys Leu Arg Cys Thr Arg 195
200 205 Cys Asp Ser Gly Glu Val Glu Leu Ser Pro Cys Thr Thr Thr Arg
Asn 210 215 220 Thr Val Cys Gln Cys Glu Glu Gly Thr Phe Arg Glu Glu
Asp Ser Pro 225 230 235 240Glu Met Cys Arg Lys Cys Arg Thr Gly Cys
Pro Arg Gly Met Val Lys 245 250 255 Val Gly Asp Cys Thr Pro Trp Ser
Asp Ile Glu Cys Val 260 265 76202PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide sequence 76Met Val His
Ala Thr Ser Pro Leu Leu Leu Leu Leu Leu Leu Ser Leu 1 5 10 15 Ala
Leu Val Ala Pro Gly Leu Ser Ala Arg Lys Cys Ser Leu Thr Gly 20 25
30 Lys Trp Thr Asn Asp Leu Gly Ser Asn Met Thr Ile Gly Ala Val Asn
35 40 45 Ser Lys Gly Glu Phe Thr Gly Thr Tyr Thr Thr Ala Val Thr
Ala Thr 50 55 60 Ser Asn Glu Ile Lys Glu Ser Pro Leu His Gly Thr
Gln Asn Thr Ile 65 70 75 80Asn Lys Arg Thr Gln Pro Thr Phe Gly Phe
Thr Val Asn Trp Lys Phe 85 90 95 Ser Glu Ser Thr Thr Val Phe Thr
Gly Gln Cys Phe Ile Asp Arg Asn 100 105 110 Gly Lys Glu Val Leu Lys
Thr Met Trp Leu Leu Arg Ser Ser Val Asn 115 120 125 Asp Ile Gly Asp
Asp Trp Lys Ala Thr Arg Val Gly Ile Asn Ile Phe 130 135 140 Thr Arg
Leu Arg Thr Gln Lys Glu Gln Leu Leu Ala Ser Leu Ile Ser 145 150 155
160Cys Lys Tyr Gly Gln Asp Tyr Ser Thr His Trp Asn Asp Leu Leu Phe
165 170 175 Cys Leu Arg Cys Thr Arg Cys Asp Ser Gly Glu Val Glu Leu
Ser Pro 180 185 190 Cys Thr Thr Thr Arg Asn Thr Val Cys Gln 195 200
77243PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide sequence 77Met Val His Ala Thr Ser Pro Leu
Leu Leu Leu Leu Leu Leu Ser Leu 1 5 10 15 Ala Leu Val Ala Pro Gly
Leu Ser Ala Arg Lys Cys Ser Leu Thr Gly 20 25 30 Lys Trp Thr Asn
Asp Leu Gly Ser Asn Met Thr Ile Gly Ala Val Asn 35 40 45 Ser Lys
Gly Glu Phe Thr Gly Thr Tyr Thr Thr Ala Val Thr Ala Thr 50 55 60
Ser Asn Glu Ile Lys Glu Ser Pro Leu His Gly Thr Gln Asn Thr Ile 65
70 75 80Asn Lys Arg Thr Gln Pro Thr Phe Gly Phe Thr Val Asn Trp Lys
Phe 85 90 95 Ser Glu Ser Thr Thr Val Phe Thr Gly Gln Cys Phe Ile
Asp Arg Asn 100 105 110 Gly Lys Glu Val Leu Lys Thr Met Trp Leu Leu
Arg Ser Ser Val Asn 115 120 125 Asp Ile Gly Asp Asp Trp Lys Ala Thr
Arg Val Gly Ile Asn Ile Phe 130 135 140 Thr Arg Leu Arg Thr Gln Lys
Glu Gln Leu Leu Ala Ser Leu Ile Ser 145 150 155 160Cys Lys Tyr Gly
Gln Asp Tyr Ser Thr His Trp Asn Asp Leu Leu Phe 165 170 175 Cys Leu
Arg Cys Thr Arg Cys Asp Ser Gly Glu Val Glu Leu Ser Pro 180 185 190
Cys Thr Thr Thr Arg Asn Thr Val Cys Gln Cys Glu Glu Gly Thr Phe 195
200 205 Arg Glu Glu Asp Ser Pro Glu Met Cys Arg Lys Cys Arg Thr Gly
Cys 210 215 220 Pro Arg Gly Met Val Lys Val Gly Asp Cys Thr Pro Trp
Ser Asp Ile 225 230 235 240Glu Cys Val 78228PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
sequence 78Met Val His Ala Thr Ser Pro Leu Leu Leu Leu Leu Leu Leu
Ser Leu 1 5 10 15 Ala Leu Val Ala Pro Gly Leu Ser Ala Arg Lys Cys
Ser Leu Thr Gly 20 25 30 Lys Trp Thr Asn Asp Leu Gly Ser Asn Met
Thr Ile Gly Ala Val Asn 35 40 45 Ser Lys Gly Glu Phe Thr Gly Thr
Tyr Thr Thr Ala Val Thr Ala Thr 50 55 60 Ser Asn Glu Ile Lys Glu
Ser Pro Leu His Gly Thr Gln Asn Thr Ile 65 70 75 80Asn Lys Arg Thr
Gln Pro Thr Phe Gly Phe Thr Val Asn Trp Lys Phe 85 90 95 Ser Glu
Ser Thr Thr Val Phe Thr Gly Gln Cys Phe Ile Asp Arg Asn 100 105 110
Gly Lys Glu Val Leu Lys Thr Met Trp Leu Leu Arg Ser Ser Val Asn 115
120 125 Asp Ile Gly Asp Asp Trp Lys Ala Thr Arg Val Gly Ile Asn Ile
Phe 130 135 140 Thr Arg Leu Arg Thr Gln Lys Glu Gln Leu Leu Ala Ser
Leu Gln Cys 145 150 155 160Glu Glu Gly Thr Phe Arg Glu Glu Asp Ser
Pro Glu Met Cys Arg Lys 165 170 175 Cys Arg Thr Gly Cys Pro Arg Gly
Met Val Lys Val Gly Asp Cys Thr 180 185 190 Pro Trp Ser Asp Ile Glu
Cys Val His Lys Glu Ser Gly Thr Lys His 195 200 205 Ser Gly Glu Ala
Pro Ala Val Glu Glu Thr Val Thr Ser Ser Pro Gly 210 215 220 Thr Pro
Ala Ser 225 79271PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide sequence 79Met Val His Ala Thr Ser
Pro Leu Leu Leu Leu Leu Leu Leu Ser Leu 1 5 10 15 Ala Leu Val Ala
Pro Gly Leu Ser Ala Arg Lys Cys Ser Leu Thr Gly 20 25 30 Lys Trp
Thr Asn Asp Leu Gly Ser Asn Met Thr Ile Gly Ala Val Asn 35 40 45
Ser Lys Gly Glu Phe Thr Gly Thr Tyr Thr Thr Ala Val Thr Ala Thr 50
55 60 Ser Asn Glu Ile Lys Glu Ser Pro Leu His Gly Thr Gln Asn Thr
Ile 65 70 75 80Asn Lys Arg Thr Gln Pro Thr Phe Gly Phe Thr Val Asn
Trp Lys Phe 85 90 95 Ser Glu Ser Thr Thr Val Phe Thr Gly Gln Cys
Phe Ile Asp Arg Asn 100 105 110 Gly Lys Glu Val Leu Lys Thr Met Trp
Leu Leu Arg Ser Ser Val Asn 115 120 125 Asp Ile Gly Asp Asp Trp Lys
Ala Thr Arg Val Gly Ile Asn Ile Phe 130 135 140 Thr Arg Leu Arg Thr
Gln Lys Glu Gln Leu Leu Ala Ser Leu Ile Ser 145 150 155 160Cys Lys
Tyr Gly Gln Asp Tyr Ser Thr His Trp Asn Asp Leu Leu Phe 165 170 175
Cys Leu Arg Cys Thr Arg Cys Asp Ser Gly Glu Val Glu Leu Ser Pro 180
185 190 Cys Thr Thr Thr Arg Asn Thr Val Cys Gln Cys Glu Glu Gly Thr
Phe 195 200 205 Arg Glu Glu Asp Ser Pro Glu Met Cys Arg Lys Cys Arg
Thr Gly Cys 210 215 220 Pro Arg Gly Met Val Lys Val Gly Asp Cys Thr
Pro Trp Ser Asp Ile 225 230 235 240Glu Cys Val His Lys Glu Ser Gly
Thr Lys His Ser Gly Glu Ala Pro 245 250 255 Ala Val Glu Glu Thr Val
Thr Ser Ser Pro Gly Thr Pro Ala Ser 260 265 270 80209PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
sequence 80Met Val His Ala Thr Ser Pro Leu Leu Leu Leu Leu Leu Leu
Ser Leu 1 5 10 15 Ala Leu Val Ala Pro Gly Leu Ser Ala Arg Lys Cys
Ser Leu Thr Gly 20 25 30 Lys Trp Thr Asn Asp Leu Gly Ser Asn Met
Thr Ile Gly Ala Val Asn 35 40 45 Ser Lys Gly Glu Phe Thr Gly Thr
Tyr Thr Thr Ala Val Thr Ala Thr 50 55 60 Ser Asn Glu Ile Lys Glu
Ser Pro Leu His Gly Thr Gln Asn Thr Ile 65 70 75 80Asn Lys Arg Thr
Gln Pro Thr Phe Gly Phe Thr Val Asn Trp Lys Phe 85 90 95 Ser Glu
Ser Thr Thr Val Phe Thr Gly Gln Cys Phe Ile Asp Arg Asn 100 105 110
Gly Lys Glu Val Leu Lys Thr Met Trp Leu Leu Arg Ser Ser Val Asn 115
120 125 Asp Ile Gly Asp Asp Trp Lys Ala Thr Arg Val Gly Ile Asn Ile
Phe 130 135
140 Thr Arg Leu Arg Thr Gln Lys Glu Gln Leu Leu Ala Ser Leu Pro Gln
145 150 155 160Gln Lys Arg Ser Ser Pro Ser Glu Gly Leu Cys Pro Pro
Gly His His 165 170 175 Ile Ser Glu Asp Gly Arg Asp Cys Ile Ser Tyr
Lys Tyr Gly Gln Asp 180 185 190 Tyr Ser Thr His Trp Asn Asp Leu Leu
Phe Cys Leu Arg Cys Thr Arg 195 200 205 Cys 81217PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
sequence 81Met Val His Ala Thr Ser Pro Leu Leu Leu Leu Leu Leu Leu
Ser Leu 1 5 10 15 Ala Leu Val Ala Pro Gly Leu Ser Ala Arg Lys Cys
Ser Leu Thr Gly 20 25 30 Lys Trp Thr Asn Asp Leu Gly Ser Asn Met
Thr Ile Gly Ala Val Asn 35 40 45 Ser Lys Gly Glu Phe Thr Gly Thr
Tyr Thr Thr Ala Val Thr Ala Thr 50 55 60 Ser Asn Glu Ile Lys Glu
Ser Pro Leu His Gly Thr Gln Asn Thr Ile 65 70 75 80Asn Lys Arg Thr
Gln Pro Thr Phe Gly Phe Thr Val Asn Trp Lys Phe 85 90 95 Ser Glu
Ser Thr Thr Val Phe Thr Gly Gln Cys Phe Ile Asp Arg Asn 100 105 110
Gly Lys Glu Val Leu Lys Thr Met Trp Leu Leu Arg Ser Ser Val Asn 115
120 125 Asp Ile Gly Asp Asp Trp Lys Ala Thr Arg Val Gly Ile Asn Ile
Phe 130 135 140 Thr Arg Leu Arg Thr Gln Lys Glu Gln Leu Leu Ala Ser
Leu Pro Gln 145 150 155 160Gln Lys Arg Ser Ser Pro Ser Glu Gly Leu
Cys Pro Pro Gly His His 165 170 175 Ile Ser Glu Asp Gly Arg Asp Cys
Ile Ser Cys Lys Tyr Gly Gln Asp 180 185 190 Tyr Ser Thr His Trp Asn
Asp Leu Leu Phe Cys Leu Arg Cys Thr Arg 195 200 205 Cys Asp Ser Gly
Glu Val Glu Leu Ser 210 215 82290PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide sequence 82Met Val His
Ala Thr Ser Pro Leu Leu Leu Leu Leu Leu Leu Ser Leu 1 5 10 15 Ala
Leu Val Ala Pro Gly Leu Ser Ala Arg Lys Cys Ser Leu Thr Gly 20 25
30 Lys Trp Thr Asn Asp Leu Gly Ser Asn Met Thr Ile Gly Ala Val Asn
35 40 45 Ser Lys Gly Glu Phe Thr Gly Thr Tyr Thr Thr Ala Val Thr
Ala Thr 50 55 60 Ser Asn Glu Ile Lys Glu Ser Pro Leu His Gly Thr
Gln Asn Thr Ile 65 70 75 80Asn Lys Arg Thr Gln Pro Thr Phe Gly Phe
Thr Val Asn Trp Lys Phe 85 90 95 Ser Glu Ser Thr Thr Val Phe Thr
Gly Gln Cys Phe Ile Asp Arg Asn 100 105 110 Gly Lys Glu Val Leu Lys
Thr Met Trp Leu Leu Arg Ser Ser Val Asn 115 120 125 Asp Ile Gly Asp
Asp Trp Lys Ala Thr Arg Val Gly Ile Asn Ile Phe 130 135 140 Thr Arg
Leu Arg Thr Gln Lys Glu Gln Leu Leu Ala Ser Leu Ala Pro 145 150 155
160Ile Thr Arg Gln Ser Leu Asp Pro Gln Arg Arg Ala Ala Pro Gln Gln
165 170 175 Lys Arg Ser Ser Pro Thr Glu Gly Leu Cys Pro Pro Gly His
His Ile 180 185 190 Ser Glu Asp Ser Arg Asp Cys Ile Ser Cys Lys Tyr
Gly Gln Asp Tyr 195 200 205 Ser Thr His Trp Asn Asp Phe Leu Phe Cys
Leu Arg Cys Thr Lys Cys 210 215 220 Asp Ser Gly Glu Val Glu Val Ser
Ser Cys Thr Thr Thr Arg Asn Thr 225 230 235 240Val Cys Gln Cys Glu
Glu Gly Thr Phe Arg Glu Glu Asp Ser Pro Glu 245 250 255 Ile Cys Arg
Lys Cys Arg Thr Gly Cys Pro Arg Gly Met Val Lys Val 260 265 270 Lys
Asp Cys Thr Pro Trp Ser Asp Ile Glu Cys Pro Gln Arg Arg Ile 275 280
285 Gln Thr 29083312PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide sequence 83Met Val His Ala Thr Ser
Pro Leu Leu Leu Leu Leu Leu Leu Ser Leu 1 5 10 15 Ala Leu Val Ala
Pro Gly Leu Ser Ala Arg Lys Cys Ser Leu Thr Gly 20 25 30 Lys Trp
Thr Asn Asp Leu Gly Ser Asn Met Thr Ile Gly Ala Val Asn 35 40 45
Ser Lys Gly Glu Phe Thr Gly Thr Tyr Thr Thr Ala Val Thr Ala Thr 50
55 60 Ser Asn Glu Ile Lys Glu Ser Pro Leu His Gly Thr Gln Asn Thr
Ile 65 70 75 80Asn Lys Arg Thr Gln Pro Thr Phe Gly Phe Thr Val Asn
Trp Lys Phe 85 90 95 Ser Glu Ser Thr Thr Val Phe Thr Gly Gln Cys
Phe Ile Asp Arg Asn 100 105 110 Gly Lys Glu Val Leu Lys Thr Met Trp
Leu Leu Arg Ser Ser Val Asn 115 120 125 Asp Ile Gly Asp Asp Trp Lys
Ala Thr Arg Val Gly Ile Asn Ile Phe 130 135 140 Thr Arg Leu Arg Thr
Gln Lys Glu Gln Leu Leu Ala Ser Leu Ala Pro 145 150 155 160Ile Thr
Arg Gln Ser Leu Asp Pro Gln Arg Arg Ala Ala Pro Gln Gln 165 170 175
Lys Arg Ser Ser Pro Thr Glu Gly Leu Cys Pro Pro Gly His His Ile 180
185 190 Ser Glu Asp Ser Arg Asp Cys Ile Ser Cys Lys Tyr Gly Gln Asp
Tyr 195 200 205 Ser Thr His Trp Asn Asp Phe Leu Phe Cys Leu Arg Cys
Thr Lys Cys 210 215 220 Asp Ser Gly Glu Val Glu Val Ser Ser Cys Thr
Thr Thr Arg Asn Thr 225 230 235 240Val Cys Gln Cys Glu Glu Gly Thr
Phe Arg Glu Glu Asp Ser Pro Glu 245 250 255 Ile Cys Arg Lys Cys Arg
Thr Gly Cys Pro Arg Gly Met Val Lys Val 260 265 270 Lys Asp Cys Thr
Pro Trp Ser Asp Ile Glu Cys Val His Lys Glu Ser 275 280 285 Gly Thr
Lys His Thr Gly Glu Val Pro Ala Val Glu Lys Thr Val Thr 290 295 300
Thr Ser Pro Gly Thr Pro Ala Ser 305 310 84243PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
sequence 84Met Val His Ala Thr Ser Pro Leu Leu Leu Leu Leu Leu Leu
Ser Leu 1 5 10 15 Ala Leu Val Ala Pro Gly Leu Ser Ala Arg Lys Cys
Ser Leu Thr Gly 20 25 30 Lys Trp Thr Asn Asp Leu Gly Ser Asn Met
Thr Ile Gly Ala Val Asn 35 40 45 Ser Lys Gly Glu Phe Thr Gly Thr
Tyr Thr Thr Ala Val Thr Ala Thr 50 55 60 Ser Asn Glu Ile Lys Glu
Ser Pro Leu His Gly Thr Gln Asn Thr Ile 65 70 75 80Asn Lys Arg Thr
Gln Pro Thr Phe Gly Phe Thr Val Asn Trp Lys Phe 85 90 95 Ser Glu
Ser Thr Thr Val Phe Thr Gly Gln Cys Phe Ile Asp Arg Asn 100 105 110
Gly Lys Glu Val Leu Lys Thr Met Trp Leu Leu Arg Ser Ser Val Asn 115
120 125 Asp Ile Gly Asp Asp Trp Lys Ala Thr Arg Val Gly Ile Asn Ile
Phe 130 135 140 Thr Arg Leu Arg Thr Gln Lys Glu Gln Leu Leu Ala Ser
Leu Ala Pro 145 150 155 160Ile Thr Arg Gln Ser Leu Asp Pro Gln Arg
Arg Ala Ala Pro Gln Gln 165 170 175 Lys Arg Ser Ser Pro Thr Glu Gly
Leu Cys Pro Pro Gly His His Ile 180 185 190 Ser Glu Asp Ser Arg Asp
Cys Ile Ser Cys Lys Tyr Gly Gln Asp Tyr 195 200 205 Ser Thr His Trp
Asn Asp Phe Leu Phe Cys Leu Arg Cys Thr Lys Cys 210 215 220 Asp Ser
Gly Glu Val Glu Val Ser Ser Cys Thr Thr Thr Arg Asn Thr 225 230 235
240Val Cys Gln 85228PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide sequence 85Met Val His Ala Thr Ser
Pro Leu Leu Leu Leu Leu Leu Leu Ser Leu 1 5 10 15 Ala Leu Val Ala
Pro Gly Leu Ser Ala Arg Lys Cys Ser Leu Thr Gly 20 25 30 Lys Trp
Thr Asn Asp Leu Gly Ser Asn Met Thr Ile Gly Ala Val Asn 35 40 45
Ser Lys Gly Glu Phe Thr Gly Thr Tyr Thr Thr Ala Val Thr Ala Thr 50
55 60 Ser Asn Glu Ile Lys Glu Ser Pro Leu His Gly Thr Gln Asn Thr
Ile 65 70 75 80Asn Lys Arg Thr Gln Pro Thr Phe Gly Phe Thr Val Asn
Trp Lys Phe 85 90 95 Ser Glu Ser Thr Thr Val Phe Thr Gly Gln Cys
Phe Ile Asp Arg Asn 100 105 110 Gly Lys Glu Val Leu Lys Thr Met Trp
Leu Leu Arg Ser Ser Val Asn 115 120 125 Asp Ile Gly Asp Asp Trp Lys
Ala Thr Arg Val Gly Ile Asn Ile Phe 130 135 140 Thr Arg Leu Arg Thr
Gln Lys Glu Gln Leu Leu Ala Ser Leu Pro Gln 145 150 155 160Gln Lys
Arg Ser Ser Pro Ile Glu Gly Leu Cys Pro Pro Gly His His 165 170 175
Ile Ser Glu Asp Ser Arg Asp Cys Ile Ser Cys Lys Tyr Gly Gln Asp 180
185 190 Tyr Ser Thr His Trp Asn Asp Phe Leu Phe Cys Leu Arg Cys Thr
Lys 195 200 205 Cys Asp Ser Gly Glu Val Glu Val Ser Ser Cys Thr Thr
Thr Arg Asn 210 215 220 Thr Val Cys Gln 225 86243PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
sequence 86Met Val His Ala Thr Ser Pro Leu Leu Leu Leu Leu Leu Leu
Ser Leu 1 5 10 15 Ala Leu Val Ala Pro Gly Leu Ser Ala Arg Lys Cys
Ser Leu Thr Gly 20 25 30 Lys Trp Thr Asn Asp Leu Gly Ser Asn Met
Thr Ile Gly Ala Val Asn 35 40 45 Ser Lys Gly Glu Phe Thr Gly Thr
Tyr Thr Thr Ala Val Thr Ala Thr 50 55 60 Ser Asn Glu Ile Lys Glu
Ser Pro Leu His Gly Thr Gln Asn Thr Ile 65 70 75 80Asn Lys Arg Thr
Gln Pro Thr Phe Gly Phe Thr Val Asn Trp Lys Phe 85 90 95 Ser Glu
Ser Thr Thr Val Phe Thr Gly Gln Cys Phe Ile Asp Arg Asn 100 105 110
Gly Lys Glu Val Leu Lys Thr Met Trp Leu Leu Arg Ser Ser Val Asn 115
120 125 Asp Ile Gly Asp Asp Trp Lys Ala Thr Arg Val Gly Ile Asn Ile
Phe 130 135 140 Thr Arg Leu Arg Thr Gln Lys Glu Gln Leu Leu Ala Ser
Leu Ala Pro 145 150 155 160Ile Thr Arg Gln Ser Leu Asp Pro Gln Arg
Arg Ala Ala Pro Gln Gln 165 170 175 Lys Arg Ser Ser Pro Ser Glu Gly
Leu Cys Pro Pro Gly His His Ile 180 185 190 Ser Glu Asp Gly Arg Asp
Cys Ile Ser Cys Lys Tyr Gly Gln Asp Tyr 195 200 205 Ser Thr His Trp
Asn Asp Leu Leu Phe Cys Leu Arg Cys Thr Arg Cys 210 215 220 Asp Ser
Gly Glu Val Glu Leu Ser Pro Cys Thr Thr Thr Arg Asn Thr 225 230 235
240Val Cys Gln 87312PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide sequence 87Met Val His Ala Thr Ser
Pro Leu Leu Leu Leu Leu Leu Leu Ser Leu 1 5 10 15 Ala Leu Val Ala
Pro Gly Leu Ser Ala Arg Lys Cys Ser Leu Thr Gly 20 25 30 Lys Trp
Thr Asn Asp Leu Gly Ser Asn Met Thr Ile Gly Ala Val Asn 35 40 45
Ser Lys Gly Glu Phe Thr Gly Thr Tyr Thr Thr Ala Val Thr Ala Thr 50
55 60 Ser Asn Glu Ile Lys Glu Ser Pro Leu His Gly Thr Gln Asn Thr
Ile 65 70 75 80Asn Lys Arg Thr Gln Pro Thr Phe Gly Phe Thr Val Asn
Trp Lys Phe 85 90 95 Ser Glu Ser Thr Thr Val Phe Thr Gly Gln Cys
Phe Ile Asp Arg Asn 100 105 110 Gly Lys Glu Val Leu Lys Thr Met Trp
Leu Leu Arg Ser Ser Val Asn 115 120 125 Asp Ile Gly Asp Asp Trp Lys
Ala Thr Arg Val Gly Ile Asn Ile Phe 130 135 140 Thr Arg Leu Arg Thr
Gln Lys Glu Gln Leu Leu Ala Ser Leu Ala Pro 145 150 155 160Ile Thr
Arg Gln Ser Leu Asp Pro Gln Arg Arg Ala Ala Pro Gln Gln 165 170 175
Lys Arg Ser Ser Pro Ser Glu Gly Leu Cys Pro Pro Gly His His Ile 180
185 190 Ser Glu Asp Gly Arg Asp Tyr Ile Ser Cys Lys Tyr Gly Gln Asp
Tyr 195 200 205 Ser Thr His Trp Asn Asp Leu Leu Phe Cys Leu Arg Cys
Thr Arg Cys 210 215 220 Asp Ser Gly Glu Val Glu Leu Ser Pro Cys Thr
Thr Thr Arg Asn Thr 225 230 235 240Val Cys Gln Cys Glu Glu Gly Thr
Phe Arg Glu Glu Asp Ser Pro Glu 245 250 255 Met Cys Arg Lys Cys Arg
Thr Gly Cys Pro Arg Gly Met Val Lys Val 260 265 270 Gly Asp Cys Thr
Pro Trp Ser Asp Ile Glu Cys Val His Lys Glu Ser 275 280 285 Gly Thr
Lys His Ser Gly Glu Ala Pro Ala Val Glu Glu Thr Val Thr 290 295 300
Ser Ser Pro Gly Thr Pro Ala Ser 305 310 88243PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
sequence 88Met Val His Ala Thr Ser Pro Leu Leu Leu Leu Leu Leu Leu
Ser Leu 1 5 10 15 Ala Leu Val Ala Pro Gly Leu Ser Ala Arg Lys Cys
Ser Leu Thr Gly 20 25 30 Lys Trp Thr Asn Asp Leu Gly Ser Asn Met
Thr Ile Gly Ala Val Asn 35 40 45 Ser Lys Gly Glu Phe Thr Gly Thr
Tyr Thr Thr Ala Val Thr Ala Thr 50 55 60 Ser Asn Glu Ile Lys Glu
Ser Pro Leu His Gly Thr Gln Asn Thr Ile 65 70 75 80Asn Lys Arg Thr
Gln Pro Thr Phe Gly Phe Thr Val Asn Trp Lys Phe 85 90 95 Ser Glu
Ser Thr Thr Val Phe Thr Gly Gln Cys Phe Ile Asp Arg Asn 100 105 110
Gly Lys Glu Val Leu Lys Thr Met Trp Leu Leu Arg Ser Ser Val Asn 115
120 125 Asp Ile Gly Asp Asp Trp Lys Ala Thr Arg Val Gly Ile Asn Ile
Phe 130 135 140 Thr Arg Leu Arg Thr Gln Lys Glu Gln Leu Leu Ala Ser
Leu Ala Leu 145 150 155 160Ile Thr Gln Gln Asp Leu Ala Pro Gln Gln
Arg Ala Ala Pro Gln Gln 165 170 175 Lys Arg Ser Ser Pro Thr Glu Gly
Leu Cys Pro Pro Gly His His Ile 180 185 190 Ser Glu Asp Ser Arg Asp
Cys Ile Ser Cys Lys Tyr Gly Gln Asp Tyr 195 200 205 Ser Thr His Trp
Asn Asp Phe Leu Phe Cys Leu Arg Cys Thr Lys Cys 210 215 220 Asp Ser
Gly Glu Val Glu Val Ser Ser Cys Thr Thr Thr Arg Asn Thr 225 230
235 240Val Cys Gln 89312PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide sequence 89Met Val His
Ala Thr Ser Pro Leu Leu Leu Leu Leu Leu Leu Ser Leu 1 5 10 15 Ala
Leu Val Ala Pro Gly Leu Ser Ala Arg Lys Cys Ser Leu Thr Gly 20 25
30 Lys Trp Thr Asn Asp Leu Gly Ser Asn Met Thr Ile Gly Ala Val Asn
35 40 45 Ser Lys Gly Glu Phe Thr Gly Thr Tyr Thr Thr Ala Val Thr
Ala Thr 50 55 60 Ser Asn Glu Ile Lys Glu Ser Pro Leu His Gly Thr
Gln Asn Thr Ile 65 70 75 80Asn Lys Arg Thr Gln Pro Thr Phe Gly Phe
Thr Val Asn Trp Lys Phe 85 90 95 Ser Glu Ser Thr Thr Val Phe Thr
Gly Gln Cys Phe Ile Asp Arg Asn 100 105 110 Gly Lys Glu Val Leu Lys
Thr Met Trp Leu Leu Arg Ser Ser Val Asn 115 120 125 Asp Ile Gly Asp
Asp Trp Lys Ala Thr Arg Val Gly Ile Asn Ile Phe 130 135 140 Thr Arg
Leu Arg Thr Gln Lys Glu Gln Leu Leu Ala Ser Leu Ala Leu 145 150 155
160Ile Thr Gln Gln Asp Leu Ala Pro Gln Gln Arg Ala Ala Pro Gln Gln
165 170 175 Lys Arg Ser Ser Pro Thr Glu Gly Leu Cys Pro Pro Gly His
His Ile 180 185 190 Ser Glu Asp Ser Arg Asp Cys Ile Ser Cys Lys Tyr
Gly Gln Asp Tyr 195 200 205 Ser Thr His Trp Asn Asp Phe Leu Phe Cys
Leu Arg Cys Thr Lys Cys 210 215 220 Asp Ser Gly Glu Val Glu Val Ser
Ser Cys Thr Thr Thr Arg Asn Thr 225 230 235 240Val Cys Gln Cys Glu
Glu Gly Thr Phe Arg Glu Glu Asp Ser Pro Glu 245 250 255 Ile Cys Arg
Lys Cys Arg Thr Gly Cys Pro Arg Gly Met Val Lys Val 260 265 270 Lys
Asp Cys Thr Pro Trp Ser Asp Ile Glu Cys Val His Lys Glu Ser 275 280
285 Gly Thr Lys His Thr Gly Glu Val Pro Ala Val Glu Lys Thr Val Thr
290 295 300 Thr Ser Pro Gly Thr Pro Ala Ser 305 310
90311PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide sequence 90Met Val His Ala Thr Ser Pro Leu
Leu Leu Leu Leu Leu Leu Ser Leu 1 5 10 15 Ala Leu Val Ala Pro Gly
Leu Ser Ala Arg Lys Cys Ser Leu Thr Gly 20 25 30 Lys Trp Thr Asn
Asp Leu Gly Ser Asn Met Thr Ile Gly Ala Val Asn 35 40 45 Ser Lys
Gly Glu Phe Thr Gly Thr Tyr Thr Thr Ala Val Thr Ala Thr 50 55 60
Ser Asn Glu Ile Lys Glu Ser Pro Leu His Gly Thr Gln Asn Thr Ile 65
70 75 80Asn Lys Arg Thr Gln Pro Thr Phe Gly Phe Thr Val Asn Trp Lys
Phe 85 90 95 Ser Glu Ser Thr Thr Val Phe Thr Gly Gln Cys Phe Ile
Asp Arg Asn 100 105 110 Gly Lys Glu Val Leu Lys Thr Met Trp Leu Leu
Arg Ser Ser Val Asn 115 120 125 Asp Ile Gly Asp Asp Trp Lys Ala Thr
Arg Val Gly Ile Asn Ile Phe 130 135 140 Thr Arg Leu Arg Thr Gln Lys
Glu Gln Leu Leu Ala Ser Leu Val Pro 145 150 155 160Val Thr Ala Asn
Pro Ala His Asn Arg Pro Ala Gly Leu Gln Arg Pro 165 170 175 Glu Glu
Ser Pro Ser Glu Gly Leu Cys Pro Pro Gly His His Ile Ser 180 185 190
Glu Asp Gly Arg Asp Cys Ile Ser Cys Lys Tyr Gly Gln Asp Tyr Ser 195
200 205 Thr His Trp Asn Asp Leu Leu Phe Cys Leu Arg Cys Thr Arg Cys
Asp 210 215 220 Ser Gly Glu Val Glu Leu Ser Pro Cys Thr Thr Thr Arg
Asn Thr Val 225 230 235 240Cys Gln Cys Glu Glu Gly Thr Phe Arg Glu
Glu Asp Ser Pro Glu Met 245 250 255 Cys Arg Lys Cys Arg Thr Gly Cys
Pro Arg Gly Met Val Lys Val Gly 260 265 270 Asp Cys Thr Pro Trp Ser
Asp Ile Glu Cys Val His Lys Glu Ser Gly 275 280 285 Thr Lys His Ser
Gly Glu Ala Pro Ala Val Glu Glu Thr Val Thr Ser 290 295 300 Ser Pro
Gly Thr Pro Ala Ser 305 310 91312PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide sequence 91Met Val His
Ala Thr Ser Pro Leu Leu Leu Leu Leu Leu Leu Ser Leu 1 5 10 15 Ala
Leu Val Ala Pro Gly Leu Ser Ala Arg Lys Cys Ser Leu Thr Gly 20 25
30 Lys Trp Thr Asn Asp Leu Gly Ser Asn Met Thr Ile Gly Ala Val Asn
35 40 45 Ser Lys Gly Glu Phe Thr Gly Thr Tyr Thr Thr Ala Val Thr
Ala Thr 50 55 60 Ser Asn Glu Ile Lys Glu Ser Pro Leu His Gly Thr
Gln Asn Thr Ile 65 70 75 80Asn Lys Arg Thr Gln Pro Thr Phe Gly Phe
Thr Val Asn Trp Lys Phe 85 90 95 Ser Glu Ser Thr Thr Val Phe Thr
Gly Gln Cys Phe Ile Asp Arg Asn 100 105 110 Gly Lys Glu Val Leu Lys
Thr Met Trp Leu Leu Arg Ser Ser Val Asn 115 120 125 Asp Ile Gly Asp
Asp Trp Lys Ala Thr Arg Val Gly Ile Asn Ile Phe 130 135 140 Thr Arg
Leu Arg Thr Gln Lys Glu Gln Leu Leu Ala Ser Leu Ala Leu 145 150 155
160Ile Thr Gln Gln Asp Leu Ala Pro Gln Gln Arg Ala Ala Pro Gln Gln
165 170 175 Lys Arg Ser Ser Pro Ser Glu Gly Leu Cys Leu Ala Gly Gln
Tyr Leu 180 185 190 Ser Glu Asp Gly Arg Asp Cys Ile Ser Cys Lys Tyr
Gly Gln Asp Tyr 195 200 205 Ser Thr His Trp Asn Asp Leu Leu Phe Cys
Leu Arg Cys Thr Arg Cys 210 215 220 Asp Ser Gly Glu Val Glu Leu Ser
Pro Cys Thr Thr Thr Arg Asn Thr 225 230 235 240Val Cys Gln Cys Glu
Glu Gly Thr Phe Arg Glu Glu Asp Ser Pro Glu 245 250 255 Met Cys Arg
Lys Cys Arg Thr Gly Cys Pro Arg Gly Met Val Lys Val 260 265 270 Gly
Asp Cys Thr Pro Trp Ser Asp Ile Glu Cys Val His Lys Glu Ser 275 280
285 Gly Thr Lys His Ser Gly Glu Ala Pro Ala Val Glu Glu Thr Val Thr
290 295 300 Ser Ser Pro Gly Thr Pro Ala Ser 305 310
92312PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide sequence 92Met Val His Ala Thr Ser Pro Leu
Leu Leu Leu Leu Leu Leu Ser Leu 1 5 10 15 Ala Leu Val Ala Pro Gly
Leu Ser Ala Arg Lys Cys Ser Leu Thr Gly 20 25 30 Lys Trp Thr Asn
Asp Leu Gly Ser Asn Met Thr Ile Gly Ala Val Asn 35 40 45 Ser Lys
Gly Glu Phe Thr Gly Thr Tyr Thr Thr Ala Val Thr Ala Thr 50 55 60
Ser Asn Glu Ile Lys Glu Ser Pro Leu His Gly Thr Gln Asn Thr Ile 65
70 75 80Asn Lys Arg Thr Gln Pro Thr Phe Gly Phe Thr Val Asn Trp Lys
Phe 85 90 95 Ser Glu Ser Thr Thr Val Phe Thr Gly Gln Cys Phe Ile
Asp Arg Asn 100 105 110 Gly Lys Glu Val Leu Lys Thr Met Trp Leu Leu
Arg Ser Ser Val Asn 115 120 125 Asp Ile Gly Asp Asp Trp Lys Ala Thr
Arg Val Gly Ile Asn Ile Phe 130 135 140 Thr Arg Leu Arg Thr Gln Lys
Glu Gln Leu Leu Ala Ser Leu Ala Leu 145 150 155 160Ile Thr Gln Gln
Asp Leu Ala Pro Gln Gln Arg Ala Ala Pro Gln Gln 165 170 175 Lys Arg
Ser Ser Pro Ser Glu Gly Leu Cys Pro Pro Gly His His Ile 180 185 190
Ser Glu Asp Gly Arg Asp Cys Ile Ser Cys Lys Tyr Gly Gln Asp Tyr 195
200 205 Ser Thr His Trp Asn Asp Leu Leu Phe Cys Leu Arg Cys Thr Arg
Cys 210 215 220 Asp Ser Gly Glu Val Glu Leu Ser Pro Cys Thr Thr Thr
Arg Asn Thr 225 230 235 240Val Cys Gln Cys Glu Glu Gly Thr Phe Arg
Glu Glu Asp Ser Pro Glu 245 250 255 Met Cys Arg Lys Cys Arg Thr Gly
Cys Pro Arg Gly Met Val Lys Val 260 265 270 Gly Asp Cys Thr Pro Trp
Ser Asp Ile Glu Cys Val His Lys Glu Ser 275 280 285 Gly Thr Lys His
Ser Gly Glu Ala Pro Ala Val Glu Glu Thr Val Thr 290 295 300 Ser Ser
Pro Gly Thr Pro Ala Ser 305 310 93313PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
sequence 93Met Val His Ala Thr Ser Pro Leu Leu Leu Leu Leu Leu Leu
Ser Leu 1 5 10 15 Ala Leu Val Ala Pro Gly Leu Ser Ala Arg Lys Cys
Ser Leu Thr Gly 20 25 30 Lys Trp Thr Asn Asp Leu Gly Ser Asn Met
Thr Ile Gly Ala Val Asn 35 40 45 Ser Lys Gly Glu Phe Thr Gly Thr
Tyr Thr Thr Ala Val Thr Ala Thr 50 55 60 Ser Asn Glu Ile Lys Glu
Ser Pro Leu His Gly Thr Gln Asn Thr Ile 65 70 75 80Asn Lys Arg Thr
Gln Pro Thr Phe Gly Phe Thr Val Asn Trp Lys Phe 85 90 95 Ser Glu
Ser Thr Thr Val Phe Thr Gly Gln Cys Phe Ile Asp Arg Asn 100 105 110
Gly Lys Glu Val Leu Lys Thr Met Trp Leu Leu Arg Ser Ser Val Asn 115
120 125 Asp Ile Gly Asp Asp Trp Lys Ala Thr Arg Val Gly Ile Asn Ile
Phe 130 135 140 Thr Arg Leu Arg Thr Gln Lys Glu Gln Leu Leu Ala Ser
Leu Ala Leu 145 150 155 160Ile Thr Gln Gln Asp Leu Ala Pro Gln Gln
Arg Ala Ala Pro Gln Gln 165 170 175 Lys Arg Ser Ser Pro Ser Glu Gly
Leu Cys Pro Pro Gly His His Ile 180 185 190 Ser Glu Asp Gly Arg Asp
Cys Ile Ser Cys Lys Tyr Gly Gln Asp Tyr 195 200 205 Ser Thr His Ser
Asn His Ser Leu Asp Ser Cys Leu Arg Cys Thr Arg 210 215 220 Cys Asp
Ser Gly Glu Val Glu Leu Ser Pro Cys Thr Thr Thr Arg Asn 225 230 235
240Thr Val Cys Gln Cys Glu Glu Gly Thr Phe Arg Glu Glu Asp Ser Pro
245 250 255 Glu Met Cys Arg Lys Cys Arg Thr Gly Cys Pro Arg Gly Met
Val Lys 260 265 270 Val Gly Asp Cys Thr Pro Trp Ser Asp Ile Glu Cys
Val His Lys Glu 275 280 285 Ser Gly Thr Lys His Ser Gly Glu Ala Pro
Ala Val Glu Glu Thr Val 290 295 300 Thr Ser Ser Pro Gly Thr Pro Ala
Ser 305 310 9415PRTHomo sapiens 94Ala Leu Ile Thr Gln Gln Asp Leu
Ala Pro Gln Gln Arg Ala Ala 1 5 10 159542PRTHomo sapiens 95Cys Lys
Tyr Gly Gln Asp Tyr Ser Thr His Trp Asn Asp Leu Leu Phe 1 5 10 15
Cys Leu Arg Cys Thr Arg Cys Asp Ser Gly Glu Val Glu Leu Ser Pro 20
25 30 Cys Thr Thr Thr Arg Asn Thr Val Cys Gln 35 40 9685PRTHomo
sapiens 96Ala Leu Ile Thr Gln Gln Asp Leu Ala Pro Gln Gln Arg Ala
Ala Pro 1 5 10 15 Gln Gln Lys Arg Ser Ser Pro Ser Glu Gly Leu Cys
Pro Pro Gly His 20 25 30 His Ile Ser Glu Asp Gly Arg Asp Cys Ile
Ser Cys Lys Tyr Gly Gln 35 40 45 Asp Tyr Ser Thr His Trp Asn Asp
Leu Leu Phe Cys Leu Arg Cys Thr 50 55 60 Arg Cys Asp Ser Gly Glu
Val Glu Leu Ser Pro Cys Thr Thr Thr Arg 65 70 75 80Asn Thr Val Cys
Gln 859714DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 97gtaggtgctg tcct 149814DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
98tgagttccac gaca 149913DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 99cttccaagcc act
1310012DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 100cargcactgt ca 1210118DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
101gtaggtgctg tccttgct 1810221DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 102ctctgtgaca ctctcctggg a
2110334DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 103gctctagatt ggagggcgtt atccaccttc cact
3410439DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 104aactagctag cagttccaga tttcaactgc tcatcagat
3910518DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 105gctcccgggt agaagtca 1810622DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
106acyagtgtgg ccttgttggc tt 2210726DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
107gctctagagg gygggaacag agtgac 2610818DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
108acgacaccgt caccggtt 1810925DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 109aagtagtcct tgaccaggca gccca
2511030DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 110gctctagagg gtgccagggg gaagaccgat
3011128DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 111gctctagagc agggcgccag ggggaaga
2811220DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 112atgaggstcc cygctcagct 2011321DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
113atggaarccc cagckcagct t 2111422DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 114atggtgttgc agacccaggt ct
2211535DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 115gaagatctca ccatgaggst cccygctcag ctyct
3511638DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 116gaagatctca ccatggaarc cccagckcag cttctctt
3811738DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 117gaagatctca ccatggtgtt gcagacccag gtcttcat
3811818DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 118crtcwccacc atgrcmwg 1811919DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
119caccatgrcc wgstyccct 1912020DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer
120accatggcct ggrctcykct 2012119DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 121caccatggcm tggrycvyt
1912221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 122caccatggcy tggryccmay t 2112329DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
123gaagatctca ccatgrccwg styccctct 2912432DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
124gaagatctca ccatggcctg grctcykcts yt 3212530DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
125gaagatctca ccatggcmtg grycvytctc 3012630DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
126gaagatctca ccatggcytg gryccmaytc 3012725DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
127caccatggas tggacctgga gvntc 2512828DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
128caccatggac atactttgyt ccacgctc 2812923DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
129caccatggar ttkggrctbh gct 2313030DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
130caccatgaar cayctgtggt tcttcctyct 3013124DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
131caccatgggg tcaaccgyca tcct 2413228DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
132caccatgtct gtctccttcc tcatcttc 2813334DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
133gaagatctca ccatggastg gacctggagv ntcc 3413437DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
134gaagatctca ccatggacat actttgytcc acgctcc 3713541DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
135gaagatctca ccatggartt kggrctbhgc tggvttttyc t
4113639DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 136gaagatctca ccatgaarca yctgtggttc ttcctyctc
3913732DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 137gaagatctca ccatggggtc aaccgycatc ct
3213837DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 138gaagatctca ccatgtctgt ctccttcctc atcttct
3713944DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 139gaagatctca ccatggactg gacctggagg atcctcttct
tggt 44140165PRTHomo sapiens 140Ala Leu Ile Thr Gln Gln Asp Leu Ala
Pro Gln Gln Arg Ala Ala Pro 1 5 10 15 Gln Gln Lys Arg Ser Ser Pro
Ser Glu Gly Leu Cys Pro Pro Gly His 20 25 30 His Ile Ser Glu Asp
Gly Arg Asp Cys Ile Ser Cys Lys Tyr Gly Gln 35 40 45 Asp Tyr Ser
Thr His Trp Asn Asp Leu Leu Phe Cys Leu Arg Cys Thr 50 55 60 Arg
Cys Asp Ser Gly Glu Val Glu Leu Ser Pro Cys Thr Thr Thr Arg 65 70
75 80Asn Thr Val Cys Gln Cys Glu Glu Gly Thr Phe Arg Glu Glu Asp
Ser 85 90 95 Pro Glu Met Cys Arg Lys Cys Arg Thr Gly Cys Pro Arg
Gly Met Val 100 105 110 Lys Val Gly Asp Cys Thr Pro Trp Ser Asp Ile
Glu Cys Val His Lys 115 120 125 Glu Ser Gly Thr Lys His Ser Gly Glu
Ala Pro Ala Val Glu Glu Thr 130 135 140 Val Thr Ser Ser Pro Gly Thr
Pro Ala Ser Arg Ser Gly Ser Ser His 145 150 155 160His His His His
His 165141119PRTMus sp. 141Met Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Lys Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Thr 20 25 30 Tyr Gly Met Ser Trp Val
Arg Gln Thr Pro Asp Lys Arg Leu Glu Leu 35 40 45 Val Ala Leu Ile
Asn Ser Gln Gly Gly Ser Thr Tyr Asn Ser Asp Ser 50 55 60 Val Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Thr Leu 65 70 75
80Tyr Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr
85 90 95 Cys Ala Arg Arg Asp Tyr Glu Ser Leu Asp Ser Trp Gly Gln
Gly Thr 100 105 110 Ser Val Thr Val Ser Ser Gly 115 142122PRTMus
sp. 142Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Pro Val Ser Leu
Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val
Glu Tyr Ser 20 25 30 Gly Thr Ser Leu Ile Gln Trp Tyr Arg Gln Lys
Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Ala Ala Ser Asn
Val Asp Ser Glu Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Ser Leu Tyr Ile His 65 70 75 80Pro Val Glu Glu Asp
Asp Ile Ala Met Tyr Phe Cys Gln Gln Ser Arg 85 90 95 Lys Val Pro
Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110 Thr
Asp Ala Ala Pro Gly Leu Glu Ala Ala 115 120 143119PRTMus sp. 143Lys
Val Gln Leu Gln Gln Ser Gly Thr Glu Leu Val Lys Pro Gly Ala 1 5 10
15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Tyr
20 25 30 Ile Ile His Trp Val Lys Gln Arg Ser Gly Gln Gly Leu Glu
Trp Ile 35 40 45 Gly Trp Phe Tyr Pro Gly Ser Gly Tyr Ile Lys Tyr
Asn Glu Lys Phe 50 55 60 Lys Asp Lys Ala Thr Met Thr Ala Asp Lys
Ser Ser Ser Thr Val Tyr 65 70 75 80Met Glu Leu Ser Arg Leu Thr Ser
Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Thr Arg His Glu Glu Asp
Gly Tyr Tyr Ala Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr
Val Ser Ala 115 144108PRTMus sp. 144Asp Ile Val Met Thr Gln Ser His
Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr
Cys Lys Ala Ser Gln Asp Val Ser Ser Ala 20 25 30 Val Ala Trp Tyr
Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Trp
Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Val Gln Ala 65
70 75 80Glu Asp Leu Ala Leu Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro
Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100
105 14533DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 145gtaagcaagc ttggctctga tcacccaaca aga
3314630DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 146gattagggat ccagaggcag gagtccctgg
3014735DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 147tagttgggat cctcaggaga tgcaatctct accgt
3514835DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 148ggtagtggat cctcactgac acactgtgtt tctgg
3514935DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 149gtaatgggat cctcagacac attcgatgtc actcc
3515033DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 150gtaatgaagc ttgccacaac aaaagaggtc cag
3315134DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 151gattgaaagc ttgatctcct gcaaatatgg acag
3415232DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 152gtaatgaagc ttgcagtgcg aagaaggcac ct
3215334DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 153gatggaggat cctcaacacc tggtgcagcg caag
3415434DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 154gtaagtggat cctcagcagg gacttagctc cact
3415529DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 155gttagtaagc ttggctccaa tcacccgac
2915631DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 156gttgatggat ccttctttgt ggacactcga t
3115734DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 157gtagttggat cctcaagaag caggagtccc aggg
3415836DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 158gtatgaggga tcctcactga cacaccgtgt ttctgg
3615934DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 159gtatggaagc ttgccacaac aaaagagatc cagc
3416035DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 160cagcgaagag cggctccaca acaaaagagg tccag
3516135DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 161ggatctcttt tgttgtgggg ccgctctctg ctggg
3516236DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 162cagcagagag cggccccaca acaaaagaga tccagc
3616334DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 163cagcggccgg aggagagccc ctcagaggga ttgt
3416433DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 164gattgaggat ccctaagagg caggagtccc tgg
3316534DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 165tgaatgaagc ttggttccag taacagctaa ccca
3416633DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 166tccctctgag gggctctcct ccggccgctg tag
3316737DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 167caggtactgg cctgctagac acaatccctc tgagggg
3716839DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 168ctagcaggcc agtacctgtc agaagacggt agagattgc
3916937DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 169caggtactgg cctgctagac acaatccctc tgagggg
3717039DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 170ctagcaggcc agtacctgtc agaagacggt agagattgc
3917141DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 171tgaatccaga gaatggttgg agtgagtgct atagtcctgt c
4117239DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 172tccaaccatt ctctggattc atgcttgcgc tgcaccagg
3917333DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 173gattgaggat ccctaagagg caggagtccc tgg
3317446DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 174tcgggtttct acgactttat cttccttaca cctggtgcag
cgcaag 4617547DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 175aaggaagata aagtcgtaga aacccgatgc
accacgacca gaaacac 4717633DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 176gattgaggat ccctaagagg
caggagtccc tgg 33177118PRTHomo sapiens 177Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asp Ile
Asn Trp Val Arg Gln Ala Thr Gly Gln Gly Leu Glu Trp Met 35 40 45
Gly Trp Met Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe 50
55 60 Gln Gly Arg Val Thr Met Thr Arg Asn Thr Ser Ile Ser Thr Ala
Tyr 65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Tyr Gly Ser Gly Ser Phe Asp Tyr Trp
Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala 115
178118PRTHomo sapiens 178Gln Val Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val
Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30 Gly Tyr Tyr Trp Ser Trp
Ile Arg Gln His Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Tyr
Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys
Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75
80Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
85 90 95 Cys Ala Arg Ser Ser Gly Trp Phe Asp Tyr Trp Gly Gln Gly
Thr Leu 100 105 110 Val Thr Val Ser Ser Ala 115 179117PRTHomo
sapiens 179Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Asp Tyr 20 25 30 Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Gly Ser
Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Ala Ala Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val 100 105 110
Thr Val Ser Ser Ala 115 180119PRTHomo sapiens 180Gln Val Gln Leu
Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser
Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr 65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Ser Ser Ser Trp Tyr Phe
Asp Leu Trp Gly Arg Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala
115 181117PRTHomo sapiens 181Gln Val Gln Leu Gln Glu Ser Gly Pro
Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr
Val Ser Gly Gly Ser Ile Ser Ser Tyr 20 25 30 Tyr Trp Ser Trp Ile
Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile
Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55
60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu
65 70 75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
Cys Ala 85 90 95 Arg Gly Tyr Ser Gly Tyr Phe Asp Tyr Trp Gly Gln
Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala 115 182117PRTHomo
sapiens 182Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro
Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser
Ile Ser Ser Gly 20 25 30 Gly Tyr Tyr Trp Ser Trp Ile Arg Gln His
Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Tyr Ile Tyr Tyr Ser
Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Thr
Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80Ser Leu Lys Leu
Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala
Arg Asn Trp Asn Phe Asp Ile Trp Gly Gln Gly Thr Met Val 100 105 110
Thr Val Ser Ser Ala 115 183114PRTHomo sapiens 183Gln Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30
Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser
Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
Ser Leu Tyr 65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Met Asp Val Trp Gly Gln
Gly Thr Thr Val Thr Val Ser 100 105 110 Ser Ala 184123PRTHomo
sapiens 184Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro
Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser
Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Tyr Ser Ser Ser Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val Trp 100 105 110
Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala 115 120 185118PRTHomo
sapiens 185Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro
Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser
Phe Ser Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly
Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn His Ser Gly Ser
Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser
Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80Lys Leu Ser Ser
Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Ser
Ser Gly Tyr Trp Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu 100 105 110
Val Thr Val Ser Ser Ala 115 186117PRTHomo sapiens 186Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25
30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp
Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Ser Leu Tyr 65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Ser Trp Tyr Trp Phe Asp
Pro Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala 115
187126PRTHomo sapiens 187Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp
Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Arg Gly Tyr Cys Thr Asn Gly Val Cys Tyr Tyr Tyr Tyr
Gly Met 100 105 110 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser
Ser Ala 115 120 125 188118PRTHomo sapiens 188Gln Val Gln Leu Gln
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser
Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30 Gly
Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu 35 40
45 Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser
50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn
Gln Phe 65 70 75 80Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr
Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Tyr Tyr Tyr Gly Met Asp Val
Trp Gly Gln Gly Thr Thr 100 105 110 Val Thr Val Ser Ser Ala 115
189120PRTHomo sapiens 189Gln Val Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val
Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30 Gly Tyr Tyr Trp Ser Trp
Ile Arg Gln His Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Tyr
Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys
Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75
80Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
85 90 95 Cys Ala Arg Gly Ser Gly Ser Tyr Trp Phe Asp Pro Trp Gly
Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala 115
120190106PRTHomo sapiens 190Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser
Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Thr Phe
85 90 95 Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105
191108PRTHomo sapiens 191Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser
Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Phe
85 90 95 Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg 100 105
192111PRTHomo sapiens 192Asp Ile Val Met Thr Gln Ser Pro Leu Ser
Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg
Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Asn Gly Tyr Asn Tyr Leu
Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu
Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75
80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala
85 90 95 Leu Gln Thr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
Arg 100 105 110 193106PRTHomo sapiens 193Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro 65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser
Phe Pro Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100
105 194106PRTHomo sapiens 194Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Gly Ile Arg Asn Asp 20 25 30 Leu Gly Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Ala Ala
Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70
75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Trp Thr
Phe 85 90 95 Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105
195108PRTHomo sapiens 195Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Gly Ile Ser Asn Tyr 20 25 30 Leu Ala Trp Phe Gln Gln
Lys Pro Gly Lys Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Ala Ala Ser
Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Leu
85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105
196114PRTHomo sapiens 196Asp Ile Val Met Thr Gln Ser Pro Asp Ser
Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys
Ser Ser Gln Ser Val Leu Tyr Ser 20 25 30 Ser Asn Asn Lys Asn Tyr
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu
Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75
80Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln
85 90 95 Tyr Tyr Ser Thr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val
Glu Ile 100 105 110 Lys Arg 197114PRTHomo sapiens 197Asp Ile Val
Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu
Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser 20 25
30 Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser
Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr 65 70 75 80Ile Ser Ser Leu Gln Ala Glu Asp Val Ala
Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Ser Thr Pro Phe Thr Phe
Gly Pro Gly Thr Lys Val Asp Ile 100 105 110 Lys Arg 198106PRTHomo
sapiens 198Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly
Ile Ser Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys
Val Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Gln Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80Glu Asp Val Ala
Thr Tyr Tyr Cys Gln Lys Tyr Asn Ser Ala Thr Phe 85 90 95 Gly Gly
Gly Thr Lys Val Glu Ile Lys Arg 100 105 199111PRTHomo sapiens
199Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu
Tyr Ser 20 25 30 Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser
Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80Ile Ser Ser Leu Gln Ala
Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Ser Thr
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110
200109PRTHomo sapiens 200Glu Ile Val Leu Thr Gln Ser Pro Gly Thr
Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg
Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala
Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75
80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln
Gln Tyr Gly Ser Ser Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg 100 105 201108PRTHomo sapiens 201Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25
30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro 65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Ala Asn Ser Phe Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys Arg 100 105 202129PRTMacaca fascicularis 202Ala Pro Ile
Thr Arg Gln Ser Leu Asp Pro Gln Arg Arg Ala Ala Pro 1 5 10 15 Gln
Gln Lys Arg Ser Ser Pro Thr Glu Gly Leu Cys Pro Pro Gly His 20 25
30 His Ile Ser Glu Asp Ser Arg Asp Cys Ile Ser Cys Lys Tyr Gly Gln
35 40 45 Asp Tyr Ser Thr His Trp Asn Asp Phe Leu Phe Cys Leu Arg
Cys Thr 50 55 60 Lys Cys Asp Ser Gly Glu Val Glu Val Ser Ser Cys
Thr Thr Thr Arg 65 70 75 80Asn Thr Val Cys Gln Cys Glu Glu Gly Thr
Phe Arg Glu Glu Asp Ser 85 90 95 Pro Glu Ile Cys Arg Lys Cys Arg
Thr Gly Cys Pro Arg Gly Met Val 100 105 110 Lys Val Lys Asp Cys Thr
Pro Trp Ser Asp Ile Glu Cys Val His Lys 115 120 125 Glu
203154PRTHomo sapiens 203Ala Leu Ile Thr Gln Gln Asp Leu Ala Pro
Gln Gln Arg Ala Ala Pro 1 5 10 15 Gln Gln Lys Arg Ser Ser Pro Ser
Glu Gly Leu Cys Pro Pro Gly His 20 25 30 His Ile Ser Glu Asp Gly
Arg Asp Cys Ile Ser Cys Lys Tyr Gly Gln 35 40 45 Asp Tyr Ser Thr
His Trp Asn Asp Leu Leu Phe Cys Leu Arg Cys Thr 50 55 60 Arg Cys
Asp Ser Gly Glu Val Glu Leu Ser Pro Cys Thr Thr Thr Arg 65 70 75
80Asn Thr Val Cys Gln Cys Glu Glu Gly Thr Phe Arg Glu Glu Asp Ser
85 90 95 Pro Glu Met Cys Arg Lys Cys Arg Thr Gly Cys Pro Arg Gly
Met Val 100 105 110 Lys Val Gly Asp Cys Thr Pro Trp Ser Asp Ile Glu
Cys Val His Lys 115 120 125 Glu Ser Gly Thr Lys His Ser Gly Glu Ala
Pro Ala Val Glu Glu Thr 130 135 140 Val Thr Ser Ser Pro Gly Thr Pro
Ala Ser 145 150 204133PRTMus sp. 204Pro Val Thr Ala Asn Pro Ala His
Asn Arg Pro Ala Gly Leu Gln Arg 1 5 10 15 Pro Glu Glu Ser Pro Ser
Arg Gly Pro Cys Leu Ala Gly Gln Tyr Leu 20 25 30 Ser Glu Gly Asn
Cys Lys Pro Cys Arg Glu Gly Ile Asp Tyr Thr Ser 35 40 45 His Ser
Asn His Ser Leu Asp Ser Cys Ile Leu Cys Thr Val Cys Lys 50 55 60
Glu Asp Lys Val Val Glu Thr Arg Cys Asn Ile Thr Thr Asn Thr Val 65
70 75 80Cys Arg Cys Lys Pro Gly Thr Phe Glu Asp Lys Asp Ser Pro Glu
Ile 85 90 95 Cys Gln Ser Cys Ser Asn Cys Thr Asp Gly Glu Glu Glu
Leu Thr Ser 100 105 110 Cys Thr Pro Arg Glu Asn Arg Lys Cys Val Ser
Lys Thr Ala Trp Ala 115 120 125 Ser Trp His Lys Leu 130
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