U.S. patent application number 16/459791 was filed with the patent office on 2019-10-17 for multi-specific antigen-binding molecule having alternative function to function of blood coagulation factor viii.
This patent application is currently assigned to Chugai Seiyaku Kabushiki Kaisha. The applicant listed for this patent is Chugai Seiyaku Kabushiki Kaisha. Invention is credited to Tomoyuki Igawa, Chifumi Imai, Takehisa Kitazawa, Tetsuo Kojima, Atsushi Muto, Yukiko Nishida, Zenjiro Sampei, Tetsuhiro Soeda, Tsukasa Suzuki, Kazutaka Yoshihashi.
Application Number | 20190315884 16/459791 |
Document ID | / |
Family ID | 46084097 |
Filed Date | 2019-10-17 |
United States Patent
Application |
20190315884 |
Kind Code |
A1 |
Igawa; Tomoyuki ; et
al. |
October 17, 2019 |
MULTI-SPECIFIC ANTIGEN-BINDING MOLECULE HAVING ALTERNATIVE FUNCTION
TO FUNCTION OF BLOOD COAGULATION FACTOR VIII
Abstract
Various bispecific antibodies that specifically bind to both
blood coagulation factor IX/activated blood coagulation factor IX
and blood coagulation factor X and functionally substitute for the
cofactor function of blood coagulation factor VIII, that is, the
function to promote activation of blood coagulation factor X by
activated blood coagulation factor IX, were produced. From these
antibodies, multispecific antigen-binding molecules having a high
activity of functionally substituting for blood coagulation factor
VIII were successfully discovered.
Inventors: |
Igawa; Tomoyuki; (Shizuoka,
JP) ; Sampei; Zenjiro; (Shizuoka, JP) ;
Kojima; Tetsuo; (Shizuoka, JP) ; Soeda;
Tetsuhiro; (Shizuoka, JP) ; Muto; Atsushi;
(Shizuoka, JP) ; Kitazawa; Takehisa; (Shizuoka,
JP) ; Nishida; Yukiko; (Shizuoka, JP) ; Imai;
Chifumi; (Shizuoka, JP) ; Suzuki; Tsukasa;
(Shizuoka, JP) ; Yoshihashi; Kazutaka; (Shizuoka,
JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Chugai Seiyaku Kabushiki Kaisha |
Tokyo |
|
JP |
|
|
Assignee: |
Chugai Seiyaku Kabushiki
Kaisha
Tokyo
JP
|
Family ID: |
46084097 |
Appl. No.: |
16/459791 |
Filed: |
July 2, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15288965 |
Oct 7, 2016 |
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16459791 |
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15132996 |
Apr 19, 2016 |
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15288965 |
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13885421 |
Aug 30, 2013 |
9334331 |
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PCT/JP2011/076486 |
Nov 17, 2011 |
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15132996 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/565 20130101;
A61P 43/00 20180101; C07K 2317/31 20130101; C07K 16/36 20130101;
A61P 7/04 20180101; C07K 2317/76 20130101; C07K 2317/94 20130101;
C07K 2317/56 20130101; C07K 2317/51 20130101; C07K 2317/75
20130101; A61P 7/00 20180101; C07K 16/40 20130101 |
International
Class: |
C07K 16/40 20060101
C07K016/40; C07K 16/36 20060101 C07K016/36 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 17, 2010 |
JP |
2010-257022 |
Claims
1. A multispecific antigen-binding molecule that functionally
substitutes for blood coagulation factor VIII, which comprises a
first antigen-binding site that recognizes blood coagulation factor
IX and/or activated blood coagulation factor IX and a second
antigen-binding site that recognizes blood coagulation factor X,
wherein the functional substitution for blood coagulation factor
VIII results from an activated blood coagulation factor X (F.Xa)
generation-promoting activity higher than the activity of a
bispecific antibody (hA69-KQ/hB26-PF/hAL-AQ) which comprises an H
chain comprising SEQ ID NOs: 165 and 166, and a commonly shared L
chain comprising SEQ ID NO: 167.
2. The multispecific antigen-binding molecule of claim 1, which
comprises a first polypeptide comprising a first antigen-binding
site that recognizes blood coagulation factor IX and/or activated
blood coagulation factor IX and a third polypeptide comprising a
third antigen-binding site that recognizes blood coagulation factor
IX and/or activated blood coagulation factor IX, as well as a
second polypeptide comprising a second antigen-binding site that
recognizes blood coagulation factor X and a fourth polypeptide
comprising a fourth antigen-binding site that recognizes blood
coagulation factor X.
3. The multispecific antigen-binding molecule of claim 2, wherein
the first polypeptide and the third polypeptide each comprises an
antigen-binding site of an H chain or L chain of an antibody
against blood coagulation factor IX or activated blood coagulation
factor IX, respectively; and the second polypeptide and the fourth
polypeptide each comprises an antigen-binding site of an H chain or
L chain of an antibody against blood coagulation factor X,
respectively.
4. The multispecific antigen-binding molecule of claim 3, wherein
the antigen-binding site of the first polypeptide comprises an
antigen-binding site which comprises H chain CDRs consisting of any
one of the amino acid sequences selected from the following (a1) to
(a11), or an antigen-binding site functionally equivalent thereto,
and the antigen-binding site of the second polypeptide comprises an
antigen-binding site which comprises H chain CDRs consisting of any
one of the amino acid sequences selected from the following (b1) to
(b11), or an antigen-binding site functionally equivalent thereto:
(a1) an antigen-binding site comprising an H chain CDR 1, 2, and 3
amino acid sequences of SEQ ID NOs: 75, 76, and 77 (H chain CDRs of
Q1), respectively; (a2) an antigen-binding site comprising an H
chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 78, 79,
and 80 (H chain CDRs of Q31), respectively; (a3) an antigen-binding
site comprising an H chain CDR 1, 2, and 3 amino acid sequences of
SEQ ID NOs: 81, 82, and 83 (H chain CDRs of Q64), respectively;
(a4) an antigen-binding site comprising an H chain CDR 1, 2, and 3
amino acid sequences of SEQ ID NOs: 84, 85, and 86 (H chain CDRs of
Q85), respectively; (a5) an antigen-binding site comprising the H
chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 87, 88,
and 89 (H chain CDRs of Q153), respectively; (a6) an
antigen-binding site comprising an H chain CDR 1, 2, and 3 amino
acid sequences of SEQ ID NOs: 90, 91, and 92 (H chain CDRs of
Q354), respectively; (a7) an antigen-binding site comprising the H
chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 93, 94,
and 95 (H chain CDRs of Q360), respectively; (a8) an
antigen-binding site comprising the of H chain CDR 1, 2, and 3
amino acid sequences of SEQ ID NOs: 96, 97, and 98 (H chain CDRs of
Q405), respectively; (a9) an antigen-binding site comprising an H
chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 99, 100,
and 101 (H chain CDRs of Q458), respectively; (a10) an
antigen-binding site comprising an H chain CDR 1, 2, and 3 amino
acid sequences of SEQ ID NOs: 102, 103, and 104 (H chain CDRs of
Q460), respectively; (a11) an antigen-binding site comprising an H
chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 105, 106,
and 107 (H chain CDRs of Q499), respectively; (b1) an
antigen-binding site comprising an H chain CDR 1, 2, and 3 amino
acid sequences of SEQ ID NOs: 108, 109, and 110 (H chain CDRs of
J232), respectively; (b2) an antigen-binding site comprising an H
chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 111, 112,
and 113 (H chain CDRs of J259), respectively; (b3) an
antigen-binding site comprising an H chain CDR 1, 2, and 3 amino
acid sequences of SEQ ID NOs: 114, 115, and 116 (H chain CDRs of
J268), respectively; (b4) an antigen-binding site comprising an H
chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 117, 118,
and 119 (H chain CDRs of J300), respectively; (b5) an
antigen-binding site comprising an H chain CDR 1, 2, and 3 amino
acid sequences of SEQ ID NOs: 120, 121, and 122 (H chain CDRs of
J321), respectively; (b6) an antigen-binding site comprising the H
chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 123, 124,
and 125 (H chain CDRs of J326), respectively; (b7) an
antigen-binding site comprising an H chain CDR 1, 2, and 3 amino
acid sequences of SEQ ID NOs: 126, 127, and 128 (H chain CDRs of
J327), respectively; (b8) an antigen-binding site comprising an H
chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 129, 130,
and 131 (H chain CDRs of J339), respectively; (b9) an
antigen-binding site comprising an H chain CDR 1, 2, and 3 amino
acid sequences of SEQ ID NOs: 132, 133, and 134 (H chain CDRs of
J344), respectively; (b10) an antigen-binding site comprising an H
chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 135, 136,
and 137 (H chain CDRs of J346), respectively; and (b11) an
antigen-binding site comprising an H chain CDR 1, 2, and 3 amino
acid sequences of SEQ ID NOs: 174, 175, and 176 (H chain CDRs of
J142), respectively.
5. The multispecific antigen-binding molecule of claim 3, wherein
the antigen-binding site of the first polypeptide comprises an
antigen-binding site which comprises an H chain variable region
consisting of any one of the amino acid sequences selected from the
following (a1) to (a11), or an antigen-binding site functionally
equivalent thereto, and the antigen-binding site of the second
polypeptide comprises an antigen-binding site which comprises an H
chain variable region consisting of any one of the amino acid
sequences selected from the following (b1) to (b11), or an
antigen-binding site functionally equivalent thereto: (a1) an
antigen-binding site comprising an H chain variable region amino
acid sequence of SEQ ID NO: 35 (H chain variable region of Q1);
(a2) an antigen-binding site comprising an H chain variable region
amino acid sequence of SEQ ID NO: 36 (H chain variable region of
Q31); (a3) an antigen-binding site comprising an H chain variable
region amino acid sequence of SEQ ID NO: 37 (H chain variable
region of Q1); (a4) an antigen-binding site comprising an H chain
variable region amino acid sequence of SEQ ID NO: 38 (H chain
variable region of Q85); (a5) an antigen-binding site comprising an
H chain variable region amino acid sequence of SEQ ID NO: 39 (H
chain variable region of Q153); (a6) an antigen-binding site
comprising an H chain variable region amino acid sequence of SEQ ID
NO: 40 (H chain variable region of Q354); (a7) an antigen-binding
site comprising an H chain variable region amino acid sequence of
SEQ ID NO: 41 (H chain variable region of Q360); (a8) an
antigen-binding site comprising an H chain variable region amino
acid sequence of SEQ ID NO: 42 (H chain variable region of Q405);
(a9) an antigen-binding site comprising an H chain variable region
amino acid sequence of SEQ ID NO: 43 (H chain variable region of
Q458); (a10) an antigen-binding site comprising an H chain variable
region amino acid sequence of SEQ ID NO: 44 (H chain variable
region of Q460); (a11) an antigen-binding site comprising an H
chain variable region amino acid sequence of SEQ ID NO: 45 (H chain
variable region of Q499); (b1) an antigen-binding site comprising
an H chain variable region amino acid sequence of SEQ ID NO: 46 (H
chain variable region of J232); (b2) an antigen-binding site
comprising an H chain variable region amino acid sequence of SEQ ID
NO: 47 (H chain variable region of J259); (b3) an antigen-binding
site comprising an H chain variable region amino acid sequence of
SEQ ID NO: 48 (H chain variable region of J268); (b4) an
antigen-binding site comprising an H chain variable region amino
acid sequence of SEQ ID NO: 49 (H chain variable region of J300);
(b5) an antigen-binding site comprising an H chain variable region
amino acid sequence of SEQ ID NO: 50 (H chain variable region of
J321); (b6) an antigen-binding site comprising an H chain variable
region amino acid sequence of SEQ ID NO: 51 (H chain variable
region of J326); (b7) an antigen-binding site comprising an H chain
variable region amino acid sequence of SEQ ID NO: 52 (H chain
variable region of J327); (b8) an antigen-binding site comprising
an H chain variable region amino acid sequence of SEQ ID NO: 53 (H
chain variable region of J339); (b9) an antigen-binding site
comprising an H chain variable region amino acid sequence of SEQ ID
NO: 54 (H chain variable region of J344); (b10) an antigen-binding
site comprising an H chain variable region amino acid sequence of
SEQ ID NO: 55 (H chain variable region of J346); and (b11) an
antigen-binding site comprising an H chain variable region amino
acid sequence of SEQ ID NO: 172 (H chain variable region of
J142).
6. The multispecific antigen-binding molecule of claim 3, wherein
the antigen-binding sites included in the third polypeptide and the
fourth polypeptide comprise an antigen-binding site which comprises
L chain CDRs consisting of any one of the amino acid sequences
selected from the following (c1) to (c10), or an antigen-binding
site functionally equivalent thereto: (c1) an antigen-binding site
comprising an L chain CDR1, 2, and 3 amino acid sequences of SEQ ID
NOs: 138, 139, and 140 (L chain CDR of L2), respectively; (c2) an
antigen-binding site comprising an L chain CDR1, 2, and 3 amino
acid sequences of SEQ ID NOs: 141, 142, and 143 (L chain CDR of
L45), respectively; (c3) an antigen-binding site comprising an L
chain CDR1, 2, and 3 amino acid sequences of SEQ ID NOs: 144, 145,
and 146 (L chain CDR of L248), respectively; (c4) an
antigen-binding site comprising an L chain CDR1, 2, and 3 amino
acid sequences of SEQ ID NOs: 147, 148, and 149 (L chain CDR of
L324), respectively; (c5) an antigen-binding site comprising an L
chain CDR1, 2, and 3 amino acid sequences of SEQ ID NOs: 150, 151,
and 152 (L chain CDR of L334), respectively; (c6) an
antigen-binding site comprising an L chain CDR1, 2, and 3 amino
acid sequences of SEQ ID NOs: 153, 154, and 155 (L chain CDR of
L377), respectively; (c7) an antigen-binding site comprising an L
chain CDR1, 2, and 3 amino acid sequences of SEQ ID NOs: 156, 157,
and 158 (L chain CDR of L404), respectively; (c8) an
antigen-binding site comprising an L chain CDR1, 2, and 3 amino
acid sequences of SEQ ID NOs: 159, 160, and 161 (L chain CDR of
L406), respectively; (c9) an antigen-binding site comprising an L
chain CDR1, 2, and 3 amino acid sequences of SEQ ID NOs: 137, 138,
and 139 (L chain CDR of L408), respectively; and (c10) an
antigen-binding site comprising an L chain CDR1, 2, and 3 amino
acid sequences of SEQ ID NOs: 177, 178, and 179 (L chain CDR of
L180), respectively.
7. The multispecific antigen-binding molecule of claim 3, wherein
the antigen-binding sites included in the third polypeptide and the
fourth polypeptide comprise an antigen-binding site which comprises
an L chain variable region consisting of any one of the amino acid
sequences selected from the following (c1) to (c10), or an
antigen-binding site functionally equivalent thereto: (c1) an
antigen-binding site comprising an L chain variable region amino
acid sequence of SEQ ID NO: 56 (L chain variable region of L2);
(c2) an antigen-binding site comprising an L chain variable region
amino acid sequence of SEQ ID NO: 57 (L chain variable region of
L45); (c3) an antigen-binding site comprising an L chain variable
region amino acid sequence of SEQ ID NO: 58 (L chain variable
region of L248); (c4) an antigen-binding site comprising an L chain
variable region amino acid sequence of SEQ ID NO: 59 (L chain
variable region of L324); (c5) an antigen-binding site comprising
an L chain variable region amino acid sequence of SEQ ID NO: 60 (L
chain variable region of L334); (c6) an antigen-binding site
comprising an L chain variable region amino acid sequence of SEQ ID
NO: 61 (L chain variable region of L377); (c7) an antigen-binding
site comprising an L chain variable region amino acid sequence of
SEQ ID NO: 62 (L chain variable region of L404); (c8) an
antigen-binding site comprising an L chain variable region amino
acid sequence of SEQ ID NO: 63 (L chain variable region of L406);
(c9) an antigen-binding site comprising an L chain variable region
amino acid sequence of SEQ ID NO: 64 (L chain variable region of
L408); and (c10) an antigen-binding site comprising an L chain
variable region amino acid sequence of SEQ ID NO: 173 (L chain
variable region of L180).
8. The multispecific antigen-binding molecule of claim 3, wherein
the first and second polypeptides further comprise an antibody H
chain constant region, and the third and fourth polypeptides
comprise an antibody L chain constant region.
9. The multispecific antigen-binding molecule of claim 3, wherein
the first and second polypeptides comprise an antibody H chain
constant region, and the third and fourth polypeptides comprise an
antibody L chain constant region, and wherein the third polypeptide
and the fourth polypeptide are a commonly shared L chain.
10. The multispecific antigen-binding molecule of claim 8, wherein
the first polypeptide comprises any one antibody H chain selected
from the following (a1) to (a14), the second polypeptide comprises
any one antibody H chain selected from the following (b1) to (b12),
and the third polypeptide and the fourth polypeptide comprise any
one antibody L chain selected from the following (c1) to (c10):
(a1) an antibody H chain consisting of the amino acid sequence of
SEQ ID NO: 1 (Q1-G4k); (a2) an antibody H chain consisting of the
amino acid sequence of SEQ ID NO: 2 (Q31-z7); (a3) an antibody H
chain consisting of the amino acid sequence of SEQ ID NO: 3
(Q64-z55); (a4) an antibody H chain consisting of the amino acid
sequence of SEQ ID NO: 10 (Q64-z7); (a5) an antibody H chain
consisting of the amino acid sequence of SEQ ID NO: 11 (Q85-G4k);
(a6) an antibody H chain consisting of the amino acid sequence of
SEQ ID NO: 12 (Q153-G4k); (a7) an antibody H chain consisting of
the amino acid sequence of SEQ ID NO: 13 (Q354-z106); (a8) an
antibody H chain consisting of the amino acid sequence of SEQ ID
NO: 14 (Q360-G4k); (a9) an antibody H chain consisting of the amino
acid sequence of SEQ ID NO: 15 (Q360-z118); (a10) an antibody H
chain consisting of the amino acid sequence of SEQ ID NO: 16
(Q405-G4k); (a11) an antibody H chain consisting of the amino acid
sequence of SEQ ID NO: 17 (Q458-z106); (a12) an antibody H chain
consisting of the amino acid sequence of SEQ ID NO: 18 (Q460-z121);
(a13) an antibody H chain consisting of the amino acid sequence of
SEQ ID NO: 19 (Q499-z118); (a14) an antibody H chain consisting of
the amino acid sequence of SEQ ID NO: 20 (Q499-z121); (b1) an
antibody H chain consisting of the amino acid sequence of SEQ ID
NO: 4 (J268-G4h); (b2) an antibody H chain consisting of the amino
acid sequence of SEQ ID NO: 5 (J321-G4h); (b3) an antibody H chain
consisting of the amino acid sequence of SEQ ID NO: 6 (J326-z107);
(b4) an antibody H chain consisting of the amino acid sequence of
SEQ ID NO: 7 (J344-z107); (b5) an antibody H chain consisting of
the amino acid sequence of SEQ ID NO: 21 (J232-G4h); (b6) an
antibody H chain consisting of the amino acid sequence of SEQ ID
NO: 22 (J259-z107); (b7) an antibody H chain consisting of the
amino acid sequence of SEQ ID NO: 23 (J300-z107); (b8) an antibody
H chain consisting of the amino acid sequence of SEQ ID NO: 24
(J327-z107); (b9) an antibody H chain consisting of the amino acid
sequence of SEQ ID NO: 25 (J327-z119); (b10) an antibody H chain
consisting of the amino acid sequence of SEQ ID NO: 26 (J339-z119);
(b11) an antibody H chain consisting of the amino acid sequence of
SEQ ID NO: 27 (J346-z107); (b12) an antibody H chain consisting of
the amino acid sequence of SEQ ID NO: 170 (J142-G4h); (c1) an
antibody L chain consisting of the amino acid sequence of SEQ ID
NO: 8 (L2-k); (c2) an antibody L chain consisting of the amino acid
sequence of SEQ ID NO: 9 (L45-k); (c3) an antibody L chain
consisting of the amino acid sequence of SEQ ID NO: 28 (L248-k);
(c4) an antibody L chain consisting of the amino acid sequence of
SEQ ID NO: 29 (L324-k); (c5) an antibody L chain consisting of the
amino acid sequence of SEQ ID NO: 30 (L334-k); (c6) an antibody L
chain consisting of the amino acid sequence of SEQ ID NO: 31
(L377-k); (c7) an antibody L chain consisting of the amino acid
sequence of SEQ ID NO: 32 (L404-k); (c8) an antibody L chain
consisting of the amino acid sequence of SEQ ID NO: 33 (L406-k);
(c9) an antibody L chain consisting of the amino acid sequence of
SEQ ID NO: 34 (L408-k); and (c10) an antibody L chain consisting of
the amino acid sequence of SEQ ID NO: 171 (L180-k).
11. The multispecific antigen-binding molecule of claim 1, wherein
the multispecific antigen-binding molecule is a multispecific
antibody.
12. A bispecific antibody of any one of the following (a) to (u):
(a) a bispecific antibody (Q1-G4k/J268-G4h/L45-k), wherein the
first polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 1, the second polypeptide is an H chain
consisting of the amino acid sequence of SEQ ID NO: 4, and the
third polypeptide and the fourth polypeptide are a commonly shared
L chain of SEQ ID NO: 9; (b) a bispecific antibody
(Q1-G4k/J321-G4h/L45-k), wherein the first polypeptide is an H
chain consisting of the amino acid sequence of SEQ ID NO: 1, the
second polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 5, and the third polypeptide and the fourth
polypeptide are a commonly shared L chain of SEQ ID NO: 9; (c) a
bispecific antibody (Q31-z7/J326-z107/L2-k), wherein the first
polypeptide is an H chain consisting of the amino acid sequence of
SEQ ID NO: 2, the second polypeptide is an H chain consisting of
the amino acid sequence of SEQ ID NO: 6, and the third polypeptide
and the fourth polypeptide are a commonly shared L chain of SEQ ID
NO: 8; (d) a bispecific antibody (Q64-z55/J344-z107/L45-k), wherein
the first polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 3, the second polypeptide is an H chain
consisting of the amino acid sequence of SEQ ID NO: 7, and the
third polypeptide and the fourth polypeptide are a commonly shared
L chain of SEQ ID NO: 9; (e) a bispecific antibody
(Q64-z7/J326-z107/L334-k), wherein the first polypeptide is an H
chain consisting of the amino acid sequence of SEQ ID NO: 10, the
second polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 6, and the third polypeptide and the fourth
polypeptide are a commonly shared L chain of SEQ ID NO: 30; (f) a
bispecific antibody (Q64-z7/J344-z107/L406-k), wherein the first
polypeptide is an H chain consisting of the amino acid sequence of
SEQ ID NO: 10, the second polypeptide is an H chain consisting of
the amino acid sequence of SEQ ID NO: 7, and the third polypeptide
and the fourth polypeptide are a commonly shared L chain of SEQ ID
NO: 33; (g) a bispecific antibody (Q85-G4k/J268-G4h/L406-k),
wherein the first polypeptide is an H chain consisting of the amino
acid sequence of SEQ ID NO: 11, the second polypeptide is an H
chain consisting of the amino acid sequence of SEQ ID NO: 4, and
the third polypeptide and the fourth polypeptide are a commonly
shared L chain of SEQ ID NO: 33; (h) a bispecific antibody
(Q85-G4k/J321-G4h/L334-k), wherein the first polypeptide is an H
chain consisting of the amino acid sequence of SEQ ID NO: 11, the
second polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 5, and the third polypeptide and the fourth
polypeptide are a commonly shared L chain of SEQ ID NO: 30; (i) a
bispecific antibody (Q153-G4k/J232-G4h/L406-k), wherein the first
polypeptide is an H chain consisting of the amino acid sequence of
SEQ ID NO: 12, the second polypeptide is an H chain consisting of
the amino acid sequence of SEQ ID NO: 21, and the third polypeptide
and the fourth polypeptide are a commonly shared L chain of SEQ ID
NO: 33; (j) a bispecific antibody (Q354-z106/J259-z107/L324-k),
wherein the first polypeptide is an H chain consisting of the amino
acid sequence of SEQ ID NO: 13, the second polypeptide is an H
chain consisting of the amino acid sequence of SEQ ID NO: 22, and
the third polypeptide and the fourth polypeptide are a commonly
shared L chain of SEQ ID NO: 29; (k) a bispecific antibody
(Q360-G4k/J232-G4h/L406-k), wherein the first polypeptide is an H
chain consisting of the amino acid sequence of SEQ ID NO: 14, the
second polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 21, and the third polypeptide and the fourth
polypeptide are a commonly shared L chain of SEQ ID NO: 33; (l) a
bispecific antibody (Q360-z118/J300-z107/L334-k), wherein the first
polypeptide is an H chain consisting of the amino acid sequence of
SEQ ID NO: 15, the second polypeptide is an H chain consisting of
the amino acid sequence of SEQ ID NO: 23, and the third polypeptide
and the fourth polypeptide are a commonly shared L chain of SEQ ID
NO: 30; (m) a bispecific antibody (Q405-G4k/J232-G4h/L248-k),
wherein the first polypeptide is an H chain consisting of the amino
acid sequence of SEQ ID NO: 16, the second polypeptide is an H
chain consisting of the amino acid sequence of SEQ ID NO: 21, and
the third polypeptide and the fourth polypeptide are a commonly
shared L chain of SEQ ID NO: 28; (n) a bispecific antibody
(Q458-z106/J346-z107/L408-k), wherein the first polypeptide is an H
chain consisting of the amino acid sequence of SEQ ID NO: 17, the
second polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 27, and the third polypeptide and the fourth
polypeptide are a commonly shared L chain of SEQ ID NO: 34; (o) a
bispecific antibody (Q460-z121/J327-z119/L334-k), wherein the first
polypeptide is an H chain consisting of the amino acid sequence of
SEQ ID NO: 18, the second polypeptide is an H chain consisting of
the amino acid sequence of SEQ ID NO: 25, and the third polypeptide
and the fourth polypeptide are a commonly shared L chain of SEQ ID
NO: 30; (p) a bispecific antibody (Q499-z118/J327-z107/L334-k),
wherein the first polypeptide is an H chain consisting of the amino
acid sequence of SEQ ID NO: 19, the second polypeptide is an H
chain consisting of the amino acid sequence of SEQ ID NO: 24, and
the third polypeptide and the fourth polypeptide are a commonly
shared L chain of SEQ ID NO: 30; (q) a bispecific antibody
(Q499-z118/J327-z107/L377-k), wherein the first polypeptide is an H
chain consisting of the amino acid sequence of SEQ ID NO: 19, the
second polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 24, and the third polypeptide and the fourth
polypeptide are a commonly shared L chain of SEQ ID NO: 31; (r) a
bispecific antibody (Q499-z118/J346-z107/L248-k), wherein the first
polypeptide is an H chain consisting of the amino acid sequence of
SEQ ID NO: 19, the second polypeptide is an H chain consisting of
the amino acid sequence of SEQ ID NO: 27, and the third polypeptide
and the fourth polypeptide are a commonly shared L chain of SEQ ID
NO: 28; (s) a bispecific antibody (Q499-z121/J327-z119/L404-k),
wherein the first polypeptide is an H chain consisting of the amino
acid sequence of SEQ ID NO: 20, the second polypeptide is an H
chain consisting of the amino acid sequence of SEQ ID NO: 25, and
the third polypeptide and the fourth polypeptide are a commonly
shared L chain of SEQ ID NO: 32; (t) a bispecific antibody
(Q499-z121/J339-z119/L377-k), wherein the first polypeptide is an H
chain consisting of the amino acid sequence of SEQ ID NO: 20, the
second polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 26, and the third polypeptide and the fourth
polypeptide are a commonly shared L chain of SEQ ID NO: 31; and (u)
a bispecific antibody (Q153-G4k/J142-G4h/L180-k), wherein the first
polypeptide is an H chain consisting of the amino acid sequence of
SEQ ID NO: 12, the second polypeptide is an H chain consisting of
the amino acid sequence of SEQ ID NO: 170, and the third
polypeptide and the fourth polypeptide are a commonly shared L
chain of SEQ ID NO: 171.
13. A nucleic acid encoding the multispecific antigen-binding
molecule of claim 1.
14. A vector inserted with the nucleic acid of claim 13.
15. A cell comprising the nucleic acid of claim 13.
16. A method for producing a multispecific antigen-binding molecule
by culturing the cell of claim 15.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 15/288,965, filed Oct. 7, 2016, which is a divisional of U.S.
application Ser. No. 15/132,996, filed Apr. 19, 2016, which is a
divisional of U.S. application Ser. No. 13/885,421, filed May 15,
2013 (now U.S. Pat. No. 9,334,331, issued on May 10, 2016), which
is the National Stage of International Patent Application Ser. No.
PCT/JP2011/076486, filed Nov. 17, 2011, which claims the benefit of
Japanese Patent Application Serial No. 2010-257022, filed on Nov.
17, 2010.
TECHNICAL FIELD
[0002] The present invention relates to multispecific
antigen-binding molecules that functionally substitute for blood
coagulation factor VIII, a cofactor that enhances enzymatic
reactions, and pharmaceutical compositions comprising such a
molecule as an active ingredient.
BACKGROUND ART
[0003] Hemophilia A is a bleeding abnormality caused by a
hereditary decrease or deficiency of blood coagulation factor VIII
(F.VIII) function. Hemophilia A patients are generally administered
with an F.VIII formulation for the bleeding (on-demand
administration). In recent years, F.VIII formulations are also
administered prophylactically to prevent bleeding events
(preventive administration; Non-patent Documents 1 and 2). The
half-life of F.VIII formulations in blood is approximately 12 to 16
hours. Therefore, for continuous prevention, F. VIII formulations
are administered to patients three times a week (Non-patent
Documents 3 and 4). In on-demand administrations, F.VIII
formulations are also additionally administered when necessary at
regular intervals to prevent rebleeding. In addition, the
administration of F.VIII formulations is done intravenously.
Therefore, there has been a strong need for pharmaceutical agents
with a lesser burden than F.VIII formulations.
[0004] Occasionally, anti-F.VIII antibodies (inhibitors) develop in
hemophilia patients. Such inhibitors cancel the effects of the
F.VIII formulations. For bleeding in patients who have developed
inhibitors (inhibitor patients), bypass formulations are
administered. Their action mechanisms are not dependent on F.VIII
function, that is, the function of catalyzing the activation of
blood coagulation factor X (F.X) by activated blood coagulation
factor IX (F.IXa). Therefore, in some cases, bypass formulations
cannot sufficiently stop the bleeding. Accordingly, there has been
a strong need for pharmaceutical agents that are not affected by
the presence of inhibitors and which can functionally substitute
for F.VIII.
[0005] Recently, as a means for solving the problem, antibodies
that functionally substitute for F.VIII and their use were
disclosed (Patent Documents 1, 2, and 3). The antibodies may be
effective for acquired hemophilia in which anti-F.VIII
autoantibodies are present and for von Willebrand disease caused by
an abnormality or deficiency of function of von Willebrand factor
(vWF), but the activity of functionally substituting for F.VIII was
not always sufficient. Therefore, as pharmaceutical agents
exhibiting a high hemostatic effect, antibodies with a higher
activity of functionally substituting for F.VIII than the
above-mentioned antibodies were desired.
PRIOR ART DOCUMENTS
Patent Document
[0006] [Patent Document 1] WO 2005/035754
[0007] [Patent Document 2] WO 2005/035756
[0008] [Patent Document 3] WO 2006/109592
Non-Patent Document
[0009] [Non-patent Document 1] Blood 58, 1-13 (1981)
[0010] [Non-patent Document 2] Nature 312, 330-337 (1984)
[0011] [Non-patent Document 3] Nature 312, 337-342 (1984)
[0012] [Non-patent Document4] Biochim.Biophys.Acta 871, 268-278
(1986)
SUMMARY OF THE INVENTION
Problems to be Solved by the Invention
[0013] An objective of the present invention is to provide
multispecific antigen-binding molecules that functionally
substitute for F.VIII, a cofactor that enhances enzymatic
reactions.
Means for Solving the Problems
[0014] As a result of dedicated research, the present inventors
succeeded in discovering bispecific antibodies having a better F.Xa
generation-promoting activity than known antibodies from among
various bispecific antibodies that specifically bind to both
F.IX/F.IXa and F.X, and substitute for the cofactor function of
F.VIII, that is, the function to promote F.X activation by F.IXa
(F.Xa generation-promoting function).
[0015] Furthermore, the present inventors succeeded in finding the
positions in the amino acid sequences of bispecific antibodies
having the activity of functionally substituting for F.VIII that
are important for improving the F.Xa generation-promoting activity
of these antibodies, and thus they successfully obtained bispecific
antibodies in which the activity of functionally substituting for
F.VIII is further increased by replacing these amino acids. They
also succeeded in obtaining bispecific antibodies which not only
have a high activity of functionally substituting for F.VIII, but
also have a low F.Xase inhibitory action. Satisfying both of these
properties is very difficult.
[0016] Specifically, the present invention relates to multispecific
antigen-binding molecules that functionally substitute for F.VIII,
a cofactor that enhances enzymatic reactions, and pharmaceutical
compositions comprising such a molecule as an active ingredient,
and specifically relates to the following: [0017] [1] a
multispecific antigen-binding molecule that functionally
substitutes for blood coagulation factor VIII, which comprises a
first antigen-binding site that recognizes blood coagulation factor
IX and/or activated blood coagulation factor IX and a second
antigen-binding site that recognizes blood coagulation factor X,
wherein the functional substitution for blood coagulation factor
VIII results from an activated blood coagulation factor X (F.Xa)
generation-promoting activity higher than the activity of a
bispecific antibody (hA69-KQ/hB26-PF/hAL-AQ) which comprises an H
chain comprising SEQ ID NOs: 165 and 166, and a commonly shared L
chain comprising SEQ ID NO: 167; [0018] [2] the multispecific
antigen-binding molecule of [1], which comprises a first
polypeptide comprising a first antigen-binding site that recognizes
blood coagulation factor IX and/or activated blood coagulation
factor IX and a third polypeptide comprising a third
antigen-binding site that recognizes blood coagulation factor IX
and/or activated blood coagulation factor IX, as well as a second
polypeptide comprising a second antigen-binding site that
recognizes blood coagulation factor X and a fourth polypeptide
comprising a fourth antigen-binding site that recognizes blood
coagulation factor X; [0019] [3] the multispecific antigen-binding
molecule of [2], wherein the first polypeptide and the third
polypeptide each comprises an antigen-binding site of an H chain or
L chain of an antibody against blood coagulation factor IX or
activated blood coagulation factor IX, respectively; and the second
polypeptide and the fourth polypeptide each comprises an
antigen-binding site of an H chain or L chain of an antibody
against blood coagulation factor X, respectively; [0020] [4] the
multispecific antigen-binding molecule of [3], wherein the
antigen-binding site of the first polypeptide comprises an
antigen-binding site which comprises H chain CDRs consisting of any
one of the amino acid sequences selected from the following (a1) to
(a11), or an antigen-binding site functionally equivalent thereto,
and the antigen-binding site of the second polypeptide comprises an
antigen-binding site which comprises H chain CDRs consisting of any
one of the amino acid sequences selected from the following (b1) to
(b11), or an antigen-binding site functionally equivalent thereto:
[0021] (a1) an antigen-binding site comprising an H chain CDR 1, 2,
and 3 amino acid sequences of SEQ ID NOs: 75, 76, and 77 (H chain
CDRs of Q1), respectively; [0022] (a2) an antigen-binding site
comprising an H chain CDR 1, 2, and 3 amino acid sequences of SEQ
ID NOs: 78, 79, and 80 (H chain CDRs of Q31), respectively; [0023]
(a3) an antigen-binding site comprising an H chain CDR 1, 2, and 3
amino acid sequences of SEQ ID NOs: 81, 82, and 83 (H chain CDRs of
Q64), respectively; [0024] (a4) an antigen-binding site comprising
an H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 84,
85, and 86 (H chain CDRs of Q85), respectively; [0025] (a5) an
antigen-binding site comprising the H chain CDR 1, 2, and 3 amino
acid sequences of SEQ ID NOs: 87, 88, and 89 (H chain CDRs of
Q153), respectively; [0026] (a6) an antigen-binding site comprising
an H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 90,
91, and 92 (H chain CDRs of Q354), respectively; [0027] (a7) an
antigen-binding site comprising the H chain CDR 1, 2, and 3 amino
acid sequences of SEQ ID NOs: 93, 94, and 95 (H chain CDRs of
Q360), respectively; [0028] (a8) an antigen-binding site comprising
the of H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs:
96, 97, and 98 (H chain CDRs of Q405), respectively; [0029] (a9) an
antigen-binding site comprising an H chain CDR 1, 2, and 3 amino
acid sequences of SEQ ID NOs: 99, 100, and 101 (H chain CDRs of
Q458), respectively; [0030] (a10) an antigen-binding site
comprising an H chain CDR 1, 2, and 3 amino acid sequences of SEQ
ID NOs: 102, 103, and 104 (H chain CDRs of Q460), respectively;
[0031] (a11) an antigen-binding site comprising an H chain CDR 1,
2, and 3 amino acid sequences of SEQ ID NOs: 105, 106, and 107 (H
chain CDRs of Q499), respectively; [0032] (b1) an antigen-binding
site comprising an H chain CDR 1, 2, and 3 amino acid sequences of
SEQ ID NOs: 108, 109, and 110 (H chain CDRs of J232), respectively;
[0033] (b2) an antigen-binding site comprising an H chain CDR 1, 2,
and 3 amino acid sequences of SEQ ID NOs: 111, 112, and 113 (H
chain CDRs of J259), respectively; [0034] (b3) an antigen-binding
site comprising an H chain CDR 1, 2, and 3 amino acid sequences of
SEQ ID NOs: 114, 115, and 116 (H chain CDRs of J268), respectively;
[0035] (b4) an antigen-binding site comprising an H chain CDR 1, 2,
and 3 amino acid sequences of SEQ ID NOs: 117, 118, and 119 (H
chain CDRs of J300), respectively; [0036] (b5) an antigen-binding
site comprising an H chain CDR 1, 2, and 3 amino acid sequences of
SEQ ID NOs: 120, 121, and 122 (H chain CDRs of J321), respectively;
[0037] (b6) an antigen-binding site comprising the H chain CDR 1,
2, and 3 amino acid sequences of SEQ ID NOs: 123, 124, and 125 (H
chain CDRs of J326), respectively; [0038] (b7) an antigen-binding
site comprising an H chain CDR 1, 2, and 3 amino acid sequences of
SEQ ID NOs: 126, 127, and 128 (H chain CDRs of J327), respectively;
[0039] (b8) an antigen-binding site comprising an H chain CDR 1, 2,
and 3 amino acid sequences of SEQ ID NOs: 129, 130, and 131 (H
chain CDRs of J339), respectively; [0040] (b9) an antigen-binding
site comprising an H chain CDR 1, 2, and 3 amino acid sequences of
SEQ ID NOs: 132, 133, and 134 (H chain CDRs of J344), respectively;
[0041] (b10) an antigen-binding site comprising an H chain CDR 1,
2, and 3 amino acid sequences of SEQ ID NOs: 135, 136, and 137 (H
chain CDRs of J346), respectively; and [0042] (b11) an
antigen-binding site comprising an H chain CDR 1, 2, and 3 amino
acid sequences of SEQ ID NOs: 174, 175, and 176 (H chain CDRs of
J142), respectively; [0043] [5] the multispecific antigen-binding
molecule of [3], wherein the antigen-binding site of the first
polypeptide comprises an antigen-binding site which comprises an H
chain variable region consisting of any one of the amino acid
sequences selected from the following (a1) to (a11), or an
antigen-binding site functionally equivalent thereto, and the
antigen-binding site of the second polypeptide comprises an
antigen-binding site which comprises an H chain variable region
consisting of any one of the amino acid sequences selected from the
following (b1) to (b11), or an antigen-binding site functionally
equivalent thereto: [0044] (a1) an antigen-binding site comprising
an H chain variable region amino acid sequence of SEQ ID NO: 35 (H
chain variable region of Q64); [0045] (a2) an antigen-binding site
comprising an H chain variable region amino acid sequence of SEQ ID
NO: 36 (H chain variable region of Q31); [0046] (a3) an
antigen-binding site comprising an H chain variable region amino
acid sequence of SEQ ID NO: 37 (H chain variable region of Q1);
[0047] (a4) an antigen-binding site comprising an H chain variable
region amino acid sequence of SEQ ID NO: 38 (H chain variable
region of Q85); [0048] (a5) an antigen-binding site comprising an H
chain variable region amino acid sequence of SEQ ID NO: 39 (H chain
variable region of Q153); [0049] (a6) an antigen-binding site
comprising an H chain variable region amino acid sequence of SEQ ID
NO: 40 (H chain variable region of Q354); [0050] (a7) an
antigen-binding site comprising an H chain variable region amino
acid sequence of SEQ ID NO: 41 (H chain variable region of Q360);
[0051] (a8) an antigen-binding site comprising an H chain variable
region amino acid sequence of SEQ ID NO: 42 (H chain variable
region of Q405); [0052] (a9) an antigen-binding site comprising an
H chain variable region amino acid sequence of SEQ ID NO: 43 (H
chain variable region of Q458); [0053] (a10) an antigen-binding
site comprising an H chain variable region amino acid sequence of
SEQ ID NO: 44 (H chain variable region of Q460); [0054] (a11) an
antigen-binding site comprising an H chain variable region amino
acid sequence of SEQ ID NO: 45 (H chain variable region of Q499);
[0055] (b1) an antigen-binding site comprising an H chain variable
region amino acid sequence of SEQ ID NO: 46 (H chain variable
region of J232); [0056] (b2) an antigen-binding site comprising an
H chain variable region amino acid sequence of SEQ ID NO: 47 (H
chain variable region of J259); [0057] (b3) an antigen-binding site
comprising an H chain variable region amino acid sequence of SEQ ID
NO: 48 (H chain variable region of J268); [0058] (b4) an
antigen-binding site comprising an H chain variable region amino
acid sequence of SEQ ID NO: 49 (H chain variable region of J300);
[0059] (b5) an antigen-binding site comprising an H chain variable
region amino acid sequence of
[0060] SEQ ID NO: 50 (H chain variable region of J321); (b6) an
antigen-binding site comprising an H chain variable region amino
acid sequence of SEQ ID NO: 51 (H chain variable region of J326);
[0061] (b7) an antigen-binding site comprising an H chain variable
region amino acid sequence of SEQ ID NO: 52 (H chain variable
region of J327); [0062] (b8) an antigen-binding site comprising an
H chain variable region amino acid sequence of SEQ ID NO: 53 (H
chain variable region of J339); [0063] (b9) an antigen-binding site
comprising an H chain variable region amino acid sequence of SEQ ID
NO: 54 (H chain variable region of J344); [0064] (b10) an
antigen-binding site comprising an H chain variable region amino
acid sequence of SEQ ID NO: 55 (H chain variable region of J346);
and [0065] (b11) an antigen-binding site comprising an H chain
variable region amino acid sequence of SEQ ID NO: 172 (H chain
variable region of J142); [0066] [6] the multispecific
antigen-binding molecule of [3], wherein the antigen-binding sites
included in the third polypeptide and the fourth polypeptide
comprise an antigen-binding site which comprises L chain CDRs
consisting of any one of the amino acid sequences selected from the
following (c1) to (c10), or an antigen-binding site functionally
equivalent thereto: [0067] (c1) an antigen-binding site comprising
an L chain CDR1, 2, and 3 amino acid sequences of SEQ ID NOs: 138,
139, and 140 (L chain CDR of L2), respectively; [0068] (c2) an
antigen-binding site comprising an L chain CDR1, 2, and 3 amino
acid sequences of SEQ ID NOs: 141, 142, and 143 (L chain CDR of
L45), respectively; [0069] (c3) an antigen-binding site comprising
an L chain CDR1, 2, and 3 amino acid sequences of SEQ ID NOs: 144,
145, and 146 (L chain CDR of L248), respectively; [0070] (c4) an
antigen-binding site comprising an L chain CDR1, 2, and 3 amino
acid sequences of SEQ ID NOs: 147, 148, and 149 (L chain CDR of
L324), respectively; [0071] (c5) an antigen-binding site comprising
an L chain CDR1, 2, and 3 amino acid sequences of SEQ ID NOs: 150,
151, and 152 (L chain CDR of L334), respectively; [0072] (c6) an
antigen-binding site comprising an L chain CDR1, 2, and 3 amino
acid sequences of SEQ ID NOs: 153, 154, and 155 (L chain CDR of
L377), respectively; [0073] (c7) an antigen-binding site comprising
an L chain CDR1, 2, and 3 amino acid sequences of SEQ ID NOs: 156,
157, and 158 (L chain CDR of L404), respectively; [0074] (c8) an
antigen-binding site comprising an L chain CDR1, 2, and 3 amino
acid sequences of SEQ ID NOs: 159, 160, and 161 (L chain CDR of
L406), respectively; [0075] (c9) an antigen-binding site comprising
an L chain CDR1, 2, and 3 amino acid sequences of SEQ ID NOs: 137,
138, and 139 (L chain CDR of L408), respectively; and [0076] (c10)
an antigen-binding site comprising an L chain CDR1, 2, and 3 amino
acid sequences of SEQ ID NOs: 177, 178, and 179 (L chain CDR of
L180), respectively; [0077] [7] the multispecific antigen-binding
molecule of [3], wherein the antigen-binding sites included in the
third polypeptide and the fourth polypeptide comprise an
antigen-binding site which comprises an L chain variable region
consisting of any one of the amino acid sequences selected from the
following (c1) to (c10), or an antigen-binding site functionally
equivalent thereto: [0078] (c1) an antigen-binding site comprising
an L chain variable region amino acid sequence of SEQ ID NO: 56 (L
chain variable region of L2); [0079] (c2) an antigen-binding site
comprising an L chain variable region amino acid sequence of SEQ ID
NO: 57 (L chain variable region of L45); [0080] (c3) an
antigen-binding site comprising an L chain variable region amino
acid sequence of SEQ ID NO: 58 (L chain variable region of L248);
[0081] (c4) an antigen-binding site comprising an L chain variable
region amino acid sequence of SEQ ID NO: 59 (L chain variable
region of L324); [0082] (c5) an antigen-binding site comprising an
L chain variable region amino acid sequence of SEQ ID NO: 60 (L
chain variable region of L334); [0083] (c6) an antigen-binding site
comprising an L chain variable region amino acid sequence of SEQ ID
NO: 61 (L chain variable region of L377); [0084] (c7) an
antigen-binding site comprising an L chain variable region amino
acid sequence of SEQ ID NO: 62 (L chain variable region of L404);
[0085] (c8) an antigen-binding site comprising an L chain variable
region amino acid sequence of SEQ ID NO: 63 (L chain variable
region of L406); [0086] (c9) an antigen-binding site comprising an
L chain variable region amino acid sequence of SEQ ID NO: 64 (L
chain variable region of L408); and [0087] (c10) an antigen-binding
site comprising an L chain variable region amino acid sequence of
SEQ ID NO: 173 (L chain variable region of L180); [0088] [8] the
multispecific antigen-binding molecule of [3], wherein the first
and second polypeptides further comprise an antibody H chain
constant region, and the third and fourth polypeptides comprise an
antibody L chain constant region; [0089] [9] the multispecific
antigen-binding molecule of [3], wherein the first and second
polypeptides comprise an antibody H chain constant region, and the
third and fourth polypeptides comprise an antibody L chain constant
region, and wherein the third polypeptide and the fourth
polypeptide are a commonly shared L chain; [0090] [10] the
multispecific antigen-binding molecule of [8] or [9], wherein the
first polypeptide comprises an antibody H chain constant region
consisting of any one of the amino acid sequences selected from the
group consisting of the following (d1) to (d6) or the group
consisting of the following (d7) to (d9), and the second
polypeptide comprises an antibody H chain constant region
consisting of any one of the amino acid sequences selected from a
group different from that of the above-mentioned first
polypeptide:
[0091] (d1) an H chain constant region of SEQ ID NO: 65 (G4k);
[0092] (d2) an H chain constant region of SEQ ID NO: 66 (z7);
[0093] (d3) an H chain constant region of SEQ ID NO: 67 (z55);
[0094] (d4) an H chain constant region of SEQ ID NO: 68 (z106);
[0095] (d5) an H chain constant region of SEQ ID NO: 69 (z118);
[0096] (d6) an H chain constant region of SEQ ID NO: 70 (z121);
[0097] (d7) an H chain constant region of SEQ ID NO: 71 (G4h);
[0098] (d8) an H chain constant region of SEQ ID NO: 72 (z107);
and
[0099] (d9) an H chain constant region of SEQ ID NO: 73 (z119);
[0100] [11] the multispecific antigen-binding molecule of [8] or
[9], wherein the third and fourth polypeptides comprise the
antibody L chain constant region consisting of the following amino
acid sequence of: [0101] (e) an L chain constant region of SEQ ID
NO: 74 (k); [0102] [12] the multispecific antigen-binding molecule
of [8] or [9], wherein the first polypeptide comprises any one
antibody H chain selected from the following (a1) to (a14), the
second polypeptide comprises any one antibody H chain selected from
the following (b1) to (b12), and the third polypeptide and the
fourth polypeptide comprise any one antibody L chain selected from
the following (c1) to (c10): [0103] (a1) an antibody H chain
consisting of the amino acid sequence of SEQ ID NO: 1 (Q1-G4k);
[0104] (a2) an antibody H chain consisting of the amino acid
sequence of SEQ ID NO: 2 (Q31-z7); [0105] (a3) an antibody H chain
consisting of the amino acid sequence of SEQ ID NO: 3 (Q64-z55);
[0106] (a4) an antibody H chain consisting of the amino acid
sequence of SEQ ID NO: 10 (Q64-z7); [0107] (a5) an antibody H chain
consisting of the amino acid sequence of SEQ ID NO: 11 (Q85-G4k);
[0108] (a6) an antibody H chain consisting of the amino acid
sequence of SEQ ID NO: 12 (Q153-G4k); [0109] (a7) an antibody H
chain consisting of the amino acid sequence of SEQ ID NO: 13
(Q354-z106); [0110] (a8) an antibody H chain consisting of the
amino acid sequence of SEQ ID NO: 14 (Q360-G4k); [0111] (a9) an
antibody H chain consisting of the amino acid sequence of SEQ ID
NO: 15 (Q360-z118); [0112] (a10) an antibody H chain consisting of
the amino acid sequence of SEQ ID NO: 16 (Q405-G4k); [0113] (a11)
an antibody H chain consisting of the amino acid sequence of SEQ ID
NO: 17 (Q458-z106); [0114] (a12) an antibody H chain consisting of
the amino acid sequence of SEQ ID NO: 18 (Q460-z121); [0115] (a13)
an antibody H chain consisting of the amino acid sequence of SEQ ID
NO: 19 (Q499-z118); [0116] (a14) an antibody H chain consisting of
the amino acid sequence of SEQ ID NO: 20 (Q499-z121); [0117] (b1)
an antibody H chain consisting of the amino acid sequence of SEQ ID
NO: 4 (J268-G4h); [0118] (b2) an antibody H chain consisting of the
amino acid sequence of SEQ ID NO: 5 (J321-G4h); [0119] (b3) an
antibody H chain consisting of the amino acid sequence of SEQ ID
NO: 6 (J326-z107); [0120] (b4) an antibody H chain consisting of
the amino acid sequence of SEQ ID NO: 7 (J344-z107); [0121] (b5) an
antibody H chain consisting of the amino acid sequence of SEQ ID
NO: 21 (J232-G4h); [0122] (b6) an antibody H chain consisting of
the amino acid sequence of SEQ ID NO: 22 (J259-z107); [0123] (b7)
an antibody H chain consisting of the amino acid sequence of SEQ ID
NO: 23 (J300-z107); [0124] (b8) an antibody H chain consisting of
the amino acid sequence of SEQ ID NO: 24 (J327-z107); [0125] (b9)
an antibody H chain consisting of the amino acid sequence of SEQ ID
NO: 25 (J327-z119); [0126] (b10) an antibody H chain consisting of
the amino acid sequence of SEQ ID NO: 26 (J339-z119); [0127] (b11)
an antibody H chain consisting of the amino acid sequence of SEQ ID
NO: 27 (J346-z107); [0128] (b12) an antibody H chain consisting of
the amino acid sequence of SEQ ID NO: 170 (J142-G4h); [0129] (c1)
an antibody L chain consisting of the amino acid sequence of SEQ ID
NO: 8 (L2-k); [0130] (c2) an antibody L chain consisting of the
amino acid sequence of SEQ ID NO: 9 (L45-k); [0131] (c3) an
antibody L chain consisting of the amino acid sequence of SEQ ID
NO: 28 (L248-k); [0132] (c4) an antibody L chain consisting of the
amino acid sequence of SEQ ID NO: 29 (L324-k); [0133] (c5) an
antibody L chain consisting of the amino acid sequence of SEQ ID
NO: 30 (L334-k); [0134] (c6) an antibody L chain consisting of the
amino acid sequence of SEQ ID NO: 31 (L377-k); [0135] (c7) an
antibody L chain consisting of the amino acid sequence of SEQ ID
NO: 32 (L404-k); [0136] (c8) an antibody L chain consisting of the
amino acid sequence of SEQ ID NO: 33 (L406-k); [0137] (c9) an
antibody L chain consisting of the amino acid sequence of SEQ ID
NO: 34 (L408-k); and [0138] (c10) an antibody L chain consisting of
the amino acid sequence of SEQ ID NO: 171 (L180-k); [0139] [13] the
multispecific antigen-binding molecule of [1], wherein the first
polypeptide comprises an antigen-binding site which binds to an
epitope overlapping with an epitope that binds to an antibody
consisting of the antibody H chain of any one of (a1) to (a14) and
the antibody L chain of any one of (c1) to (c10) of [12], and the
second polypeptide comprises an antigen-binding site which binds to
an epitope overlapping with an epitope that binds to an antibody
consisting of the antibody H chain of any one of (b1) to (b12) and
the antibody L chain of any one of (c1) to (c10) of [12]; [0140]
[14] the multispecific antigen-binding molecule of [8] or [9],
wherein the first polypeptide comprises any one antibody H chain
selected from the following (e1) to (e3), the second polypeptide
comprises any one antibody H chain selected from the following (f1)
to (f3), and the third polypeptide and the fourth polypeptide
comprise any one antibody L chain selected from the following (g1)
to (g4): [0141] (e1) an H chain of an antibody which binds to an
epitope overlapping with an epitope bound by an antibody consisting
of an antibody H chain of any one of (a1) to (a14) and an antibody
L chain of any one of (c1) to (c10), of [12]; [0142] (e2) an
antibody H chain, wherein at least one amino acid residue selected
from the amino acid residues at positions 34, 35, 49, 61, 62, 96,
98, 100, 100b, and 102 by Kabat numbering in any one antibody H
chain selected from (e1) is substituted with another amino acid;
[0143] (e3) an antibody H chain, wherein by Kabat numbering, the
amino acid residue at position 34 is isoleucine, the amino acid
residue at position 35 is asparagine, glutamine, or serine, the
amino acid residue at position 49 is serine, the amino acid residue
at position 61 is arginine, the amino acid residue at position 62
is glutamic acid, the amino acid residue at position 96 is serine
or threonine, the amino acid residue at position 98 is lysine or
arginine, the amino acid residue at position 100 is phenylalanine
or tyrosine, the amino acid residue at position 100b is glycine, or
the amino acid residue at position 102 is tyrosine in any antibody
H chain selected from (e1); [0144] (f1) an H chain of an antibody
which binds to an epitope overlapping with an epitope bound by an
antibody consisting of an antibody H chain of any of (b1) to (b12)
of [12] and an antibody L chain of any of (c1) to (c10) of [12];
[0145] (f2) an antibody H chain, wherein at least one amino acid
residue selected from the amino acid residues at positions 35, 53,
73, 76, 96, 98, 100, and 100a by Kabat numbering in any antibody H
chain of (f1) is substituted with another amino acid; [0146] (f3)
an antibody H chain, wherein by Kabat numbering, the amino acid
residue at position 35 is aspartic acid, the amino acid residue at
position 53 is arginine, the amino acid residue at position 73 is
lysine, the amino acid residue at position 76 is glycine, the amino
acid residue at position 96 is lysine or arginine, the amino acid
residue at position 98 is tyrosine, the amino acid residue at
position 100 is tyrosine, or the amino acid residue at position
100a is histidine in any one antibody H chain selected from (f1);
[0147] (g1) an L chain of an antibody which binds to an epitope
overlapping with an epitope bound by an antibody which consists of
an antibody H chain of any one of (a1) to (a14) and an antibody L
chain of any one of (c1) to (c10), of [12]; [0148] (g2) an L chain
of an antibody which binds to an epitope overlapping with an
epitope bound by an antibody which consists of an antibody H chain
of any one of (b1) to (b12) and an antibody L chain of any one of
(c1) to (c10), of [12]; [0149] (g3) an antibody L chain, wherein at
least one amino acid residue selected from the amino acid residues
at positions 27, 30, 31, 32, 50, 52, 53, 54, 55, 92, 93, 94, and 95
by Kabat numbering in the antibody L chain of either (g1) or (g2)
is substituted with another amino acid; and [0150] (g4) an antibody
L chain, wherein by Kabat numbering, the amino acid residue at
position 27 is lysine or arginine, the amino acid residue at
position 30 is glutamic acid, the amino acid residue at position 31
is arginine, the amino acid residue at position 32 is glutamine,
the amino acid residue at position 50 is arginine or glutamine, the
amino acid residue at position 52 is serine, the amino acid residue
at position 53 is arginine, the amino acid residue at position 54
is lysine, the amino acid residue at position 55 is glutamic acid,
the amino acid residue at position 92 is serine, the amino acid
residue at position 93 is serine, the amino acid residue at
position 94 is proline, or the amino acid residue at position 95 is
proline in the antibody L chain of either (g1) or (g2); [0151] [15]
the multispecific antigen-binding molecule of any one of [1] to
[14], wherein the multispecific antigen-binding molecule is a
multispecific antibody; [0152] [16] a bispecific antibody of any
one of the following (a) to (u): [0153] (a) a bispecific antibody
(Q1-G4k/J268-G4h/L45-k), wherein the first polypeptide is an H
chain consisting of the amino acid sequence of SEQ ID NO: 1, the
second polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 4, and the third polypeptide and the fourth
polypeptide are a commonly shared L chain of SEQ ID NO: 9; [0154]
(b) a bispecific antibody (Q1-G4k/J321-G4h/L45-k), wherein the
first polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 1, the second polypeptide is an H chain
consisting of the amino acid sequence of SEQ ID NO: 5, and the
third polypeptide and the fourth polypeptide are a commonly shared
L chain of SEQ ID NO: 9; [0155] (c) a bispecific antibody
(Q31-z7/J326-z107/L2-k), wherein the first polypeptide is an H
chain consisting of the amino acid sequence of SEQ ID NO: 2, the
second polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 6, and the third polypeptide and the fourth
polypeptide are a commonly shared L chain of SEQ ID NO: 8; [0156]
(d) a bispecific antibody (Q64-z55/J344-z107/L45-k), wherein the
first polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 3, the second polypeptide is an H chain
consisting of the amino acid sequence of SEQ ID NO: 7, and the
third polypeptide and the fourth polypeptide are a commonly shared
L chain of SEQ ID NO: 9; [0157] (e) a bispecific antibody
(Q64-z7/J326-z107/L334-k), wherein the first polypeptide is an H
chain consisting of the amino acid sequence of SEQ ID NO: 10, the
second polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 6, and the third polypeptide and the fourth
polypeptide are a commonly shared L chain of SEQ ID NO: 30; [0158]
(f) a bispecific antibody (Q64-z7/J344-z107/L406-k), wherein the
first polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 10, the second polypeptide is an H chain
consisting of the amino acid sequence of SEQ ID NO: 7, and the
third polypeptide and the fourth polypeptide are a commonly shared
L chain of SEQ ID NO: 33; [0159] (g) a bispecific antibody
(Q85-G4k/J268-G4h/L406-k), wherein the first polypeptide is an H
chain consisting of the amino acid sequence of SEQ ID NO: 11, the
second polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 4, and the third polypeptide and the fourth
polypeptide are a commonly shared L chain of SEQ ID NO: 33; [0160]
(h) a bispecific antibody (Q85-G4k/J321-G4h/L334-k), wherein the
first polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 11, the second polypeptide is an H chain
consisting of the amino acid sequence of SEQ ID NO: 5, and the
third polypeptide and the fourth polypeptide are a commonly shared
L chain of SEQ ID NO: 30; [0161] (i) a bispecific antibody
(Q153-G4k/J232-G4h/L406-k), wherein the first polypeptide is an H
chain consisting of the amino acid sequence of SEQ ID NO: 12, the
second polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 21, and the third polypeptide and the fourth
polypeptide are a commonly shared L chain of SEQ ID NO: 33; [0162]
(j) a bispecific antibody (Q354-z106/J259-z107/L324-k), wherein the
first polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 13, the second polypeptide is an H chain
consisting of the amino acid sequence of SEQ ID NO: 22, and the
third polypeptide and the fourth polypeptide are a commonly shared
L chain of SEQ ID NO: 29; [0163] (k) a bispecific antibody
(Q360-G4k/J232-G4h/L406-k), wherein the first polypeptide is an H
chain consisting of the amino acid sequence of SEQ ID NO: 14, the
second polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 21, and the third polypeptide and the fourth
polypeptide are a commonly shared L chain of SEQ ID NO: 33; [0164]
(l) a bispecific antibody (Q360-z118/J300-z107/L334-k), wherein the
first polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 15, the second polypeptide is an H chain
consisting of the amino acid sequence of SEQ ID NO: 23, and the
third polypeptide and the fourth polypeptide are a commonly shared
L chain of SEQ ID NO: 30; [0165] (m) a bispecific antibody
(Q405-G4k/J232-G4h/L248-k), wherein the first polypeptide is an H
chain consisting of the amino acid sequence of SEQ ID NO: 16, the
second polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 21, and the third polypeptide and the fourth
polypeptide are a commonly shared L chain of SEQ ID NO: 28; [0166]
(n) a bispecific antibody (Q458-z106/J346-z107/L408-k), wherein the
first polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 17, the second polypeptide is an H chain
consisting of the amino acid sequence of SEQ ID NO: 27, and the
third polypeptide and the fourth polypeptide are a commonly shared
L chain of SEQ ID NO: 34; [0167] (o) a bispecific antibody
(Q460-z121/J327-z119/L334-k), wherein the first polypeptide is an H
chain consisting of the amino acid sequence of SEQ ID NO: 18, the
second polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 25, and the third polypeptide and the fourth
polypeptide are a commonly shared L chain of SEQ ID NO: 30; [0168]
(p) a bispecific antibody (Q499-z118/J327-z107/L334-k), wherein the
first polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 19, the second polypeptide is an H chain
consisting of the amino acid sequence of SEQ ID NO: 24, and the
third polypeptide and the fourth polypeptide are a commonly shared
L chain of SEQ ID NO: 30; [0169] (q) a bispecific antibody
(Q499-z118/J327-z107/L377-k), wherein the first polypeptide is an H
chain consisting of the amino acid sequence of SEQ ID NO: 19, the
second polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 24, and the third polypeptide and the fourth
polypeptide are a commonly shared L chain of SEQ ID NO: 31;
[0170] (r) a bispecific antibody (Q499-z118/J346-z107/L248-k),
wherein the first polypeptide is an H chain consisting of the amino
acid sequence of SEQ ID NO: 19, the second polypeptide is an H
chain consisting of the amino acid sequence of SEQ ID NO: 27, and
the third polypeptide and the fourth polypeptide are a commonly
shared L chain of SEQ ID NO: 28; [0171] (s) a bispecific antibody
(Q499-z121/J327-z119/L404-k), wherein the first polypeptide is an H
chain consisting of the amino acid sequence of SEQ ID NO: 20, the
second polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 25, and the third polypeptide and the fourth
polypeptide are a commonly shared L chain of SEQ ID NO: 32; [0172]
(t) a bispecific antibody (Q499-z121/J339-z119/L377-k), wherein the
first polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 20, the second polypeptide is an H chain
consisting of the amino acid sequence of SEQ ID NO: 26, and the
third polypeptide and the fourth polypeptide are a commonly shared
L chain of SEQ ID NO: 31; and [0173] (u) a bispecific antibody
(Q153-G4k/J142-G4h/L180-k), wherein the first polypeptide is an H
chain consisting of the amino acid sequence of SEQ ID NO: 12, the
second polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 170, and the third polypeptide and the
fourth polypeptide are a commonly shared L chain of SEQ ID NO: 171;
[0174] [17] a nucleic acid encoding the multispecific
antigen-binding molecule of any one of [1] to [15] or the
bispecific antibody of [16]; [0175] [18] a vector inserted with the
nucleic acid of [17]; [0176] [19] a cell comprising the nucleic
acid of [17] or the vector of [18]; [0177] [20] a method for
producing the multispecific antigen-binding molecule of any one of
[1] to [15] or the bispecific antibody of [16] by culturing the
cell of [19]; [0178] [21] a pharmaceutical composition comprising
the multispecific antigen-binding molecule of any one of [1] to
[15] or the bispecific antibody of [16], and a pharmaceutically
acceptable carrier; [0179] [22] the composition of [21], which is a
pharmaceutical composition used for prevention and/or treatment of
bleeding, a disease accompanying bleeding, or a disease caused by
bleeding; [0180] [23] the composition of [22], wherein the
bleeding, the disease accompanying bleeding, or the disease caused
by bleeding is a disease that develops and/or progresses due to a
decrease or deficiency in the activity of blood coagulation factor
VIII and/or activated blood coagulation factor VIII; [0181] [24]
the composition of [23], wherein the disease that develops and/or
progresses due to a decrease or deficiency in the activity of blood
coagulation factor VIII and/or activated blood coagulation factor
VIII is hemophilia A; [0182] [25] the composition of [23], wherein
the disease that develops and/or progresses due to a decrease or
deficiency in the activity of blood coagulation factor VIII and/or
activated blood coagulation factor VIII is a disease showing
emergence of an inhibitor against blood coagulation factor VIII
and/or activated blood coagulation factor VIII; [0183] [26] the
composition of [23], wherein the disease that develops and/or
progresses due to a decrease or deficiency in the activity of blood
coagulation factor VIII and/or activated blood coagulation factor
VIII is acquired hemophilia; [0184] [27] the composition of [23],
wherein the disease that develops and/or progresses due to a
decrease in the activity of blood coagulation factor VIII and/or
activated blood coagulation factor VIII is von Willebrand disease;
[0185] [28] a method for preventing and/or treating bleeding, a
disease accompanying bleeding, or a disease caused by bleeding,
which comprises the step of administering the multispecific
antigen-binding molecule of any one of [1] to [15] or the
bispecific antibody of [16], or the composition of any one of [21]
to [27]; and [0186] [29] a kit for use in the prevention and/or
treatment method of [28], which comprises at least the
multispecific antigen-binding molecule of any one of [1] to [15] or
the bispecific antibody of [16], or the composition of any one of
[21] to [27].
[0187] Furthermore, the present invention relates to: [0188] [30]
use of the multispecific antigen-binding molecule of any one of [1]
to [15], the bispecific antibody of [16], or the composition of any
one of [21] to [27] in the manufacture of an agent for preventing
and/or treating bleeding, a disease accompanying bleeding, or a
disease caused by bleeding; and [0189] [31] the multispecific
antigen-binding molecule of any one of [1] to [15], the bispecific
antibody of [16], or the composition of any one of [21] to [27] for
preventing and/or treating bleeding, a disease accompanying
bleeding, or a disease caused by bleeding.
[0190] The present invention also relates to bispecific antibodies
that functionally substitute for F.VIII, a cofactor that enhances
enzymatic reactions, and pharmaceutical compositions comprising the
antibody as an active ingredient, and more specifically relates to:
[0191] [32] a bispecific antibody that functionally substitutes for
blood coagulation factor VIII, which comprises a first
antigen-binding site that recognizes blood coagulation factor IX
and/or activated blood coagulation factor IX and a second
antigen-binding site that recognizes blood coagulation factor X,
wherein the bispecific antibody is any of the following (a) to (u):
[0192] (a) a bispecific antibody (Q1-G4k/J268-G4h/L45-k), wherein
the first polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 1, the second polypeptide is an H chain
consisting of the amino acid sequence of SEQ ID NO: 4, and the
third polypeptide and the fourth polypeptide are a commonly shared
L chain of SEQ ID NO: 9; [0193] (b) a bispecific antibody
(Q1-G4k/J321-G4h/L45-k), wherein the first polypeptide is an H
chain consisting of the amino acid sequence of SEQ ID NO: 1, the
second polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 5, and the third polypeptide and the fourth
polypeptide are a commonly shared L chain of SEQ ID NO: 9; [0194]
(c) a bispecific antibody (Q31-z7/J326-z107/L2-k), wherein the
first polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 2, the second polypeptide is an H chain
consisting of the amino acid sequence of SEQ ID NO: 6, and the
third polypeptide and the fourth polypeptide are a commonly shared
L chain of SEQ ID NO: 8; [0195] (d) a bispecific antibody
(Q64-z55/J344-z107/L45-k), wherein the first polypeptide is an H
chain consisting of the amino acid sequence of SEQ ID NO: 3, the
second polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 7, and the third polypeptide and the fourth
polypeptide are a commonly shared L chain of SEQ ID NO: 9; [0196]
(e) a bispecific antibody (Q64-z7/J326-z107/L334-k), wherein the
first polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 10, the second polypeptide is an H chain
consisting of the amino acid sequence of SEQ ID NO: 6, and the
third polypeptide and the fourth polypeptide are a commonly shared
L chain of SEQ ID NO: 30; [0197] (f) a bispecific antibody
(Q64-z7/J344-z107/L406-k), wherein the first polypeptide is an H
chain consisting of the amino acid sequence of SEQ ID NO: 10, the
second polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 7, and the third polypeptide and the fourth
polypeptideare a commonly shared L chain of SEQ ID NO: 33; [0198]
(g) a bispecific antibody (Q85-G4k/J268-G4h/L406-k), wherein the
first polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 11, the second polypeptide is an H chain
consisting of the amino acid sequence of SEQ ID NO: 4, and the
third polypeptide and the fourth polypeptide are a commonly shared
L chain of SEQ ID NO: 33; [0199] (h) a bispecific antibody
(Q85-G4k/J321-G4h/L334-k), wherein the first polypeptide is an H
chain consisting of the amino acid sequence of SEQ ID NO: 11, the
second polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 5, and the third polypeptide and the fourth
polypeptide are a commonly shared L chain of SEQ ID NO: 30; [0200]
(i) a bispecific antibody (Q153-G4k/J232-G4h/L406-k), wherein the
first polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 12, the second polypeptide is an H chain
consisting of the amino acid sequence of SEQ ID NO: 21, and the
third polypeptide and the fourth polypeptide are a commonly shared
L chain of SEQ ID NO: 33; [0201] (j) a bispecific antibody
(Q354-z106/J259-z107/L324-k), wherein the first polypeptide is an H
chain consisting of the amino acid sequence of SEQ ID NO: 13, the
second polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 22, and the third polypeptide and the fourth
polypeptide are a commonly shared L chain of SEQ ID NO: 29; [0202]
(k) a bispecific antibody (Q360-G4k/J232-G4h/L406-k), wherein the
first polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 14, the second polypeptide is an H chain
consisting of the amino acid sequence of SEQ ID NO: 21, and the
third polypeptide and the fourth polypeptide are a commonly shared
L chain of SEQ ID NO: 33; [0203] (l) a bispecific antibody
(Q360-z118/J300-z107/L334-k), wherein the first polypeptide is an H
chain consisting of the amino acid sequence of SEQ ID NO: 15, the
second polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 23, and the third polypeptide and the fourth
polypeptide are a commonly shared L chain of SEQ ID NO: 30; [0204]
(m) a bispecific antibody (Q405-G4k/J232-G4h/L248-k), wherein the
first polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 16, the second polypeptide is an H chain
consisting of the amino acid sequence of SEQ ID NO: 21, and the
third polypeptide and the fourth polypeptide are a commonly shared
L chain of SEQ ID NO: 28; [0205] (n) a bispecific antibody
(Q458-z106/J346-z107/L408-k), wherein the first polypeptide is an H
chain consisting of the amino acid sequence of SEQ ID NO: 17, the
second polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 27, and the third polypeptide and the fourth
polypeptide are a commonly shared L chain of SEQ ID NO: 34; [0206]
(o) a bispecific antibody (Q460-z121/J327-z119/L334-k), wherein the
first polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 18, the second polypeptide is an H chain
consisting of the amino acid sequence of SEQ ID NO: 25, and the
third polypeptide and the fourth polypeptide are a commonly shared
L chain of SEQ ID NO: 30; [0207] (p) a bispecific antibody
(Q499-z118/J327-z107/L334-k), wherein the first polypeptide is an H
chain consisting of the amino acid sequence of SEQ ID NO: 19, the
second polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 24, and the third polypeptide and the fourth
polypeptide are a commonly shared L chain of SEQ ID NO: 30; [0208]
(q) a bispecific antibody (Q499-z118/J327-z107/L377-k), wherein the
first polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 19, the second polypeptide is an H chain
consisting of the amino acid sequence of SEQ ID NO: 24, and the
third polypeptide and the fourth polypeptide are a commonly shared
L chain of SEQ ID NO: 31; [0209] (r) a bispecific antibody
(Q499-z118/J346-z107/L248-k), wherein the first polypeptide is an H
chain consisting of the amino acid sequence of SEQ ID NO: 19, the
second polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 27, and the third polypeptide and the fourth
polypeptide are a commonly shared L chain of SEQ ID NO: 28; [0210]
(s) a bispecific antibody (Q499-z121/J327-z119/L404-k), wherein the
first polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 20, the second polypeptide is an H chain
consisting of the amino acid sequence of SEQ ID NO: 25, and the
third polypeptide and the fourth polypeptide are a commonly shared
L chain of SEQ ID NO: 32; [0211] (t) a bispecific antibody
(Q499-z121/J339-z119/L377-k), wherein the first polypeptide is an H
chain consisting of the amino acid sequence of SEQ ID NO: 20, the
second polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 26, and the third polypeptide and the fourth
polypeptide are a commonly shared L chain of SEQ ID NO: 31; and
[0212] (u) a bispecific antibody (Q153-G4k/J142-G4h/L180-k),
wherein the first polypeptide is an H chain consisting of the amino
acid sequence of SEQ ID NO: 12, the second polypeptide is an H
chain consisting of the amino acid sequence of SEQ ID NO: 170, and
the third polypeptide and the fourth polypeptide are a commonly
shared L chain of SEQ ID NO: 171; [0213] [33] a nucleic acid
encoding the bispecific antibody of [32]; [0214] [34] a vector
inserted with the nucleic acid of [33]; [0215] [35] a cell
comprising the nucleic acid of [33] or the vector of [34]; [0216]
[36] a method for producing the bispecific antibody of [32] by
culturing the cell of [35]; [0217] [37] a pharmaceutical
composition comprising the bispecific antibody of [32], and a
pharmaceutically acceptable carrier; [0218] [38] the composition of
[37], which is a pharmaceutical composition used for prevention
and/or treatment of bleeding, a disease accompanying bleeding, or a
disease caused by bleeding; [0219] [39] the composition of [38],
wherein the bleeding, the disease accompanying bleeding, or the
disease caused by bleeding is a disease that develops and/or
progresses due to a decrease or deficiency in the activity of blood
coagulation factor VIII and/or activated blood coagulation factor
VIII; [0220] [40] the composition of [39], wherein the disease that
develops and/or progresses due to a decrease or deficiency in the
activity of blood coagulation factor VIII and/or activated blood
coagulation factor VIII is hemophilia A; [0221] [41] the
composition of [39], wherein the disease that develops and/or
progresses due to a decrease or deficiency in the activity of blood
coagulation factor VIII and/or activated blood coagulation factor
VIII is a disease showing emergence of an inhibitor against blood
coagulation factor VIII and/or activated blood coagulation factor
VIII; [0222] [42] the composition of [39], wherein the disease that
develops and/or progresses due to a decrease or deficiency in the
activity of blood coagulation factor VIII and/or activated blood
coagulation factor VIII is acquired hemophilia; [0223] [43] the
composition of [39], wherein the disease that develops and/or
progresses due to a decrease in the activity of blood coagulation
factor VIII and/or activated blood coagulation factor VIII is von
Willebrand disease; [0224] [44] a method for preventing and/or
treating bleeding, a disease accompanying bleeding, or a disease
caused by bleeding, which comprises the step of administering the
bispecific antibody of [32] or the composition of any one of [37]
to [43]; [0225] [45] a kit for use in the prevention and/or
treatment method of [44], which comprises the bispecific antibody
of [32], or the composition of any one of [37] to [43]; [0226] [46]
use of the bispecific antibody of [32] or the composition of any
one of [37] to [43] in the manufacture of an agent for preventing
and/or treating bleeding, a disease accompanying bleeding, or a
disease caused by bleeding; and [0227] [47] the bispecific antibody
of [32] or the composition of any one of [37] to [43] for
preventing and/or treating bleeding, a disease accompanying
bleeding, or a disease caused by bleeding.
Effects of the Invention
[0228] The present invention provides antibodies that recognize
both an enzyme and its substrate, which are multispecific
antigen-binding molecules having a high activity of functionally
substituting for F.VIII. Furthermore, the present invention
provides antibodies that recognize both an enzyme and its
substrate, which are multispecific antigen-binding molecules having
a high activity of functionally substituting for F.VIII and a low
F.Xase inhibitory action. Since humanized antibodies are generally
thought to have high stability in blood and low immunogenicity,
multispecific antibodies of the present invention may be very
promising as pharmaceuticals.
BRIEF DESCRIPTION OF THE DRAWINGS
[0229] FIG. 1 describes the F.Xase inhibitory action. [0230] (a)
F.VIIIa forms a complex with F.IXa (F.Xase) and activates F.X.
[0231] (b) A bispecific antibody binds to F.IXa and F.X and
activates F.X. [0232] (c) Both F.VIIIa and the bispecific antibody
activate F.X without competition. [0233] (d) Binding of the
bispecific antibody to F.IXa and/or F.X inhibits the formation of
the complex formed between F.Xase and F.X. [0234] (e) Binding of
the bispecific antibody to F.IXa and/or F.X inhibits the activity
of F.Xase.
[0235] FIG. 2 describes the screening. Approximately 200 types each
of genes for antibodies against human F.IXa and human F.X were
produced, and they were incorporated into animal cell expression
vectors. 40,000 or more bispecific antibodies as a combination of
an anti-F.IXa antibody and anti-F.X antibody were transiently
expressed. F.Xa generation-promoting activity and F.Xase inhibitory
action were evaluated to screen for bispecific antibodies having a
high F.Xa generation-promoting activity and a low F.Xase inhibitory
action. Furthermore, by substituting amino acids when necessary,
prototype antibodies were produced.
[0236] FIG. 3 shows the F.Xa generation-promoting activities of
hA69-KQ/hB26-PF/hAL-AQ, Q1-G4k/J268-G4h/L45-k,
Q1-G4k/J321-G4h/L45-k, Q31-z7/J326-z107/L2-k, and
Q64-z55/J344-z107/L45-k. The concentrations of the antibody
solutions were 300, 30, and 3 .mu.g/mL (the concentrations after
mixing Human Factor IXa, Novact (registered trademark) M, Human
Factor X, and the antibody solution were 100, 10, and 1 .mu.g/mL),
the enzyme reaction and color development were performed for ten
minutes and 50 minutes, respectively. As a result, these antibodies
showed a higher F.Xa generation-promoting activity compared to
hA69-KQ/hB26-PF/hAL-AQ described in WO 2006/109592.
[0237] FIG. 4 shows the F.Xa generation-promoting activity of
hA69-KQ/hB26-PF/hAL-AQ, prototype antibodies, and modified
antibodies with amino acid substitutions. The concentrations of the
antibody solutions were 300, 30, and 3 .mu.g/mL (the concentrations
after mixing Human Factor IXa, Novact (registered trademark) M,
Human Factor X, and the antibody solution were 100, 10, and 1
.mu.g/mL), the enzyme reaction and color development were performed
for two minutes and 20 minutes, respectively. As a result, these
modified antibodies showed a higher F.Xa generation-promoting
activity compared to the prototype antibodies.
[0238] FIG. 5 shows the F.Xase inhibitory action of
hA69-KQ/hB26-PF/hAL-AQ, prototype antibodies, and modified
antibodies with amino acid substitutions. The figure shows the
effects of hA69-KQ/hB26-PF/hAL-AQ, Q1-G4k/J268-G4h/L45-k,
Q31-z73326-z107/L2-k, Q1-G4k/J321-G4h/L45-k,
Q64-z55/J344-z107/L45-k, Q85-G4k/J268-G4h/L406-k,
Q85-G4k/J321-G4h/L334-k, Q64-z73344-z107/L406-k,
Q64-z7/J326-z107/L334-k, Q153-G4k/J142-G4h/L180-k,
Q405-G4k/J232-G4h/L248-k, Q360-G4k/J232-G4h/L406-k,
Q153-G4k/J232-G4h/L406-k, Q458-z106/J346-z107/L408-k,
Q360-z118/J300-z107/L334-k, Q499-z118/J327-z107/L377-k,
Q499-z121/J327-z119/L404-k, Q499-z121/J339-z119/L377-k,
Q499-z118/J346-z107/L248-k, Q354-z1063259-z107/L324-k,
Q460-z121/J327-z119/L334-k, and Q499-z118/J327-z107/L334-k on F.X
activation by F.IXa in the presence of F.VIIIa. The F.Xase
inhibitory actions of the antibodies are indicated as the value
obtained by subtracting the absorbance of the antibody-free
reaction solution from the absorbance of the antibody-supplemented
reaction solution. The concentrations of the antibody solutions
were 300 and 30 pg/mL (the concentrations after mixing Human Factor
IXa, F.VIIIa, Human Factor X, and the antibody solution were 100
and 10 pg/mL), the enzyme reaction and color development were
performed for six minutes and 14 minutes, respectively. The more
positive the value of F.Xase inhibitory action shown on the
horizontal axis, the weaker the F.Xase inhibitory action is. As a
result, hA69-KQ/hB26-PF/hAL-AQ described in WO 2006/109592 showed
strong F.Xase inhibitory action. All of the antibodies of the
present invention showed weaker F.Xase inhibitory action compared
to hA69-KQ/hB26-PF/hAL-AQ, or did not show inhibitory action.
[0239] FIG. 6A shows the amino acid sequences of the prototype
antibodies and the modified antibodies with amino acid
substitutions. When the sequence name is not indicated in the Ref
column, the variable region sequence of the Name column is
mentioned. A "- (hyphen)" is shown where an amino acid is absent at
the number by Kabat numbering. A " (dot)" is shown where amino acid
is the same when comparing the variable region of the Name column
and the Ref column, and the amino acid of the variable region of
the Name column is shown where the amino acids are different. Amino
acids found to be important for improvement of F.Xa
generation-promoting activity were indicated by framing them.
[0240] FIG. 6B is a continuation of FIG. 6A.
[0241] FIG. 6C is a continuation of FIG. 6B.
[0242] FIG. 6D is a continuation of FIG. 6C.
[0243] The amino acid sequences shown in FIGS. 6A through 6D are Q1
(SEQ ID NO: 35), Q31 (SEQ ID NO: 36), Q64 (SEQ ID NO: 37), Q85 (SEQ
ID NO: 38), Q153 (SEQ ID NO: 39), Q354 (SEQ ID NO: 40), Q360 (SEQ
ID NO: 41), Q405 (SEQ ID NO: 42), Q458 (SEQ ID NO: 43), Q460 (SEQ
ID NO: 44), Q499 (SEQ ID NO: 45), J268 (SEQ ID NO: 48), J321 (SEQ
ID NO: 50), J326 (SEQ ID NO: 51), J344 (SEQ ID NO: 54), J232 (SEQ
ID NO: 46), J259 (SEQ ID NO: 47), J346 (SEQ ID NO: 55), J300 (SEQ
ID NO: 49), J327 (SEQ ID NO: 52), J339 (SEQ ID NO: 53), J142 (SEQ
ID NO: 172), L2 (SEQ ID NO: 56), L45 (SEQ ID NO: 57), L248 (SEQ ID
NO: 58), L324 (SEQ ID NO: 59), L334 (SEQ ID NO: 60), L377 (SEQ ID
NO: 61), L404 (SEQ ID NO: 62), L406 (SEQ ID NO: 63), L408 (SEQ ID
NO: 64), and L180 (SEQ ID NO: 173).
MODE FOR CARRYING OUT THE INVENTION
[0244] Multispecific antigen-binding molecules described herein
comprise a first antigen-binding site and a second antigen-binding
site that can specifically bind to at least two different types of
antigens. While the first antigen-binding site and the second
antigen-binding site are not particularly limited as long as they
have an activity to bind to F.IX and/or F.IXa, and F.X,
respectively, examples include sites necessary for binding with
antigens, such as antibodies, scaffold molecules (antibody-like
molecules) or peptides, or fragments containing such sites.
Scaffold molecules are molecules that exhibit function by binding
to target molecules, and any polypeptide may be used as long as
they are conformationally stable polypeptides that can bind to at
least one target antigen. Examples of such polypeptides include
antibody variable regions, fibronectin (WO 2002/032925), protein A
domain (WO 1995/001937), LDL receptor A domain (WO 2004/044011, WO
2005/040229), ankyrin (WO 2002/020565), and such, and also
molecules described in documents by Nygren et al. (Current Opinion
in Structural Biology, 7: 463-469 (1997); and Journal of Immunol
Methods, 290: 3-28 (2004)), Binz et al. (Nature Biotech 23:
1257-1266 (2005)), and Hosse et al. (Protein Science 15:
14-27(2006)). Furthermore, as mentioned in Curr Opin Mol Ther. 2010
Aug; 12(4): 487-95 and Drugs. 2008; 68(7): 901-12, peptide
molecules that can bind to target antigens may be used.
[0245] Herein, multispecific antigen-binding molecules are not
particularly limited as long as they are molecules that can bind to
at least two different types of antigens, but examples include
polypeptides containing the above-mentioned antigen-binding sites,
such as antibodies and scaffold molecules as well as their
fragments, and aptamers comprising nucleic acid molecules and
peptides, and they may be single molecules or multimers thereof
Preferred multispecific antigen-binding molecules include
multispecific antibodies that can bind specifically to at least two
different antigens. Particularly preferred examples of antibodies
which have an activity of functionally substituting for F.VIII of
the present invention include bispecific antibodies (BsAb) that can
bind specifically to two different antigens (they may also be
called dual specific antibodies).
[0246] In the present invention, the term "commonly shared L chain"
refers to an L chain that can link with two or more different H
chains, and show binding ability to each antigen. Herein, the term
"different H chain(s)" preferably refers to H chains of antibodies
against different antigens, but is not limited thereto, and also
refers to H chains whose amino acid sequences are different from
each other. Commonly shared L chain can be obtained, for example,
according to the method described in WO 2006/109592.
[0247] The multispecific antigen-binding molecules of the present
invention (preferably bispecific antibodies) are antibodies having
specificity to two or more different antigens, or molecules
comprising fragments of such antibodies. The antibodies of the
present invention are not particularly limited, but are preferably
monoclonal antibodies. Monoclonal antibodies used in the present
invention include not only monoclonal antibodies derived from
animals such as humans, mice, rats, hamsters, rabbits, sheep,
camels, and monkeys, but also include artificially modified gene
recombinant antibodies such as chimeric antibodies, humanized
antibodies, and bispecific antibodies.
[0248] Furthermore, the L chains of an antibody which will become a
multispecific antigen-binding molecule of the present invention may
be different, but preferably have commonly shared L chains.
[0249] Multispecific antigen-binding molecules of the present
invention are preferably recombinant antibodies produced using
genetic recombination techniques (See, for example, Borrebaeck CAK
and Larrick JW, THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the
United Kingdom by MACMILLAN PUBLISHERS LTD, 1990). Recombinant
antibodies can be obtained by cloning DNAs encoding antibodies from
hybridomas or antibody-producing cells, such as sensitized
lymphocytes, that produce antibodies, inserting them into suitable
vectors, and then introducing them into hosts (host cells) to
produce the antibodies.
[0250] Furthermore, antibodies of the present invention may include
not only whole antibodies but also antibody fragments and
low-molecular-weight antibodies (minibodies), and modified
antibodies.
[0251] For example, antibody fragments or minibodies include
diabodies (Dbs), linear antibodies, and single chain antibody
(hereinafter, also denoted as scFvs) molecules. Herein, an "Fv"
fragment is defined as the smallest antibody fragment that
comprises a complete antigen recognition site and binding site.
[0252] An "Fv" fragment is a dimer (VH-VL dimer) in which an H
chain variable region (VH) and an L chain variable region (VL) are
strongly linked by non-covalent binding. The three complementarity
determining regions (CDRs) of each of the variable regions interact
with each other to form an antigen-binding site on the surface of
the VH-VL dimer. Six CDRs confer the antigen-binding site to an
antibody. However, one variable region (or half of the Fv
comprising only three CDRs specific to an antigen) alone can
recognize and bind to an antigen, though its affinity is lower than
that of the entire binding site.
[0253] An Fab fragment (also called F(ab)) further comprises an L
chain constant region and an H chain constant region (CH1). An Fab'
fragment differs from an Fab fragment in that it additionally
comprises several residues derived from the carboxyl terminus of
the H chain CH1 region, comprising one or more cysteines from the
hinge region of the antibody. Fab'-SH refers to an Fab' in which
one or more cysteine residues of its constant region comprise a
free thiol group. An F(ab') fragment is produced by cleavage of
disulfide bonds between the cysteine residues in the hinge region
of F(ab').sub.2 pepsin digest. Other chemically bound antibody
fragments are also known to those skilled in the art.
[0254] Diabodies are bivalent minibodies constructed by gene fusion
(Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448
(1993); EP 404,097; WO 93/11161). Diabodies are dimers consisting
of two polypeptide chains, in which each polypeptide chain
comprises an L chain variable region (VL) and an H chain variable
region (VH) linked with a linker short enough to prevent
association of these two domains within the same chain, for
example, a linker of preferably 2 to 12 amino acids, more
preferably 3 to 10 amino acids, particularly about 5 amino acids.
The polypeptide chain form a dimer since the linker between the VL
and VH encoded on the same polypeptide is too short to form a
single chain variable region fragment. Therefore, diabodies
comprise two antigen-binding sites.
[0255] A single-chain antibody or an scFv antibody fragment
comprises the VH and VL regions of an antibody, and these regions
exist in a single polypeptide chain. In general, an Fv polypeptide
further comprises a polypeptide linker between the VH and VL
regions, and this enables an scFv to form a structure necessary for
antigen binding (for a review on scFvs, see Pluckthun "The
Pharmacology of Monoclonal Antibodies" Vol. 113 (Rosenburg and
Moore ed. (Springer Verlag, New York) pp.269-315, 1994). In the
context of the present invention, linkers are not particularly
limited so long as they do not inhibit the expression of the
antibody variable regions linked at their ends.
[0256] IgG-type bispecific antibodies can be secreted from hybrid
hybridomas (quadromas) produced by fusing two kinds of hybridomas
that produce IgG antibodies (Milstein C et al. Nature 1983, 305:
537-540). They can also be secreted by taking the L chain and H
chain genes constituting the two kinds of IgGs of interest, a total
of four kinds of genes, and introducing them into cells to
coexpress the genes.
[0257] In this case, by introducing suitable amino acid
substitutions to the CH3 regions of the H chains, IgGs having a
heterogeneous combination of H chains can be preferentially
secreted (Ridgway JB et al. Protein Engineering 1996, 9: 617-621;
Merchant AM et al. Nature Biotechnology 1998, 16: 677-681; WO
2006/106905; Davis JH et al. Protein Eng Des Sel. 2010, 4:
195-202).
[0258] Regarding the L chains, since diversity of L chain variable
regions is lower than that of H chain variable regions, commonly
shared L chains that can confer binding ability to both H chains
may be obtained. The antibodies of the present invention comprise
commonly shared L chains. Bispecific IgGs can be efficiently
expressed by introducing the genes of the commonly shared L chain
and both H chains into cells.
[0259] Bispecific antibodies may be produced by chemically
crosslinking Fab's. Bispecific F(ab').sub.2 can be produced, for
example, by preparing Fab' from an antibody, using it to produce a
maleimidized Fab' with ortho-phenylenedi-maleimide (o-PDM), and
then reacting this with Fab' prepared from another antibody to
crosslink Fab's derived from different antibodies (Keler T et al.
Cancer Research 1997, 57: 4008-4014). The method of chemically
linking an Fab'-thionitrobenzoic acid (TNB) derivative and an
antibody fragment such as Fab'-thiol (SH) is also known (Brennan M
et al. Science 1985, 229: 81-83).
[0260] Instead of a chemical crosslink, a leucine zipper derived
from Fos and Jun may also be used. Preferential formation of
heterodimers by Fos and Jun is utilized, even though they also form
homodimers. Fab' to which Fos leucine zipper is added, and another
Fab' to which Jun leucine zipper is added are expressed and
prepared. Monomeric Fab'-Fos and Fab'-Jun reduced under mild
conditions are mixed and reacted to form bispecific F(ab').sub.2
(Kostelny SA et al. J. of Immunology, 1992, 148: 1547-53). This
method can be applied not only to Fab's but also to scFvs, Fvs, and
such.
[0261] Furthermore, bispecific antibodies including sc(Fv)2 such as
IgG-scFv (Protein Eng Des Sel. 2010 Apr; 23(4): 221-8) and BiTE
(Drug Discov Today. 2005 Sep 15; 10(18): 1237-44.),
[0262] DVD-Ig (Nat Biotechnol. 2007 Nov; 25(11): 1290-7. Epub 2007
Oct 14.; and MAbs. 2009 Jul; 1(4): 339-47. Epub 2009 Jul 10.), and
also others (IDrugs 2010, 13: 698-700) including two-in-one
antibodies (Science. 2009 Mar 20; 323(5921): 1610-4; and
Immunotherapy. 2009 Sep; 1(5): 749-51.), Tri-Fab, tandem scFv, and
diabodies are known (MAbs. 2009 November; 1(6): 539-547). In
addition, even when using molecular forms such as scFv-Fc and
scaffold-Fc, bispecific antibodies can be produced efficiently by
preferentially secreting a heterologous combination of Fcs (Ridgway
JB et al., Protein Engineering 1996, 9: 617-621; Merchant AM et al.
Nature Biotechnology 1998, 16: 677-681; WO 2006/106905; and Davis
JH et al., Protein Eng Des Sel. 2010, 4: 195-202.).
[0263] A bispecific antibody may also be produced using a diabody.
A bispecific diabody is a heterodimer of two cross-over scFv
fragments. More specifically, it is produced by forming a
heterodimer using VH(A)-VL(B) and VH(B)-VL(A) prepared by linking
VHs and VLs derived from two kinds of antibodies, A and B, using a
relatively short linker of about 5 residues (Holliger P et al. Proc
Natl. Acad. Sci. USA 1993, 90: 6444-6448).
[0264] The desired structure can be achieved by linking the two
scFvs with a flexible and relatively long linker comprising about
15 residues (single chain diabody: Kipriyanov SM et al. J. of
Molecular Biology. 1999, 293: 41-56), and conducting appropriate
amino acid substitutions (knobs-into-holes: Zhu Z et al. Protein
Science. 1997, 6: 781-788; VH/VL interface engineering: Igawa T et
al. Protein Eng Des Sel. 2010, 8: 667-77).
[0265] An sc(Fv).sub.2 that can be produced by linking two types of
scFvs with a flexible and relatively long linker, comprising about
15 residues, may also be a bispecific antibody (Mallender WD et al.
J. of Biological Chemistry, 1994, 269: 199-206).
[0266] Examples of modified antibodies include antibodies linked to
various molecules such as polyethylene glycol (PEG). The antibodies
of the present invention include such modified antibodies. In the
context of the present invention, the substance to which the
modified antibodies are linked is not limited. Such modified
antibodies can be obtained by chemically modifying obtained
antibodies. Such methods are well established in the art.
[0267] The antibodies of the present invention include human
antibodies, mouse antibodies, rat antibodies, or such, and their
origins are not limited. They may also be genetically modified
antibodies, such as chimeric or humanized antibodies.
[0268] Methods for obtaining human antibodies are known in the art.
For example, transgenic animals carrying the entire repertoire of
human antibody genes can be immunized with desired antigens to
obtain desired human antibodies (see International Patent
Application WO 93/12227, WO 92/03918, WO 94/02602, WO 94/25585, WO
96/34096, and WO 96/33735).
[0269] Genetically modified antibodies can also be produced using
known methods. Specifically, for example, chimeric antibodies may
comprise H chain and L chain variable regions of an immunized
animal antibody, and H chain and L chain constant regions of a
human antibody. Chimeric antibodies can be obtained by linking DNAs
encoding the variable regions of the antibody derived from the
immunized animal, with DNAs encoding the constant regions of a
human antibody, inserting this into an expression vector, and then
introducing it into host cells to produce the antibodies.
[0270] Humanized antibodies are modified antibodies often referred
to as "reshaped" human antibodies. A humanized antibody is
constructed by transferring the CDRs of an antibody derived from an
immunized animal to the complementarity determining regions of a
human antibody. Conventional genetic recombination techniques for
such purposes are known (see European Patent Application
Publication No. EP 239400; International Publication No. WO
96/02576; Sato K et al., Cancer Research 1993, 53: 851-856;
International Publication No. WO 99/51743).
[0271] The multispecific antigen-binding molecules of the present
invention are those that recognize F.IX and/or F.IXa, and F.X, and
functionally substitute for cofactor function of F.VIII, and
characterized in that the molecules have a higher F.Xa
generation-promoting activity compared to hA69-KQ/hB26-PF/hAL-AQ
(described in WO 2006/109592) which is known as a bispecific
antibody that functionally substitutes for F.VIII. Furthermore,
antibodies of the present invention usually have a structure which
comprises a variable region of an anti-F.IXa antibody and a
variable region of an anti-F.X antibody.
[0272] More specifically, the present invention provides a
multispecific antigen-binding molecule that functionally
substitutes for F.VIII, which comprises a first antigen-binding
site that recognizes F.IX and/or F.IXa and a second antigen-binding
site that recognizes F.X, wherein the function that substitutes for
the function of F.VIII is caused by a higher F.Xa
generation-promoting activity compared to the activity of the
bispecific antibody (hA69-KQ/hB26-PF/hAL-AQ) which comprises H
chains consisting of SEQ ID NOs: 165 and 166, and a commonly shared
L chain consisting of SEQ ID NO: 167.
[0273] A multispecific antigen-binding molecule of the present
invention comprises a first polypeptide and a third polypeptide
comprising an antigen-binding site that recognizes F.IX and/or
F.IXa, and a second polypeptide and a fourth polypeptide comprising
an antigen-binding site that recognizes F.X. The first polypeptide
and the third polypeptide, and the second polypeptide and the
fourth polypeptide each include the antigen-binding site of the
antibody H chain and the antigen-binding site of the antibody L
chain.
[0274] For example, in a multispecific antigen-binding molecule of
the present invention, the first polypeptide and the third
polypeptide include an antigen-binding site of an H chain and L
chain of an antibody against F.IX or F.IXa, respectively; and the
second polypeptide and the fourth polypeptide comprise an
antigen-binding site of an H chain and L chain of an antibody
against F.X, respectively.
[0275] At this time, the antigen-binding sites of the antibody L
chain included in the first polypeptide and the third polypeptide,
and the second polypeptide and the fourth polypeptide may be
commonly shared L chains.
[0276] A polypeptide comprising an antigen-binding site of an
antibody L chain in the present invention is preferably a
polypeptide which comprises all or a part of the sequence of the
antibody L chain which binds to F.IX, F.IXa and/or F.X.
[0277] Preferred embodiments of the antigen-binding site of the
first polypeptide of an antibody of the present invention
specifically include antigen-binding sites comprising the amino
acid sequences of: [0278] Q1 H chain each CDR1, 2, and 3 sequence
(SEQ ID NOs: 75, 76, and 77, respectively); [0279] Q31 H chain each
CDR1, 2, and 3 sequences (SEQ ID NOs: 78, 79, and 80,
respectively); [0280] Q64 H chain each CDR1, 2, and 3 sequence (SEQ
ID NOs: 81, 82, and 83, respectively); [0281] Q85 H chain each
CDR1, 2, and 3 sequence (SEQ ID NOs: 84, 85, and 86, respectively);
[0282] Q153 H chain each CDR1, 2, and 3 sequence (SEQ ID NOs: 87,
88, and 89, respectively); [0283] Q354 H chain each CDR1, 2, and 3
sequence (SEQ ID NOs: 90, 91, and 92, respectively); [0284] Q360 H
chain each CDR1, 2, and 3 sequence (SEQ ID NOs: 93, 94, and 95,
respectively); [0285] Q405 H chain each CDR1, 2, and 3 sequence
(SEQ ID NOs: 96, 97, and 98, respectively); [0286] Q458 H chain
each CDR1, 2, and 3 sequence (SEQ ID NOs: 99, 100, and 101,
respectively); [0287] Q460 H chain each CDR1, 2, and 3 sequence
(SEQ ID NOs: 102, 103, and 104, respectively); and [0288] Q499 H
chain each CDR1, 2, and 3 sequence (SEQ ID NOs: 105, 106, and 107,
respectively) mentioned in the later-described Examples, or
antigen-binding sites that are functionally equivalent to them.
[0289] Preferred embodiments of the antigen-binding site of a
second polypeptide specifically include, for example,
antigen-binding sites comprising the amino acid sequences of:
[0290] J232 H chain each CDR1, 2, and 3 sequence (SEQ ID NOs: 108,
109, and 110, respectively); [0291] J259 H chain each CDR1, 2, and
3 sequence (SEQ ID NOs: 111, 112, and 113, respectively); [0292]
J268 H chain each CDR1, 2, and 3 sequence (SEQ ID NOs: 114, 115,
and 116, respectively); [0293] J300 H chain each CDR1, 2, and 3
sequence (SEQ ID NOs: 117, 118, and 119, respectively); [0294] J321
H chain each CDR1, 2, and 3 sequence (SEQ ID NOs: 120, 121, and
122, respectively); [0295] J326 H chain each CDR1, 2, and 3
sequence (SEQ ID NOs: 123, 124, and 125, respectively); [0296] J327
H chain each CDR1, 2, and 3 sequence (SEQ ID NOs: 126, 127, and
128, respectively); [0297] J339 H chain each CDR1, 2, and 3
sequence (SEQ ID NOs: 129, 130, and 131, respectively); [0298] J344
H chain each CDR1, 2, and 3 sequence (SEQ ID NOs: 132, 133, and
134, respectively); [0299] J346 H chain each CDR1, 2, and 3
sequence (SEQ ID NOs: 135, 136, and 137, respectively); and [0300]
J142 H chain each CDR1, 2, and 3 sequence (SEQ ID NOs: 174, 175,
and 176, respectively) mentioned in the later-described Examples,
or antigen-binding sites that are functionally equivalent to
them.
[0301] More specifically, the present invention provides
multispecific antigen-binding molecules, wherein the
antigen-binding site of the first polypeptide comprises an
antigen-binding site which comprises H chain CDRs consisting of any
one of the amino acid sequences selected from the following (a1) to
(a11), or an antigen-binding site functionally equivalent thereto,
and the antigen-binding site of the second polypeptide comprises an
antigen-binding site which comprises H chain CDRs consisting of any
one of the amino acid sequences selected from the following (b1) to
(b11), or an antigen-binding site functionally equivalent thereto:
[0302] (a1) an antigen-binding site comprising an H chain CDR 1, 2,
and 3 amino acid sequences of SEQ ID NOs: 75, 76, and 77 (H chain
CDRs of Q1), respectively; [0303] (a2) an antigen-binding site
comprising an H chain CDR 1, 2, and 3 amino acid sequences of SEQ
ID NOs: 78, 79, and 80 (H chain CDRs of Q31), respectively; [0304]
(a3) an antigen-binding site comprising an H chain CDR 1, 2, and 3
amino acid sequences of SEQ ID NOs: 81, 82, and 83 (H chain CDRs of
Q64), respectively; [0305] (a4) an antigen-binding site comprising
an H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 84,
85, and 86 (H chain CDRs of Q85), respectively; [0306] (a5) an
antigen-binding site comprising the H chain CDR 1, 2, and 3 amino
acid sequences of SEQ ID NOs: 87, 88, and 89 (H chain CDRs of
Q153), respectively; [0307] (a6) an antigen-binding site comprising
an H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 90,
91, and 92 (H chain CDRs of Q354), respectively; [0308] (a7) an
antigen-binding site comprising the H chain CDR 1, 2, and 3 amino
acid sequences of SEQ ID NOs: 93, 94, and 95 (H chain CDRs of
Q360), respectively; [0309] (a8) an antigen-binding site comprising
the of H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs:
96, 97, and 98 (H chain CDRs of Q405), respectively; [0310] (a9) an
antigen-binding site comprising an H chain CDR 1, 2, and 3 amino
acid sequences of SEQ ID NOs: 99, 100, and 101 (H chain CDRs of
Q458), respectively; [0311] (a10) an antigen-binding site
comprising an H chain CDR 1, 2, and 3 amino acid sequences of SEQ
ID NOs: 102, 103, and 104 (H chain CDRs of Q460), respectively;
[0312] (a11) an antigen-binding site comprising an H chain CDR 1,
2, and 3 amino acid sequences of SEQ ID NOs: 105, 106, and 107 (H
chain CDRs of Q499), respectively; [0313] (b1) an antigen-binding
site comprising an H chain CDR 1, 2, and 3 amino acid sequences of
SEQ ID NOs: 108, 109, and 110 (H chain CDRs of J232), respectively;
[0314] (b2) an antigen-binding site comprising an H chain CDR 1, 2,
and 3 amino acid sequences of SEQ ID NOs: 111, 112, and 113 (H
chain CDRs of J259), respectively; [0315] (b3) an antigen-binding
site comprising an H chain CDR 1, 2, and 3 amino acid sequences of
SEQ ID NOs: 114, 115, and 116 (H chain CDRs of J268), respectively;
[0316] (b4) an antigen-binding site comprising an H chain CDR 1, 2,
and 3 amino acid sequences of SEQ ID NOs: 117, 118, and 119 (H
chain CDRs of J300), respectively; [0317] (b5) an antigen-binding
site comprising an H chain CDR 1, 2, and 3 amino acid sequences of
SEQ ID NOs: 120, 121, and 122 (H chain CDRs of J321), respectively;
[0318] (b6) an antigen-binding site comprising the H chain CDR 1,
2, and 3 amino acid sequences of SEQ ID NOs: 123, 124, and 125 (H
chain CDRs of J326), respectively; [0319] (b7) an antigen-binding
site comprising an H chain CDR 1, 2, and 3 amino acid sequences of
SEQ ID NOs: 126, 127, and 128 (H chain CDRs of J327), respectively;
[0320] (b8) an antigen-binding site comprising an H chain CDR 1, 2,
and 3 amino acid sequences of SEQ ID NOs: 129, 130, and 131 (H
chain CDRs of J339), respectively; [0321] (b9) an antigen-binding
site comprising an H chain CDR 1, 2, and 3 amino acid sequences of
SEQ ID NOs: 132, 133, and 134 (H chain CDRs of J344), respectively;
[0322] (b10) an antigen-binding site comprising an H chain CDR 1,
2, and 3 amino acid sequences of SEQ ID NOs: 135, 136, and 137 (H
chain CDRs of J346), respectively; and [0323] (b11) an
antigen-binding site comprising an H chain CDR 1, 2, and 3 amino
acid sequences of SEQ ID NOs: 174, 175, and 176 (H chain CDRs of
J142), respectively.
[0324] Preferred embodiments of the antigen-binding site of the
third and fourth polypeptides specifically include, for example,
antigen-binding sites comprising the amino acid sequences of:
[0325] L2 L chain each CDR1, 2, and 3 sequence (SEQ ID NOs: 138,
139, and 140, respectively); [0326] L45 L chain each CDR1, 2, and 3
sequence (SEQ ID NOs: 141, 142, and 143, respectively); [0327] L248
L chain each CDR1, 2, and 3 sequence (SEQ ID NOs: 144, 145, and
146, respectively); [0328] L324 L chain each CDR1, 2, and 3
sequence (SEQ ID NOs: 147, 148, and 149, respectively); [0329] L334
L chain each CDR1, 2, and 3 sequence (SEQ ID NOs: 150, 151, and
152, respectively); [0330] L377 L chain each CDR1, 2, and 3
sequence (SEQ ID NOs: 153, 154, and 155, respectively); [0331] L404
L chain each CDR1, 2, and 3 sequence (SEQ ID NOs: 156, 157, and
158, respectively); [0332] L406 L chain each CDR1, 2, and 3
sequence (SEQ ID NOs: 159, 160, and 161, respectively); [0333] L408
L chain each CDR1, 2, and 3 sequence (SEQ ID NOs: 162, 163, and
164, respectively); and [0334] L180 L chain each CDR1, 2, and 3
sequence (SEQ ID NOs: 177, 178, and 179, respectively) mentioned in
the later-described Examples, or antigen-binding sites that are
functionally equivalent to them.
[0335] More specifically, the present invention provides
multispecific antigen-binding molecules, wherein the
antigen-binding sites included in the third polypeptide and the
fourth polypeptide comprise an antigen-binding site which comprises
L chain CDRs consisting of any one of the amino acid sequences
selected from the following (c1) to (c10), or an antigen-binding
site functionally equivalent thereto: [0336] (c1) an
antigen-binding site comprising an L chain CDR1, 2, and 3 amino
acid sequences of SEQ ID NOs: 138, 139, and 140 (L chain CDR of
L2), respectively; [0337] (c2) an antigen-binding site comprising
an L chain CDR1, 2, and 3 amino acid sequences of SEQ ID NOs: 141,
142, and 143 (L chain CDR of L45), respectively; [0338] (c3) an
antigen-binding site comprising an L chain CDR1, 2, and 3 amino
acid sequences of SEQ ID NOs: 144, 145, and 146 (L chain CDR of
L248), respectively; [0339] (c4) an antigen-binding site comprising
an L chain CDR1, 2, and 3 amino acid sequences of SEQ ID NOs: 147,
148, and 149 (L chain CDR of L324), respectively; [0340] (c5) an
antigen-binding site comprising an L chain CDR1, 2, and 3 amino
acid sequences of SEQ ID NOs: 150, 151, and 152 (L chain CDR of
L334), respectively; [0341] (c6) an antigen-binding site comprising
an L chain CDR1, 2, and 3 amino acid sequences of SEQ ID NOs: 153,
154, and 155 (L chain CDR of L377), respectively; [0342] (c7) an
antigen-binding site comprising an L chain CDR1, 2, and 3 amino
acid sequences of SEQ ID NOs: 156, 157, and 158 (L chain CDR of
L404), respectively; [0343] (c8) an antigen-binding site comprising
an L chain CDR1, 2, and 3 amino acid sequences of SEQ ID NOs: 159,
160, and 161 (L chain CDR of L406), respectively; [0344] (c9) an
antigen-binding site comprising an L chain CDR1, 2, and 3 amino
acid sequences of SEQ ID NOs: 137, 138, and 139 (L chain CDR of
L408), respectively; and [0345] (c10) an antigen-binding site
comprising an L chain CDR1, 2, and 3 amino acid sequences of SEQ ID
NOs: 177, 178, and 179 (L chain CDR of L180), respectively.
[0346] The amino acid sequences of the H chain variable regions of
Q1, Q31, Q64, Q85, Q153, Q354, Q360, Q405, Q458, Q460, and Q499 of
the present invention are indicated by the following SEQ ID NOs,
respectively.
[0347] Q1: SEQ ID NO: 35
[0348] Q31: SEQ ID NO: 36
[0349] Q64: SEQ ID NO: 37
[0350] Q85: SEQ ID NO: 38
[0351] Q153: SEQ ID NO: 39
[0352] Q354: SEQ ID NO: 40
[0353] Q360: SEQ ID NO: 41
[0354] Q405: SEQ ID NO: 42
[0355] Q458: SEQ ID NO: 43
[0356] Q460: SEQ ID NO: 44
[0357] Q499: SEQ ID NO: 45
[0358] The amino acid sequences of the H chain variable regions of
J232, J259, J268, J300, J321, J326, J327, J339, J344, J346, and
J142 of the present invention are indicated by the following SEQ ID
NOs, respectively.
[0359] J232: SEQ ID NO: 46
[0360] J259: SEQ ID NO: 47
[0361] J268: SEQ ID NO: 48
[0362] J300: SEQ ID NO: 49
[0363] J321: SEQ ID NO: 50
[0364] J326: SEQ ID NO: 51
[0365] J327: SEQ ID NO: 52
[0366] J339: SEQ ID NO: 53
[0367] J344: SEQ ID NO: 54
[0368] J346: SEQ ID NO: 55
[0369] J142: SEQ ID NO: 172
[0370] More specifically, the present invention provides
multispecific antigen-binding molecules, wherein the
antigen-binding site of the first polypeptide comprises an
antigen-binding site which comprises an H chain variable region
consisting of any one of the amino acid sequences selected from the
following (a1) to (a11), or an antigen-binding site functionally
equivalent thereto, and the antigen-binding site of the second
polypeptide comprises an antigen-binding site which comprises an H
chain variable region consisting of any one of the amino acid
sequences selected from the following (b1) to (b11), or an
antigen-binding site functionally equivalent thereto: [0371] (a1)
an antigen-binding site comprising an H chain variable region amino
acid sequence of SEQ ID NO: 35 (H chain variable region of Q1);
[0372] (a2) an antigen-binding site comprising an H chain variable
region amino acid sequence of SEQ ID NO: 36 (H chain variable
region of Q31); [0373] (a3) an antigen-binding site comprising an H
chain variable region amino acid sequence of SEQ ID NO: 37 (H chain
variable region of Q64); [0374] (a4) an antigen-binding site
comprising an H chain variable region amino acid sequence of SEQ ID
NO: 38 (H chain variable region of Q85); [0375] (a5) an
antigen-binding site comprising an H chain variable region amino
acid sequence of SEQ ID NO: 39 (H chain variable region of Q153);
[0376] (a6) an antigen-binding site comprising an H chain variable
region amino acid sequence of SEQ ID NO: 40 (H chain variable
region of Q354); [0377] (a7) an antigen-binding site comprising an
H chain variable region amino acid sequence of SEQ ID NO: 41 (H
chain variable region of Q360); [0378] (a8) an antigen-binding site
comprising an H chain variable region amino acid sequence of SEQ ID
NO: 42 (H chain variable region of Q405); [0379] (a9) an
antigen-binding site comprising an H chain variable region amino
acid sequence of SEQ ID NO: 43 (H chain variable region of Q458);
[0380] (a10) an antigen-binding site comprising an H chain variable
region amino acid sequence of SEQ ID NO: 44 (H chain variable
region of Q460); [0381] (a11) an antigen-binding site comprising an
H chain variable region amino acid sequence of SEQ ID NO: 45 (H
chain variable region of Q499); [0382] (b1) an antigen-binding site
comprising an H chain variable region amino acid sequence of SEQ ID
NO: 46 (H chain variable region of J232); [0383] (b2) an
antigen-binding site comprising an H chain variable region amino
acid sequence of SEQ ID NO: 47 (H chain variable region of J259);
[0384] (b3) an antigen-binding site comprising an H chain variable
region amino acid sequence of SEQ ID NO: 48 (H chain variable
region of J268); [0385] (b4) an antigen-binding site comprising an
H chain variable region amino acid sequence of SEQ ID NO: 49 (H
chain variable region of J300); [0386] (b5) an antigen-binding site
comprising an H chain variable region amino acid sequence of SEQ ID
NO: 50 (H chain variable region of J321); [0387] (b6) an
antigen-binding site comprising an H chain variable region amino
acid sequence of SEQ ID NO: 51 (H chain variable region of J326);
[0388] (b7) an antigen-binding site comprising an H chain variable
region amino acid sequence of SEQ ID NO: 52 (H chain variable
region of J327); [0389] (b8) an antigen-binding site comprising an
H chain variable region amino acid sequence of SEQ ID NO: 53 (H
chain variable region of J339); [0390] (b9) an antigen-binding site
comprising an H chain variable region amino acid sequence of SEQ ID
NO: 54 (H chain variable region of J344); [0391] (b10) an
antigen-binding site comprising an H chain variable region amino
acid sequence of SEQ ID NO: 55 (H chain variable region of J346);
and [0392] (b11) an antigen-binding site comprising an H chain
variable region amino acid sequence of SEQ ID NO: 172 (H chain
variable region of J142).
[0393] In addition, the amino acid sequences of the L chain
variable regions of L2, L45, L248, L324, L334, L377, L404, L406,
L408, and L180 of the present invention are indicated by the
following SEQ ID NOs, respectively.
[0394] L2: SEQ ID NO: 56
[0395] L45: SEQ ID NO: 57
[0396] L248: SEQ ID NO: 58
[0397] L324: SEQ ID NO: 59
[0398] L334: SEQ ID NO: 60
[0399] L377: SEQ ID NO: 61
[0400] L404: SEQ ID NO: 62
[0401] L406: SEQ ID NO: 63
[0402] L408: SEQ ID NO: 64
[0403] L180: SEQ ID NO: 173
[0404] More specifically, the present invention provides
multispecific antigen-binding molecules, wherein the
antigen-binding sites included in the third polypeptide and the
fourth polypeptide comprise an antigen-binding site which comprises
an L chain variable region consisting of any one of the amino acid
sequences selected from the following (c1) to (c10), or an
antigen-binding site functionally equivalent thereto: [0405] (c1)
an antigen-binding site comprising an L chain variable region amino
acid sequence of SEQ ID NO: 56 (L chain variable region of L2);
[0406] (c2) an antigen-binding site comprising an L chain variable
region amino acid sequence of SEQ ID NO: 57 (L chain variable
region of L45); [0407] (c3) an antigen-binding site comprising an L
chain variable region amino acid sequence of SEQ ID NO: 58 (L chain
variable region of L248); [0408] (c4) an antigen-binding site
comprising an L chain variable region amino acid sequence of SEQ ID
NO: 59 (L chain variable region of L324); [0409] (c5) an
antigen-binding site comprising an L chain variable region amino
acid sequence of SEQ ID NO: 60 (L chain variable region of L334);
[0410] (c6) an antigen-binding site comprising an L chain variable
region amino acid sequence of SEQ ID NO: 61 (L chain variable
region of L377); [0411] (c7) an antigen-binding site comprising an
L chain variable region amino acid sequence of SEQ ID NO: 62 (L
chain variable region of L404); [0412] (c8) an antigen-binding site
comprising an L chain variable region amino acid sequence of SEQ ID
NO: 63 (L chain variable region of L406); [0413] (c9) an
antigen-binding site comprising an L chain variable region amino
acid sequence of SEQ ID NO: 64 (L chain variable region of L408);
and [0414] (c10) an antigen-binding site comprising an L chain
variable region amino acid sequence of SEQ ID NO: 173 (L chain
variable region of L180).
[0415] The amino acid sequences of CDR1 to 3 and FR1 to 4 in each
of the sequences are as described in FIGS. 3A to D
[0416] When producing a full-length antibody using the variable
regions disclosed in the present invention, without particular
limitations, constant regions well known to those skilled in the
art may be used. For example, constant regions described in
"Sequences of proteins of immunological interest", (1991), U.S.
Department of Health and Human Services. Public Health Service
National Institutes of Health, or "An efficient route to human
bispecific IgG", (1998). Nature Biotechnology vol. 16, 677-681 can
be used. Preferred examples of the antibody constant regions of the
present invention include the constant regions of IgG antibodies.
When using the constant region of an IgG antibody, its type is not
limited, and a constant region of IgG subclass such as IgG1, IgG2,
IgG3, or IgG4 may be used. Furthermore, amino acid mutations may be
introduced into the constant region of these IgG subclasses. Amino
acid mutations to be introduced may be, for example, those that
enhance or decrease binding to Fcy receptors (Proc Natl Acad Sci U
S A. 2006 Mar 14; 103(11): 4005-10; and MAbs. 2009 Nov; 1(6):
572-9), or enhance or decrease binding to FcRn (J Biol Chem. 2001
Mar 2; 276(9): 6591-604; Int Immunol. 2006 Dec; 18(12): 1759-69;
and J Biol Chem. 2006 Aug 18; 281(33): 23514-24), but are not
limited thereto. Two types of H chains must be heterologously
associated to produce a bispecific antibody. The knobs-into-holes
technology (J Immunol Methods. 2001 Feb 1; 248(1-2): 7-15; and J
Biol Chem. 2010 Jul 2; 285(27): 20850-9), the electrostatic
repulsion technology (WO 2006/106905), the SEEDbody technology
(Protein Eng Des Sel. 2010 Apr; 23(4): 195-202), and such may be
used for heterologous association of two types of H chains via a
CH3 domain. Furthermore, the antibodies of the present invention
may be those with a modified or deficient sugar chain. Examples of
antibodies having modified sugar chains include
glycosylation-engineered antibodies (such as WO 99/54342),
antibodies with defucosylated sugar chains (WO 00/61739, WO
02/31140, WO 2006/067847, WO 2006/067913, etc.), and antibodies
having a sugar chain with bisecting GlcNAc (such as WO 02/79255).
Known examples of methods for producing sugar chain-deficient IgG
antibodies include the method of introducing a mutation to
asparagine at position 297 in the EU numbering (J Clin Pharmacol.
2010 May; 50(5): 494-506), and the method of producing IgG using
Escherichia coli (J Immunol Methods. 2002 May 1; 263(1-2): 133-47;
and J Biol Chem. 2010 Jul 2; 285(27): 20850-9). Furthermore,
heterogeneity accompanying deletion of C-terminal lysine in IgG,
and heterogeneity accompanying mispairing of disulfide bonds in the
hinge region of IgG2 can be decreased by introducing amino acid
deletions/substitutions (WO 2009/041613).
[0417] The present invention provides, for example, multispecific
antigen-binding molecules, wherein the first and second
polypeptides comprise an antibody H chain constant region, and the
third and fourth polypeptides comprise an antibody L chain constant
region.
[0418] Furthermore, the present invention provides multispecific
antigen-binding molecules, wherein the first polypeptide comprises
an antibody H chain constant region consisting of any one of the
amino acid sequences selected from the group consisting of the
following (d1) to (d6) or the group consisting of the following
(d7) to (d9), and the second polypeptide comprises an antibody H
chain constant region consisting of any one of the amino acid
sequences selected from a group different from that of the
above-mentioned first polypeptide:
[0419] (d1) an H chain constant region of SEQ ID NO: 65 (G4k);
[0420] (d2) an H chain constant region of SEQ ID NO: 66 (z7);
[0421] (d3) an H chain constant region of SEQ ID NO: 67 (z55);
[0422] (d4) an H chain constant region of SEQ ID NO: 68 (z106);
[0423] (d5) an H chain constant region of SEQ ID NO: 69 (z118);
[0424] (d6) an H chain constant region of SEQ ID NO: 70 (z121);
[0425] (d7) an H chain constant region of SEQ ID NO: 71 (G4h);
[0426] (d8) an H chain constant region of SEQ ID NO: 72 (z107);
and
[0427] (d9) an H chain constant region of SEQ ID NO: 73 (z119).
[0428] Furthermore, the present invention provides a multispecific
antigen-binding molecule, wherein the third and fourth polypeptides
comprise an antibody L chain constant region consisting of the
following amino acid sequence of: [0429] (e) an L chain constant
region of SEQ ID NO: 74 (k).
[0430] In the present invention, the phrase "functionally
substitute for F.VIII" means that F.IX and/or F.IXa, and F.X is
recognized, and activation of F.X is promoted (F.Xa generation is
promoted).
[0431] In the present invention, "F.Xa generation-promoting
activity" can be confirmed by evaluating the multispecific
antigen-binding molecules of the present invention using, for
example, a measurement system comprising F.XIa (F.IX activating
enzyme), F.IX, F.X, F synthetic substrate S-2222 (synthetic
substrate of F.Xa), and phospholipids. This measurement system
shows the correlation between the severity of the disease and
clinical symptoms in hemophilia A cases (Rosen S, Andersson M,
Blomback M et al. Clinical applications of a chromogenic substrate
method for determination of FVIII activity. Thromb Haemost 1985,
54: 811-23). That is, in the present measurement system, test
substances that show higher F.Xa generation-promoting activity are
expected to show better hemostatic effects against bleeding
episodes in hemophilia A. With these results, if a multispecific
antigen-binding molecule having activity of functionally
substituting for F.VIII is a molecule having a higher activity than
hA69-KQ/hB26-PF/hAL-AQ, it may yield excellent blood
coagulation-promoting activity, and excellent effects may be
obtained as a pharmaceutical component for preventing and/or
treating bleeding, a disease accompanying bleeding, or a disease
caused by bleeding. To obtain excellent effects as the
above-mentioned pharmaceutical component, for example, F.Xa
generation-promoting activity measured under the conditions
described in [Example 2] is preferably not less than that of
hA69-KQ/hB26-PF/hAL-AQ, and in particular, the activity is more
preferably the same as or not less than that of
Q153-G4k/J142-G4h/L180-k. Herein, the "F.Xa generation-promoting
activity" is the value obtained by subtracting the change in
absorbance upon 20 minutes in a solvent from the change in
absorbance upon 20 minutes in an antibody solution.
[0432] A preferred embodiment of the present invention is a
multispecific antibody that functionally substitutes for F.VIII,
which recognizes F.IX and/or F.IXa, and F.X.
[0433] The above-mentioned multispecific antibodies of the present
invention are preferably antibodies which comprise H chain CDRs of
anti-F.IX/F.IXa antibodies or CDRs functionally equivalent to them,
and H chain CDRs of anti-F.X antibodies or CDRs functionally
equivalent to them.
[0434] Furthermore, the antibodies of the present invention are
preferably antibodies comprising an antigen-binding site having:
[0435] H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs
75, 76, and 77 (H chain CDRs of Q1), respectively; [0436] H chain
CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs 78, 79, and 80
(H chain CDRs of Q31), respectively; [0437] H chain CDR 1, 2, and 3
amino acid sequences of SEQ ID NOs 81, 82, and 83 (H chain CDRs of
Q64), respectively; [0438] H chain CDR 1, 2, and 3 amino acid
sequences of SEQ ID NOs 84, 85, and 86 (H chain CDRs of Q85),
respectively; [0439] H chain CDR 1, 2, and 3 amino acid sequences
of SEQ ID NOs 87, 88, and 89 (H chain CDRs of Q153), respectively;
[0440] H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs
90, 91, and 92 (H chain CDRs of Q354), respectively; [0441] H chain
CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs 93, 94, and 95
(H chain CDRs of Q360), respectively; [0442] H chain CDR 1, 2, and
3 amino acid sequences of SEQ ID NOs 96, 97, and 98 (H chain CDRs
of Q405), respectively; [0443] H chain CDR 1, 2, and 3 amino acid
sequences of SEQ ID NOs 99, 100, and 101 (H chain CDRs of Q458),
respectively; [0444] H chain CDR 1, 2, and 3 amino acid sequences
of SEQ ID NOs 102, 103, and 104 (H chain CDRs of Q460),
respectively; or [0445] H chain CDR 1, 2, and 3 amino acid
sequences of SEQ ID NOs 105, 106, and 107 (H chain CDRs of Q499),
respectively, in an anti-F.IX/IXa antibody, or an antigen-binding
site functionally equivalent to it, and an antigen-binding site
comprising: [0446] H chain CDR 1, 2, and 3 amino acid sequences of
SEQ ID NOs 108, 109, and 110 (H chain CDRs of J232), respectively;
[0447] H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs
111, 112, and 113 (H chain CDRs of J259), respectively; [0448] H
chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs 114, 115,
and 116 (H chain CDRs of J268), respectively; [0449] H chain CDR 1,
2, and 3 amino acid sequences of SEQ ID NOs 117, 118, and 119 (H
chain CDRs of J300), respectively; [0450] H chain CDR 1, 2, and 3
amino acid sequences of SEQ ID NOs 120, 121, and 122 (H chain CDRs
of J321), respectively; [0451] H chain CDR 1, 2, and 3 amino acid
sequences of SEQ ID NOs 123, 124, and 125 (H chain CDRs of J326),
respectively; [0452] H chain CDR 1, 2, and 3 amino acid sequences
of SEQ ID NOs 126, 127, and 128 (H chain CDRs of J327),
respectively; [0453] H chain CDR 1, 2, and 3 amino acid sequences
of SEQ ID NOs 129, 130, and 131 (H chain CDRs of J339),
respectively; [0454] H chain CDR 1, 2, and 3 amino acid sequences
of SEQ ID NOs 132, 133, and 134 (H chain CDRs of J334),
respectively; the amino acid sequences of H chain CDR 1, 2, and 3
amino acid sequences of SEQ ID NOs 135, 136, and 137 (H chain CDRs
of J346), respectively; or [0455] H chain CDR 1, 2, and 3 amino
acid sequences of SEQ ID NOs 174, 175, and 176 (H chain CDRs of
J142), respectively, in an anti-F.X antibody, or an antigen-binding
site functionally equivalent to it.
[0456] In the present invention, "antigen-binding sites are
functionally equivalent" means that the activities of functionally
substituting for F.VIII possessed by the multispecific
antigen-binding molecules having the antigen-binding sites are
equivalent.
[0457] In the present invention, the term "equivalent" does not
necessarily have to mean the same degree of activity, and the
activity may be enhanced, or the activity may be decreased as long
as there is an activity higher than that of hA69-KQ/hB26-PF/hAL-AQ
according to the measurement system described above, or preferably
F.Xa generation-promoting activity measured under the conditions
described in [Example 2] is equivalent to or not less than that of
Q153-G4k/J142-G4h/L180-k.
[0458] The above-mentioned antibodies may have one or more amino
acid substitutions, deletions, additions, and/or insertions in the
variable region (CDR sequences and/or FR sequences) of the amino
acid sequence as long as they have an activity higher than that of
hA69-KQ/hB26-PF/hAL-AQ according to the measurement system
described above at page 35, lines 11-30, or preferably F.Xa
generation-promoting activity measured under the conditions
described in [Example 2] is equivalent to or not less than that of
Q153-G4k/J142-G4h/L180-k. A method of introducing mutations into
proteins is well known to those skilled in the art as a method for
introducing one or more amino acid substitutions, deletions,
additions, and/or insertions into an amino acid sequence. For
example, those skilled in the art can prepare a desired mutant
functionally equivalent to a multispecific polypeptide multimer
having the activity of functionally substituting for F.VIII by
introducing appropriate mutations into the amino acid sequence
using site-directed mutagenesis (Hashimoto-Gotoh, T, Mizuno, T,
Ogasahara, Y, and Nakagawa, M. (1995) An
oligodeoxyribonucleotide-directed dual amber method for
site-directed mutagenesis. Gene 152: 271-275; Zoller, MJ, and
Smith, M. (1983) Oligonucleotide-directed mutagenesis of DNA
fragments cloned into M13 vectors. Methods Enzymol. 100: 468-500;
Kramer, W, Drutsa, V, Jansen, HW, Kramer, B, Pflugfelder, M, and
Fritz, HJ (1984) The gapped duplex DNA approach to
oligonucleotide-directed mutation construction. Nucleic Acids Res.
12, 9441-9456; Kramer W, and Fritz HJ (1987)
Oligonucleotide-directed construction of mutations via gapped
duplex DNA Methods. Enzymol. 154: 350-367; and Kunkel, TA (1985)
Rapid and efficient site-specific mutagenesis without phenotypic
selection. Proc Natl Acad Sci U S A. 82: 488-492) and such.
[0459] As such, antibodies of the present invention also include
antibodies with one or more amino acid mutations in the variable
region, and having an activity higher than that of
hA69-KQ/hB26-PF/hAL-AQ according to the measurement system
described above at page 35, lines 11-30, or preferably F.Xa
generation-promoting activity measured under the conditions
described in [Example 2] is equivalent to or not less than that of
Q153-G4k/J142-G4h/L180-k.
[0460] When an amino acid residue is altered, the amino acid is
preferably mutated for a different amino acid(s) that conserves the
properties of the amino acid side-chain. Examples of amino acid
side chain properties are: hydrophobic amino acids (A, I, L, M, F,
P, W, Y, and V), hydrophilic amino acids (R, D, N, C, E, Q, G, H,
K, S, and T), amino acids containing aliphatic side chains (G, A,
V, L, I, and P), amino acids containing hydroxyl group-containing
side chains (S, T, and Y), amino acids containing sulfur-containing
side chains (C and M), amino acids containing carboxylic acid- and
amide-containing side chains (D, N, E, and Q), amino acids
containing basic side chains (R, K, and H), and amino acids
containing aromatic side chains (H, F, Y, and W) (amino acids are
represented by one-letter codes in parentheses). Amino acid
substitutions within each group are called conservative
substitutions. It is already known that a polypeptide containing a
modified amino acid sequence in which one or more amino acid
residues in a given amino acid sequence are deleted, added, and/or
substituted with other amino acids can retain the original
biological activity (Mark, D. F. et al., Proc. Natl. Acad. Sci.
USA; (1984) 81: 5662-6; Zoller, M. J. and Smith, M., Nucleic Acids
Res. (1982) 10: 6487-500; Wang, A. et al., Science (1984) 224:
1431-3; Dalbadie-McFarland, G. et al., Proc. Natl. Acad. Sci. USA
(1982) 79: 6409-13). Such mutants have an amino acid identity of at
least 70%, more preferably at least 75%, even more preferably at
least 80%, still more preferably at least 85%, yet more preferably
at least 90%, and most preferably at least 95%, with the variable
regions (for example, CDR sequences, FR sequences, or whole
variable regions) of the present invention. Herein, sequence
identity is defined as the percentage of residues identical to
those in the original amino acid sequence of the heavy chain
variable region or light chain variable region, determined after
the sequences are aligned and gaps are appropriately introduced to
maximize the sequence identity as necessary. The identity of amino
acid sequences can be determined by the method described below.
[0461] Alternatively, the amino acid sequences of variable regions
that have a substitution, deletion, addition, and/or insertion of
one or more amino acids in the amino acid sequence of the variable
regions (CDR sequences and/or FR sequences) and have an activity
higher than that of hA69-KQ/hB26-PF/hAL-AQ according to the
measurement system described above at page 35, lines 11-30, or
preferably F.Xa generation-promoting activity measured under the
conditions described in [Example 2] is equivalent to or not less
than that of Q153-G4k/J142-G4h/L180-k can be obtained from nucleic
acids that hybridize under stringent conditions to nucleic acid
composed of the nucleotide sequence encoding the amino acid
sequence of the variable regions. Stringent hybridization
conditions to isolate a nucleic acid that hybridizes under
stringent conditions to a nucleic acid that includes the nucleotide
sequence encoding the amino acid sequence of the variable regions
include, for example, the conditions of 6 M urea, 0.4% SDS,
0.5.times. SSC, and 37.degree. C., or hybridization conditions with
stringencies equivalent thereto. With more stringent conditions,
for example, the conditions of 6 M urea, 0.4% SDS, 0.1.times. SSC,
and 42.degree. C., isolation of nucleic acids with a much higher
homology can be expected. The sequences of the isolated nucleic
acids can be determined by the known methods described below. The
overall nucleotide sequence homology of the isolated nucleic acid
is at least 50% or higher sequence identity, preferably 70% or
higher, more preferably 90% or higher (for example, 95%, 96%, 97%,
98%, 99%, or higher).
[0462] Nucleic acids that hybridize under stringent conditions to a
nucleic acid composed of the nucleotide sequence encoding the amino
acid sequence of the variable regions can also be isolated using,
instead of the above-described methods using hybridization
techniques, gene amplification methods such as polymerase chain
reaction (PCR) using primers synthesized based on the information
of nucleotide sequence encoding the amino acid sequence of the
variable regions.
[0463] The identity of one nucleotide sequence or amino acid
sequence to another can be determined using the algorithm BLAST, by
Karlin and Altschul (Proc. Natl. Acad. Sci. USA (1993) 90: 5873-7).
Programs such as BLASTN and BLASTX were developed based on this
algorithm (Altschul et al., J. Mol. Biol. (1990) 215: 403-10). To
analyze nucleotide sequences according to BLASTN based on BLAST,
the parameters are set, for example, as score=100 and
wordlength=12. On the other hand, parameters used for the analysis
of amino acid sequences by BLASTX based on BLAST include, for
example, score=50 and wordlength=3. Default parameters for each
program are used when using the BLAST and Gapped BLAST programs.
Specific techniques for such analyses are known in the art (see the
website of the National Center for Biotechnology Information
(NCBI), Basic Local Alignment Search Tool (BLAST);
http://www.ncbi.nlm.nih.gov).
[0464] The present invention also provides antibodies that bind to
an epitope overlapping with an epitope bound by the antibodies
described above.
[0465] Whether an antibody recognizes an epitope overlapping with
an epitope that is recognized by another antibody can be confirmed
by the competition between the two antibodies against the epitope.
Competition between the antibodies can be evaluated by competitive
binding assays using means such as enzyme-linked immunosorbent
assay (ELISA), fluorescence energy transfer method (FRET), and
fluorometric microvolume assay technology (FMAT (Registered
trademark)). The amount of antibodies bound to an antigen
indirectly correlate with the binding ability of candidate
competitor antibodies (test antibodies) that competitively bind to
the overlapping epitope. In other words, as the amount of or the
affinity of test antibodies against the overlapping epitope
increases, the amount of antibodies bound to the antigen decreases,
and the amount of test antibodies bound to the antigen increases.
Specifically, appropriately labeled antibodies and antibodies to be
evaluated are simultaneously added to the antigens, and the thus
bound antibodies are detected using the label. The amount of
antibodies bound to the antigen can be easily determined by
labeling the antibodies beforehand. This label is not particularly
limited, and the labeling method is selected according to the assay
technique used. The labeling method includes fluorescent labeling,
radiolabeling, enzymatic labeling, and such.
[0466] For example, fluorescently labeled antibodies and unlabeled
antibodies or test antibodies are simultaneously added to beads
immobilized with F.IX, F.IXa or F.X, and the labeled antibodies are
detected by fluorometric microvolume assay technology.
[0467] Herein, the "antibody that binds to the overlapping epitope"
refers to an antibody that can reduce the binding of the labeled
antibody by at least 50% at a concentration that is usually 100
times higher, preferably 80 times higher, more preferably 50 times
higher, even more preferably 30 times higher, and still more
preferably 10 times higher than a concentration at which the
non-labeled antibody reduces the binding of the labeled antibody by
50% (IC5o).
[0468] Multispecific antigen-binding molecules, which have
antigen-binding sites of antibodies that bind to epitopes
overlapping with epitopes bound by the above-mentioned antibodies,
may yield an excellent activity of functionally substituting for
F.VIII. Furthermore, in antigen-binding sites of antibodies that
bind to epitopes overlapping with epitopes bound by the
above-mentioned antibodies, one or more amino acids may be altered
to obtain a better activity of functionally substituting for
F.VIII. Multispecific antigen-binding molecules having a better
activity of functionally substituting for F.VIII can be obtained by
altering the amino acids of the antigen-binding sites and selecting
multispecific antigen-binding molecules having an activity higher
than that of hA69-KQ/hB26-PF/hAL-AQ according to the measurement
system described above, or preferably having an F.Xa
generation-promoting activity measured under the conditions
described in [Example 2] that is equivalent to or not less than
that of Q153-G4k/J142-G4h/L180-k. To obtain an excellent activity
of functionally substituting for F.VIII of the present invention,
the following amino acid alterations are particularly preferred.
[0469] (1) At least one amino acid residue selected from the amino
acid residues at positions 34, 35, 49, 61, 62, 96, 98, 100, 100b,
and 102 by Kabat numbering in the H chain of the antibody that
recognizes F.IX and/or F.IXa is substituted with a different amino
acid. [0470] (2) At least one amino acid residue selected from the
amino acid residues at positions 35, 53, 73, 76, 96, 98, 100, and
1009a by Kabat numbering in the H chain of the antibody that
recognizes F.X is substituted with a different amino acid. [0471]
(3) At least one amino acid residue selected from the amino acid
residues at positions 27, 30, 31, 32, 50, 52, 53, 54, 55, 92, 93,
94, and 95 by Kabat numbering in the antibody L chain is
substituted with a different amino acid.
[0472] Furthermore, in the present invention, preferred antibody
amino acids for obtaining a better activity of functionally
substituting for F.VIII include those mentioned in (4) to (6)
below. Regarding these amino acids, the antibody H chain may
originally have such amino acids, or antibody H chain amino acids
may be modified to have such a sequence. [0473] (4) An antibody H
chain which recognizes F.IX and/or F.IXa, wherein, by Kabat
numbering, the amino acid residue at position 34 is isoleucine, the
amino acid residue at position 35 is asparagine, glutamine, or
serine, the amino acid residue at position 49 is serine, the amino
acid residue at position 61 is arginine, the amino acid residue at
position 62 is glutamic acid, the amino acid residue at position 96
is serine or threonine, the amino acid residue at position 98 is
lysine or arginine, the amino acid residue at position 100 is
phenylalanine or tyrosine, the amino acid residue at position 100b
is glycine, or the amino acid residue at position 102 is tyrosine.
[0474] (5) An antibody H chain which recognizes F.X, wherein, by
Kabat numbering, the amino acid residue at position 35 is aspartic
acid, the amino acid residue at position 53 is arginine, the amino
acid residue at position 73 is lysine, the amino acid residue at
position 76 is glycine, the amino acid residue at position 96 is
lysine or arginine, the amino acid residue at position 98 is
tyrosine, the amino acid residue at position 100 is tyrosine, or
the amino acid residue at position 100a is histidine. [0475] (6) An
antibody L chain, wherein, by Kabat numbering, the amino acid
residue at position 27 is lysine or arginine, the amino acid
residue at position 30 is glutamic acid, the amino acid residue at
position 31 is arginine, the amino acid residue at position 32 is
glutamine, the amino acid residue at position 50 is arginine or
glutamine, the amino acid residue at position 52 is serine, the
amino acid residue at position 53 is arginine, the amino acid
residue at position 54 is lysine, the amino acid residue at
position 55 is glutamic acid, the amino acid residue at position 92
is serine, the amino acid residue at position 93 is serine, the
amino acid residue at position 94 is proline, or the amino acid
residue at position 95 is proline.
[0476] Among the above-mentioned antibody amino acid residues of
(1) to (6), favorable positions of amino acid residues for
obtaining a particularly excellent F.VIII-like activity are shown
in the following (1) to (3). [0477] (1) Amino acid residues at
positions 34, 35, 61, 98, 100, and 100b, particularly amino acid
residues at positions 61 and 100, by Kabat numbering in the H chain
of the antibody that recognizes F.IX and/or F.IXa. [0478] (2) Amino
acid residues at positions 35, 53, 73, 96, 98, 100, and 100a by
Kabat numbering in the H chain of the antibody that recognizes F.X.
[0479] (3) Amino acid residues at positions 27, 30, 31, 32, 50, 52,
53, 93, 94, and 95, and particularly amino acid residues at
positions 27, 30, 31, 50, 53, 94, and 95, by Kabat numbering in the
antibody L chain.
[0480] Specifically, the present invention provides multispecific
antigen-binding molecules, wherein a first polypeptide comprises
any of the antibody H chains selected from the following (a1) to
(a14) and any of the antibody L chains selected from the following
(c1) to (c10), and the second polypeptide comprises any of the
antibody H chains selected from the following (b1) to (b12) and any
of the antibody L chains selected from the following (c1) to (c10):
[0481] (a1) an antibody H chain consisting of the amino acid
sequence of SEQ ID NO: 1 (Q1-G4k); [0482] (a2) an antibody H chain
consisting of the amino acid sequence of SEQ ID NO: 2 (Q31-z7);
[0483] (a3) an antibody H chain consisting of the amino acid
sequence of SEQ ID NO: 3 (Q64-z55); [0484] (a4) an antibody H chain
consisting of the amino acid sequence of SEQ ID NO: 10 (Q64-z7);
[0485] (a5) an antibody H chain consisting of the amino acid
sequence of SEQ ID NO: 11 (Q85-G4k); [0486] (a6) an antibody H
chain consisting of the amino acid sequence of SEQ ID NO: 12
(Q153-G4k); [0487] (a7) an antibody H chain consisting of the amino
acid sequence of SEQ ID NO: 13 (Q354-z106); [0488] (a8) an antibody
H chain consisting of the amino acid sequence of SEQ ID NO: 14
(Q360-G4k); [0489] (a9) an antibody H chain consisting of the amino
acid sequence of SEQ ID NO: 15 (Q360-z118); [0490] (a10) an
antibody H chain consisting of the amino acid sequence of SEQ ID
NO: 16 (Q405-G4k); [0491] (a11) an antibody H chain consisting of
the amino acid sequence of SEQ ID NO: 17 (Q458-z106); [0492] (a12)
an antibody H chain consisting of the amino acid sequence of SEQ ID
NO: 18 (Q460-z121); [0493] (a13) an antibody H chain consisting of
the amino acid sequence of SEQ ID NO: 19 (Q499-z118); [0494] (a14)
an antibody H chain consisting of the amino acid sequence of SEQ ID
NO: 20 (Q499-z121); [0495] (b1) an antibody H chain consisting of
the amino acid sequence of SEQ ID NO: 4 (J268-G4h); [0496] (b2) an
antibody H chain consisting of the amino acid sequence of SEQ ID
NO: 5 (J321-G4h); [0497] (b3) an antibody H chain consisting of the
amino acid sequence of SEQ ID NO: 6 (J326-z107); [0498] (b4) an
antibody H chain consisting of the amino acid sequence of SEQ ID
NO: 7 (J344-z107); [0499] (b5) an antibody H chain consisting of
the amino acid sequence of SEQ ID NO: 21 (J232-G4h); [0500] (b6) an
antibody H chain consisting of the amino acid sequence of SEQ ID
NO: 22 (J259-z107); [0501] (b7) an antibody H chain consisting of
the amino acid sequence of SEQ ID NO: 23 (J300-z107); [0502] (b8)
an antibody H chain consisting of the amino acid sequence of SEQ ID
NO: 24 (J327-z107); [0503] (b9) an antibody H chain consisting of
the amino acid sequence of SEQ ID NO: 25 (J327-z119); [0504] (b10)
an antibody H chain consisting of the amino acid sequence of SEQ ID
NO: 26 (J339-z119); [0505] (b11) an antibody H chain consisting of
the amino acid sequence of SEQ ID NO: 27 (J346-z107); [0506] (b12)
an antibody H chain consisting of the amino acid sequence of SEQ ID
NO: 170 (J142-G4h); [0507] (c1) an antibody L chain consisting of
the amino acid sequence of SEQ ID NO: 8 (L2-k); [0508] (c2) an
antibody L chain consisting of the amino acid sequence of SEQ ID
NO: 9 (L45-k); [0509] (c3) an antibody L chain consisting of the
amino acid sequence of SEQ ID NO: 28 (L248-k); [0510] (c4) an
antibody L chain consisting of the amino acid sequence of SEQ ID
NO: 29 (L324-k); [0511] (c5) an antibody L chain consisting of the
amino acid sequence of SEQ ID NO: 30 (L334-k); [0512] (c6) an
antibody L chain consisting of the amino acid sequence of SEQ ID
NO: 31 (L377-k); [0513] (c7) an antibody L chain consisting of the
amino acid sequence of SEQ ID NO: 32 (L404-k); [0514] (c8) an
antibody L chain consisting of the amino acid sequence of SEQ ID
NO: 33 (L406-k); [0515] (c9) an antibody L chain consisting of the
amino acid sequence of SEQ ID NO: 34 (L408-k); and [0516] (c10) an
antibody L chain consisting of the amino acid sequence of SEQ ID
NO: 171 (L180-k).
[0517] The present invention also provides multispecific
antigen-binding molecules, wherein the first polypeptide comprises
an antigen-binding site which binds to an epitope overlapping with
an epitope that binds to an antibody consisting of the antibody H
chain of any one of (a1) to (a14) and the antibody L chain of any
one of (c1) to (c10) described above, and the second polypeptide
comprises an antigen-binding site which binds to an epitope
overlapping with an epitope that binds to an antibody consisting of
the antibody H chain of any one of (b1) to (b12) and the antibody L
chain of any one of (c1) to (c10) described above.
[0518] Furthermore, the present invention provides multispecific
antigen-binding molecules, wherein the first polypeptide comprises
any one antibody H chain selected from the following (e1) to (e3),
the second polypeptide comprises any one antibody H chain selected
from the following (f1) to (f3), and the third polypeptide and the
fourth polypeptide comprise any one antibody L chain selected from
the following (g1) to (g4): [0519] (e1) an H chain of an antibody
which binds to an epitope overlapping with an epitope bound by an
antibody consisting of an antibody H chain of any one of (a1) to
(a14) and an antibody L chain of any one of (c1) to (c10) described
above; [0520] (e2) an antibody H chain, wherein at least one amino
acid residue selected from the amino acid residues at positions 34,
35, 49, 61, 62, 96, 98, 100, 100b, and 102 by Kabat numbering in
any one antibody H chain selected from (e1) described above is
substituted with another amino acid; [0521] (e3) an antibody H
chain, wherein by Kabat numbering, the amino acid residue at
position 34 is isoleucine, the amino acid residue at position 35 is
asparagine, glutamine, or serine, the amino acid residue at
position 49 is serine, the amino acid residue at position 61 is
arginine, the amino acid residue at position 62 is glutamic acid,
the amino acid residue at position 96 is serine or threonine, the
amino acid residue at position 98 is lysine or arginine, the amino
acid residue at position 100 is phenylalanine or tyrosine, the
amino acid residue at position 100b is glycine, or the amino acid
residue at position 102 is tyrosine in any antibody H chain
selected from (e1) described above; [0522] (f1) an H chain of an
antibody which binds to an epitope overlapping with an epitope
bound by an antibody consisting of an antibody H chain of any of
(b1) to (b12) described above and an antibody L chain of any of
(c1) to (c10) described above; [0523] (f2) an antibody H chain,
wherein at least one amino acid residue selected from the amino
acid residues at positions 35, 53, 73, 76, 96, 98, 100, and 100a by
Kabat numbering in any antibody H chain of (f1) described above is
substituted with another amino acid; [0524] (f3) an antibody H
chain, wherein by Kabat numbering, the amino acid residue at
position 35 is aspartic acid, the amino acid residue at position 53
is arginine, the amino acid residue at position 73 is lysine, the
amino acid residue at position 76 is glycine, the amino acid
residue at position 96 is lysine or arginine, the amino acid
residue at position 98 is tyrosine, the amino acid residue at
position 100 is tyrosine, or the amino acid residue at position
100a is histidine in any one antibody H chain selected from (f1)
described above; [0525] (g1) an L chain of an antibody which binds
to an epitope overlapping with an epitope bound by an antibody
which consists of an antibody H chain of any one of (a1) to (a14)
and an antibody L chain of any one of (c1) to (c10) described
above; [0526] (g2) an L chain of an antibody which binds to an
epitope overlapping with an epitope bound by an antibody which
consists of an antibody H chain of any one of (b1) to (b12) and an
antibody L chain of any one of (c1) to (c10) described above;
[0527] (g3) an antibody L chain, wherein at least one amino acid
residue selected from the amino acid residues at positions 27, 30,
31, 32, 50, 52, 53, 54, 55, 92, 93, 94, and 95 by Kabat numbering
in the antibody L chain of either (g1) or (g2) described above is
substituted with another amino acid; and [0528] (g4) an antibody L
chain, wherein by Kabat numbering, the amino acid residue at
position 27 is lysine or arginine, the amino acid residue at
position 30 is glutamic acid, the amino acid residue at position 31
is arginine, the amino acid residue at position 32 is glutamine,
the amino acid residue at position 50 is arginine or glutamine, the
amino acid residue at position 52 is serine, the amino acid residue
at position 53 is arginine, the amino acid residue at position 54
is lysine, the amino acid residue at position 55 is glutamic acid,
the amino acid residue at position 92 is serine, the amino acid
residue at position 93 is serine, the amino acid residue at
position 94 is proline, or the amino acid residue at position 95 is
proline in the antibody L chain of either (g1) or (g2) described
above.
[0529] Amino acid substitutions can be performed on the antibodies
(clones) of the present invention to avoid deamidation, methionine
oxidation, and such, or to structurally stabilize the
antibodies.
[0530] The method for obtaining multispecific antigen-binding
molecules of the present invention is not particularly limited, and
may be any method. Bispecific antibodies can be generated according
to the methods described in WO 2006/109592, WO 2005/035756, WO
2006/106905, or WO 2007/114325, which are known as examples of the
method for producing the bispecific antibodies; and then desired
antibodies having a cofactor function-substituting activity can be
selected and obtained.
[0531] For example, the bispecific antibody described in any of the
following (a) to (u) is provided by the present invention: [0532]
(a) a bispecific antibody (Q1-G4k/J268-G4h/L45-k), wherein the
first polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 1, the second polypeptide is an H chain
consisting of the amino acid sequence of SEQ ID NO: 4, and the
third polypeptide and the fourth polypeptide are a commonly shared
L chain of SEQ ID NO: 9; [0533] (b) a bispecific antibody
(Q1-G4k/J321-G4h/L45-k), wherein the first polypeptide is an H
chain consisting of the amino acid sequence of SEQ ID NO: 1, the
second polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 5, and the third polypeptide and the fourth
polypeptide are a commonly shared L chain of SEQ ID NO: 9; [0534]
(c) a bispecific antibody (Q31-z7/J326-z107/L2-k), wherein the
first polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 2, the second polypeptide is an H chain
consisting of the amino acid sequence of SEQ ID NO: 6, and the
third polypeptide and the fourth polypeptide are a commonly shared
L chain of SEQ ID NO: 8; [0535] (d) a bispecific antibody
(Q64-z553344-z107/L45-k), wherein the first polypeptide is an H
chain consisting of the amino acid sequence of SEQ ID NO: 3, the
second polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 7, and the third polypeptide and the fourth
polypeptide are a commonly shared L chain of SEQ ID NO: 9; [0536]
(e) a bispecific antibody (Q64-z7/J326-z107/L334-k), wherein the
first polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 10, the second polypeptide is an H chain
consisting of the amino acid sequence of SEQ ID NO: 6, and the
third polypeptide and the fourth polypeptide are a commonly shared
L chain of SEQ ID NO: 30; [0537] (f) a bispecific antibody
(Q64-z7/J344-z107/L406-k), wherein the first polypeptide is an H
chain consisting of the amino acid sequence of SEQ ID NO: 10, the
second polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 7, and the third polypeptide and the fourth
polypeptide are a commonly shared L chain of SEQ ID NO: 33; [0538]
(g) a bispecific antibody (Q85-G4k/J268-G4h/L406-k), wherein the
first polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 11, the second polypeptide is an H chain
consisting of the amino acid sequence of SEQ ID NO: 4, and the
third polypeptide and the fourth polypeptide are a commonly shared
L chain of SEQ ID NO: 33; [0539] (h) a bispecific antibody
(Q85-G4k/J321-G4h/L334-k), wherein the first polypeptide is an H
chain consisting of the amino acid sequence of SEQ ID NO: 11, the
second polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 5, and the third polypeptide and the fourth
polypeptide are a commonly shared L chain of SEQ ID NO: 30; [0540]
(i) a bispecific antibody (Q153-G4k/J232-G4h/L406-k), wherein the
first polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 12, the second polypeptide is an H chain
consisting of the amino acid sequence of SEQ ID NO: 21, and the
third polypeptide and the fourth polypeptide are a commonly shared
L chain of SEQ ID NO: 33; [0541] (j) a bispecific antibody
(Q354-z106/J259-z107/L324-k), wherein the first polypeptide is an H
chain consisting of the amino acid sequence of SEQ ID NO: 13, the
second polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 22, and the third polypeptide and the fourth
polypeptide are a commonly shared L chain of SEQ ID NO: 29; [0542]
(k) a bispecific antibody (Q360-G4k/J232-G4h/L406-k), wherein the
first polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 14, the second polypeptide is an H chain
consisting of the amino acid sequence of SEQ ID NO: 21, and the
third polypeptide and the fourth polypeptide are a commonly shared
L chain of SEQ ID NO: 33; [0543] (l) a bispecific antibody
(Q360-z118/J300-z107/L334-k), wherein the first polypeptide is an H
chain consisting of the amino acid sequence of SEQ ID NO: 15, the
second polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 23, and the third polypeptide and the fourth
polypeptide are a commonly shared L chain of SEQ ID NO: 30; [0544]
(m) a bispecific antibody (Q405-G4k/J232-G4h/L248-k), wherein the
first polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 16, the second polypeptide is an H chain
consisting of the amino acid sequence of SEQ ID NO: 21, and the
third polypeptide and the fourth polypeptide are a commonly shared
L chain of SEQ ID NO: 28; [0545] (n) a bispecific antibody
(Q458-z106/J346-z107/L408-k), wherein the first polypeptide is an H
chain consisting of the amino acid sequence of SEQ ID NO: 17, the
second polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 27, and the third polypeptide and the fourth
polypeptide are a commonly shared L chain of SEQ ID NO: 34; [0546]
(o) a bispecific antibody (Q460-z121/J327-z119/L334-k), wherein the
first polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 18, the second polypeptide is an H chain
consisting of the amino acid sequence of SEQ ID NO: 25, and the
third polypeptide and the fourth polypeptide are a commonly shared
L chain of SEQ ID NO: 30; [0547] (p) a bispecific antibody
(Q499-z118/J327-z107/L334-k), wherein the first polypeptide is an H
chain consisting of the amino acid sequence of SEQ ID NO: 19, the
second polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 24, and the third polypeptide and the fourth
polypeptide are a commonly shared L chain of SEQ ID NO: 30; [0548]
(q) a bispecific antibody (Q499-z118/J327-z107/L377-k), wherein the
first polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 19, the second polypeptide is an H chain
consisting of the amino acid sequence of SEQ ID NO: 24, and the
third polypeptide and the fourth polypeptide are a commonly shared
L chain of SEQ ID NO: 31; [0549] (r) a bispecific antibody
(Q499-z118/J346-z107/L248-k), wherein the first polypeptide is an H
chain consisting of the amino acid sequence of SEQ ID NO: 19, the
second polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 27, and the third polypeptide and the fourth
polypeptide are a commonly shared L chain of SEQ ID NO: 28; [0550]
(s) a bispecific antibody (Q499-z121/J327-z119/L404-k), wherein the
first polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 20, the second polypeptide is an H chain
consisting of the amino acid sequence of SEQ ID NO: 25, and the
third polypeptide and the fourth polypeptide are a commonly shared
L chain of SEQ ID NO: 32; [0551] (t) a bispecific antibody
(Q499-z121/J339-z119/L377-k), wherein the first polypeptide is an H
chain consisting of the amino acid sequence of SEQ ID NO: 20, the
second polypeptide is an H chain consisting of the amino acid
sequence of SEQ ID NO: 26, and the third polypeptide and the fourth
polypeptide are a commonly shared L chain of SEQ ID NO: 31; and
[0552] (u) a bispecific antibody (Q153-G4k/J142-G4h/L180-k),
wherein the first polypeptide is an H chain consisting of the amino
acid sequence of SEQ ID NO: 12, the second polypeptide is an H
chain consisting of the amino acid sequence of SEQ ID NO: 170, and
the third polypeptide and the fourth polypeptide are a commonly
shared L chain of SEQ ID NO: 171.
[0553] Amino acid sequences, molecular weights, isoelectric points,
or presence or absence and form of sugar chains of the antibodies
of the present invention vary depending on cells or hosts that
produce the antibodies or purification methods described later.
However, as long as the obtained antibodies have functions
equivalent to the antibodies of the present invention, they are
included in the present invention. For example, when an antibody of
the present invention is expressed in prokaryotic cells such as E.
coli, a methionine residue will be added to the N terminus of the
original antibody amino acid sequence. Antibodies of the present
invention also comprise such antibodies.
[0554] Bispecific antibodies of the present invention can be
produced by methods known to those skilled in the art.
[0555] Based on the obtained sequence of the anti-F.IX/F.IXa
antibody or anti-F.X antibody, the anti-F.IX/F.IXa antibody or
anti-F.X antibody can be prepared, for example, by genetic
recombination techniques known to those skilled in the art.
Specifically, a polynucleotide encoding an antibody can be
constructed based on the sequence of the anti-F.IX/F.IXa antibody
or anti-F.X antibody, inserted into an expression vector, and then
expressed in appropriate host cells (see for example, Co, M. S. et
al., J. Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz, A.
H., Methods Enzymol. (1989) 178, 476-496; Pluckthun, A. and Skerra,
A., Methods Enzymol. (1989) 178, 497-515; Lamoyi, E., Methods
Enzymol. (1986) 121, 652-663; Rousseaux, J. et al., Methods
Enzymol. (1986) 121, 663-669; Bird, R. E. and Walker, B. W., Trends
Biotechnol. (1991) 9, 132-137).
[0556] The vectors include M13 vectors, pUC vectors, pBR322,
pBluescript, and pCR-Script. Alternatively, when aiming to subclone
and excise cDNA, the vectors include, for example, pGEM-T, pDIRECT,
and pT7, in addition to the vectors described above. Expression
vectors are particularly useful when using vectors for producing
the antibodies of the present invention. For example, when aiming
for expression in E. coli such as JM109, DHS.alpha., HB101, and
XL1-Blue, the expression vectors not only have the characteristics
that allow vector amplification in E. coli, but must also carry a
promoter that allows efficient expression in E. coli, for example,
lacZ promoter (Ward et al., Nature (1989) 341: 544-546; FASEB J.
(1992) 6: 2422-2427), araB promoter (Better et al., Science (1988)
240: 1041-1043), T7 promoter or such. Such vectors include
pGEX-5X-1 (Pharmacia), "QlAexpress system" (Qiagen), pEGFP, or pET
(in this case, the host is preferably BL21 that expresses T7 RNA
polymerase) in addition to the vectors described above.
[0557] The expression plasmid vectors may contain signal sequences
for antibody secretion. As a signal sequence for antibody
secretion, a pelB signal sequence (Lei, S. P. et al J. Bacteriol.
(1987) 169: 4379) may be used when a protein is secreted into the
E. coli periplasm. The vector can be introduced into host cells by
calcium chloride or electroporation methods, for example.
[0558] In addition to vectors for E. coli, the vectors for
producing the antibodies of the present invention include mammalian
expression vectors (for example, pcDNA3 (Invitrogen), pEF-BOS
(Nucleic Acids. Res. 1990, 18(17): p5322), pEF, and pCDM8), insect
cell-derived expression vectors (for example, the "Bac-to-BAC
baculovirus expression system" (Gibco-BRL) and pBacPAK8),
plant-derived expression vectors (for example, pMH1 and pMH2),
animal virus-derived expression vectors (for example, pHSV, pMV,
and pAdexLcw), retroviral expression vectors (for example,
pZlPneo), yeast expression vectors (for example, "Pichia Expression
Kit" (Invitrogen), pNV11, and SP-Q01), and Bacillus subtilis
expression vectors (for example, pPL608 and pKTH50), for
example.
[0559] When aiming for expression in animal cells such as CHO, COS,
and NIH3T3 cells, the expression plasmid vectors must have a
promoter essential for expression in cells, for example, SV40
promoter (Mulligan et al., Nature (1979) 277: 108), MMLV-LTR
promoter, EF1.alpha. promoter (Mizushima et al., Nucleic Acids Res.
(1990) 18: 5322), and CMV promoter, and more preferably they have a
gene for selecting transformed cells (for example, a drug
resistance gene that allows evaluation using an agent (neomycin,
G418, or such)). Vectors with such characteristics include pMAM,
pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13, for example.
[0560] In addition, the following method can be used for stable
gene expression and gene amplification in cells: CHO cells
deficient in a nucleic acid synthesis pathway are introduced with a
vector that carries a DHFR gene which compensates for the
deficiency (for example, pSV2-dhfr (Molecular Cloning 2.sup.nd
edition, Cold Spring Harbor Laboratory Press, 1989)), and the
vector is amplified using methotrexate (MTX). Alternatively, the
following method can be used for transient gene expression: COS
cells with a gene expressing SV40 T antigen on their chromosome are
transformed with a vector with an SV40 replication origin (pcD and
such). Replication origins derived from polyoma virus, adenovirus,
bovine papilloma virus (BPV), and such can also be used. To amplify
gene copy number in host cells, the expression vectors may further
carry selection markers such as aminoglycoside transferase (APH)
gene, thymidine kinase (TK) gene, E. coli xanthine-guanine
phosphoribosyltransferase (Ecogpt) gene, and dihydrofolate
reductase (dhfr) gene.
[0561] The antibodies of the present invention obtained by the
methods described above can be isolated from inside host cells or
from outside the cells (the medium, or such), and purified to
homogeneity. The antibodies can be isolated and purified by methods
routinely used for isolating and purifying antibodies, and the type
of method is not limited. For example, the antibodies can be
isolated and purified by appropriately selecting and combining
column chromatography, filtration, ultrafiltration, salting-out,
solvent precipitation, solvent extraction, distillation,
immunoprecipitation, SDS-polyacrylamide gel electrophoresis,
isoelectrofocusing, dialysis, recrystallization, and such.
[0562] The chromatographies include, for example, affinity
chromatography, ion exchange chromatography, hydrophobic
chromatography, gel filtration, reverse phase chromatography, and
adsorption chromatography (Strategies for Protein Purification and
Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak
et al., Cold Spring Harbor Laboratory Press, 1996). The
chromatographic methods described above can be conducted using
liquid chromatography, for example, HPLC and FPLC. Columns that can
be used for affinity chromatography include protein A columns and
protein G columns. Columns using protein A include, for example,
Hyper D, POROS, and Sepharose FF (GE Amersham Biosciences). The
present invention includes antibodies that are highly purified
using these purification methods.
[0563] The obtained antibodies can be purified to homogeneity.
Separation and purification of the antibodies can be performed
using conventional separation and purification methods used for
ordinary proteins. For example, the antibodies can be separated and
purified by appropriately selecting and combining column
chromatography such as affinity chromatography, filtration,
ultrafiltration, salting-out, dialysis, SDS polyacrylamide gel
electrophoresis, isoelectric focusing, and such, without limitation
(Antibodies : A Laboratory Manual. Ed Harlow and David Lane, Cold
Spring Harbor Laboratory, 1988). Columns used for affinity
chromatography include, for example, protein A columns and protein
G columns.
[0564] In one embodiment of antibodies of the present invention,
since the antibodies of the present invention functionally
substitute for cofactor F.VIII, they are expected to become
effective pharmaceutical agents against diseases resulting from
decrease in activity (function) of this cofactor. Examples of the
above-mentioned diseases include bleeding, diseases accompanying
bleeding, or a disease caused by bleeding. In particular, there may
have excellent therapeutic effects on hemophilias, in which
bleeding disorders are caused by a deficiency or decrease of
F.VIII/F.VIIIa function. Among the hemophilias, they are expected
to become excellent therapeutic agents for hemophilia A, in which
bleeding disorders are caused by a hereditary deficiency or
decrease of F.VIII/F.VIIIa function.
[0565] The present invention provides (pharmaceutical) compositions
comprising the antibodies of the present invention and
pharmaceutically acceptable carriers. For example, the antibodies
of the present invention that recognize both F.IX or F.IXa and F.X,
and functionally substitute for F.VIII are expected to become
pharmaceuticals (pharmaceutical compositions) or pharmaceutical
agents for preventing and/or treating bleeding, diseases
accompanying bleeding, diseases caused by bleeding, and the
like.
[0566] In the context of the present invention, bleeding, diseases
accompanying bleeding, and/or diseases caused by bleeding
preferably refer to diseases that develop and/or progress due to
reduction or deficiency in activity of F.VIII and/or activated
coagulation factor VIII (F.VIIIa). Such diseases include the
above-described hemophilia A, diseases in which an inhibitor
against F.VIII /F.VIIIa appear, acquired hemophilia, von
Willebrand's disease, and such, but are not particularly limited
thereto.
[0567] Pharmaceutical compositions used for therapeutic or
preventive purposes, which comprise antibodies of the present
invention as active ingredients, can be formulated by mixing, if
necessary, with suitable pharmaceutically acceptable carriers,
vehicles, and such that are inactive against the antibodies. For
example, sterilized water, physiological saline, stabilizers,
excipients, antioxidants (such as ascorbic acid), buffers (such as
phosphate, citrate, histidine, and other organic acids),
antiseptics, surfactants (such as PEG and Tween), chelating agents
(such as EDTA), and binders may be used. They may also comprise
other low-molecular-weight polypeptides, proteins such as serum
albumin, gelatin, and immunoglobulins, amino acids such as glycine,
glutamine, asparagine, glutamic acid, asparagic acid, methionine,
arginine, and lysine, sugars and carbohydrates such as
polysaccharides and monosaccharides, and sugar alcohols such as
mannitol and sorbitol. When preparing an aqueous solution for
injection, physiological saline and isotonic solutions comprising
glucose and other adjuvants such as D-sorbitol, D-mannose,
D-mannitol, and sodium chloride may be used, and if necessary, in
combination with appropriate solubilizers such as alcohol (for
example, ethanol), polyalcohols (such as propylene glycol and PEG),
and nonionic surfactants (such as polysorbate 80, polysorbate 20,
poloxamer 188, and HCO-50). By mixing hyaluronidase into the
formulation, a larger fluid volume can be administered
subcutaneously (Expert Opin Drug Deliv. 2007 Jul; 4(4):
427-40).
[0568] If necessary, antibodies of the present invention may be
encapsulated in microcapsules (e.g., those made of
hydroxymethylcellulose, gelatin, and poly(methylmetacrylate)), or
incorporated as components of colloidal drug delivery systems
(e.g., liposomes, albumin microspheres, microemulsion,
nanoparticles, and nanocapsules) (see, for example, "Remington's
Pharmaceutical Science 16th edition", Oslo Ed. (1980)). Methods for
preparing the pharmaceutical agents as controlled-release
pharmaceutical agents are also well known, and such methods may be
applied to the antibodies of the present invention (Langer et al. ,
J. Biomed. Mater. Res. 15: 267-277 (1981); Langer, Chemtech. 12:
98-105 (1982); U.S. Patent No. 3,773,919; European Patent
Application Publication No. EP 58,481; Sidman et al., Biopolymers
22: 547-556 (1983); EP 133,988).
[0569] The dose of a pharmaceutical composition of the present
invention may be appropriately determined by considering the dosage
form, method of administration, patient age and body weight,
symptoms of the patient, type of the disease, and degree of
progress of the disease, and is ultimately decided by physicians.
Generally, the daily dose for an adult is 0.1 mg to 2,000 mg at
once or in several portions. The dose is more preferably 0.2 to
1,000 mg/day, even more preferably 0.5 to 500 mg/day, still more
preferably 1 to 300 mg/day, yet more preferably 3 to 100 mg/day,
and most preferably 5 to 50 mg/day. These doses may vary, depending
on the patient body weight and age, and the method of
administration; however, selection of suitable dosage is well
within the purview of those skilled in the art. Similarly, the
dosing period may be appropriately determined depending on the
therapeutic progress.
[0570] Furthermore, the present invention provides genes or nucleic
acids encoding the antibodies of the present invention. In
addition, gene therapy may be performed by incorporating genes or
nucleic acids encoding the antibodies of the present invention into
vectors for gene therapy. In addition to direct administration
using naked plasmids, methods of administration include
administration after packaging into liposomes and such, forming a
variety of virus vectors such as retrovirus vectors, adenovirus
vectors, vaccinia virus vectors, poxvirus vectors, adeno-associated
virus vectors, and HVJ vectors (see Adolph "Viral Genome Methods"
CRC Press, Florida (1996)), or coating with carrier beads such as
colloidal gold particles (WO 93/17706, and such). However, so long
as the antibodies are expressed in vivo and their activities are
exercised, any method can be used for administration. Preferably, a
sufficient dose can be administered by a suitable parenteral route
(such as injecting or infusing intravenously, intraperitoneally,
subcutaneously, intradermally, intramuscularly, into adipose
tissues or mammary glands; inhalation; gas-driven particle
bombardment (using electron gun and such); or mucosal route such as
nasal drops). Alternatively, the genes encoding the antibodies of
the present invention may be administered into blood cells, bone
marrow cells, and such ex vivo using liposome transfection,
particle bombardment (U.S. Patent No. 4,945,050), or viral
infection, and the cells may be reintroduced into patients. Any
gene encoding an antibody of the present invention may be used in
gene therapy, and its examples include genes comprising nucleotide
sequences encoding the CDRs of Q1, Q31, Q64, Q85, Q153, Q354, Q360,
Q405, Q458, Q460, Q499, J232, J259, J268, J300, J321, J326, J327,
J339, J344, J346, J142, L2, L45, L248, L324, L334, L377, L404,
L406, L408, and L180 described above.
[0571] The present invention also provides methods for preventing
and/or treating bleeding, diseases accompanying bleeding, and/or
diseases caused by bleeding, such methods comprising the step of
administering the antibodies or compositions of the present
invention. The antibodies or compositions can be administered, for
example, by the above-mentioned methods.
[0572] Furthermore, the present invention provides kits to be used
for the above-mentioned methods, such kits comprising at least an
antibody or composition of the present invention. In addition, the
kits may include, packaged therewith, a syringe, injection needle,
pharmaceutically acceptable medium, alcohol-soaked cotton, adhesive
bandage, instructions describing the method of use, and the
like.
[0573] The present invention also relates to use of a multispecific
antigen-binding molecule, a bispecific antibody, or a composition
of the present invention in the manufacture of an agent for
preventing and/or treating bleeding, a disease accompanying
bleeding, or a disease caused by bleeding.
[0574] Furthermore, the present invention relates to a
multispecific antigen-binding molecule, a bispecific antibody, or a
composition of the present invention for preventing and/or treating
bleeding, a disease accompanying bleeding, or a disease caused by
bleeding.
[0575] All prior art references cited herein are incorporated by
reference into this description.
EXAMPLES
[0576] Herein below, the present invention will be specifically
described with reference to the Examples, but it is not to be
construed as being limited thereto.
Example 1
Production of Bispecific Antibodies Having F.Xa
Generation-Promoting Activity
[0577] In WO 2006/109592, hA69-KQ/hB26-PF/hAL-AQ was obtained as a
bispecific antibody having an activity of functionally substituting
for F.VIII. However, there was the possibility that this antibody
has an inhibiting action on the reaction in which F.IXa activates
F.X using F.VIIIa as a cofactor.
[0578] As shown in FIG. 1, antibodies that bind to F.IX/F.IXa or
F.X may inhibit the formation of the F.IXa-F.VIIIa complex (Factor
Xase (F.Xase)), or inhibit F.Xase activity (activation of F.X).
Hereafter, inhibition of F.Xase formation and/or action of
inhibiting F.Xase activity will be mentioned as F.Xase inhibitory
action. F.Xase inhibitory action is the inhibition of a coagulation
reaction in which F.VIIIa serves as the cofactor, which may
suppress the remaining F.VIII function in a patient or the function
of the administered F.VIII formulation. Therefore, it is desirable
that F.Xa generation-promoting activity, which is the objective of
the bispecific antibody, is high, while F.Xase inhibitory action is
low. In particular, for patients maintaining F.VIII function and
patients receiving treatment with a F.VIII formulation, it is more
desirable that F.Xa generation-promoting activity and F.Xase
inhibitory action are separated as much as possible.
[0579] However, F.Xase inhibitory action is due to the binding to
the antigen (F.IXa and/or F.X), which is fundamental property of
the antibody. On the other hand, a bispecific antibody having F.Xa
generation-promoting action (functionally substituting for F.VIII)
also needs to bind to the antigens (F.IXa and F.X). Therefore, it
is predicted that it is extremely difficult to obtain bispecific
antibodies that do not have an F.Xase inhibitory action but have an
F.Xa generation-promoting activity (functionally substituting for
F.VIII). Similarly, it is predicted that it is extremely difficult
to decrease an F.Xase inhibitory action while increasing the target
F.Xa generation-promoting activity by introducing amino acid
substitutions in a bispecific antibody.
[0580] The present inventors prepared genes for approximately 200
types of antibodies against human F.IXa and human F.X,
respectively, using a method known to those skilled in the art,
which is the method of obtaining antibody genes from
antibody-producing cells of animals immunized with an antigen
(human F.IXa or human F.X), and introducing amino acid
substitutions, when necessary. Each antibody gene was incorporated
into an animal cell expression vector.
[0581] 40,000 or more bispecific antibodies as anti-F.IXa antibody
and anti-F.X antibody combinations were transiently expressed by
simultaneously transfecting the anti-human F.IXa antibody H chain
expression vector, the anti-human F.X antibody H chain expression
vector, and the commonly shared antibody L chain expression vector
into mammalian cells such as HEK293H cells. As a comparative
control, bispecific antibody hA69-KQ/hB26-PF/hAL-AQ (SEQ ID NOs:
165/166/167) described in WO 2006/109592 was prepared. Since the
mutations mentioned in WO 2006/106905 or WO 1996/027011 were
introduced into the CH3 domain of each H chain, it was thought that
bispecific antibodies were mainly expressed. Antibodies in the cell
culture supernatant were purified by a method known to those
skilled in the art using Protein A.
[0582] The present inventors measured the F.Xa generation-promoting
activity of these antibodies by the method described below. All
reactions were performed at room temperature.
[0583] Five .mu.L of antibody solution diluted with Tris-buffered
saline containing 0.1% bovine serum albumin (hereafter referred to
as TBSB) was mixed with 2.5 .mu.L of 27 ng/mL Human Factor IXa beta
(Enzyme Research Laboratories) and 2.5 .mu.L of 6 IU/mL of human
blood coagulation factor IX (Novact (registered trademark) M
(Kaketsuken)), and then incubated in a 384-well plate at room
temperature for 30 minutes.
[0584] The enzyme reaction in this mixed solution was initiated by
adding 5 .mu.L of 24.7 .mu.g/mL of Human Factor X (Enzyme Research
Laboratories), and ten minutes later, 5 .mu.L of 0.5 M EDTA was
added to stop the reaction. The coloring reaction was initiated by
adding 5 .mu.L of coloring substrate solution. After a 50-minute
coloring reaction, the change in absorbance at 405 nm was measured
using the SpectraMax 340PC.sup.384 (Molecular Devices). F.Xa
generation-promoting activity was indicated as the value obtained
by subtracting the absorbance of the antibody-free reaction
solution from the absorbance of the antibody-supplemented reaction
solution.
[0585] TBCP (TBSB containing 93.75 .mu.M synthetic phospholipid
solution (SYSMEX CO.), 7.5 mM CaC12, and 1.5 mM MgC12) was used as
the solvent for Human Factor IXa, Novact (registered trademark) M,
and Human Factor X. A coloring substrate solution
N-benzoyl-L-isoleucyl-L-glutamyl-glycyl-L-arginine-p-nitroaniline
hydrochloride (S-2222.TM.; CHROMOGENIX) was dissolved in purified
water at 1.47 mg/mL, and then used in this assay.
[0586] To evaluate the F.Xase inhibitory action of the antibodies,
the present inventors measured the effects on F.X activation by
F.IXa in the presence of F.VIIIa using the following method. All
reactions were performed at room temperature.
[0587] Five .mu.L of antibody solution diluted with Tris-buffered
saline containing 0.1% bovine serum albumin (hereafter referred to
as TBSB) was mixed with 2.5 .mu.L of 80.9 ng/mL Human Factor IXa
beta (Enzyme Research Laboratories), and then incubated in a
384-well plate at room temperature for 30 minutes.
[0588] 2.5 .mu.L of 1.8 IU/mL of F.VIIIa (production method will be
descried later) was further added, and 30 seconds later, the enzyme
reaction in this mixed solution was initiated by adding 5 .mu.L of
24.7 .mu.g/mL of Human Factor X (Enzyme Research Laboratories). Six
minutes later, 5 .mu.L of 0.5 M EDTA was added to stop the
reaction. The coloring reaction was initiated by adding 5 .mu.L of
coloring substrate solution. After a 14-minute coloring reaction,
the change in absorbance at 405 nm was measured using the
SpectraMax 340PC.sup.384 (Molecular Devices). F.Xase inhibitory
action of an antibody was indicated as the value obtained by
subtracting the absorbance of the antibody-free reaction solution
from the absorbance of the antibody-supplemented reaction
solution.
[0589] F.VIIIa was prepared by mixing 5.4 IU/mL of Kogenate
(registered trademark) FS (Bayer HealthCare) and 1.11 .mu.g/mL of
Human alpha Thrombin (Enzyme Research Laboratories) at a volume
ratio of 1:1, incubating at room temperature for one minute, and
then adding 7.5 U/mL of Hirudin (Merck KgaA) at a quantity that is
half the volume of the mixture solution. The prepared solution was
defined as 1.8 IU/mL of FVIIIa, and one minute after addition of
Hirudin, this was used for assays.
[0590] TBCP (TBSB containing 93.75 .mu.M phospholipid solution
(SYSMEX CO.), 7.5 mM CaCl.sub.2, and 1.5 mM MgCl.sub.2) was used
for the solvent for Human Factor IXa, Human Factor X, Kogenate
(registered trademark) FS, Human alpha Thrombin, and Hirudin. A
coloring substrate solution S-2222.TM. (CHROMOGENIX) was dissolved
in purified water at 1.47 mg/mL, and then used in this assay.
[0591] The F.Xa generation-promoting activities of each of the
bispecific antibodies are indicated in FIGS. 3 and 4, and the
F.Xase inhibitory actions of each of the bispecific antibodies are
indicated in FIG. 5. Various amino acid substitutions that increase
the F.Xa generation-promoting activity have been found, but as
expected, most of the amino acid substitutions that increase the
F.Xa generation-promoting activity increased F.Xase inhibitory
action as well, and suppressing F.Xase inhibitory action while
increasing F.Xa generation-promoting activity was very
difficult.
[0592] Under such circumstances, the inventors of the present
application obtained Q1-G4k/J268-G4h/L45-k, Q1-G4k/J321-G4h/L45-k,
Q31-z7/J326-z107/L2-k, Q64-z55/J344-z107/L45-k as bispecific
antibodies with a high F.Xa generation-promoting activity and a low
F.Xase inhibitory action. In addition, Q1-G4k (SEQ ID NO: 1),
Q31-z7 (SEQ ID NO: 2), and Q64-z55 (SEQ ID NO: 3) were obtained as
anti-human F.IXa antibody H chains, J268-G4h (SEQ ID NO: 4),
J321-G4h (SEQ ID NO: 5), J326-z107 (SEQ ID NO: 6), and J344-z107
(SEQ ID NO: 7) were obtained as prototype anti-human F.X antibody H
chains, and L2-k (SEQ ID NO: 8) and L45-k (SEQ ID NO: 9) were
obtained as prototype commonly shared antibody L chains. The
character before the hyphen in the sequence name indicates the
variable region and the character after the hyphen indicates the
constant region. Each bispecific antibody name is indicated by
listing the sequence names of each chain to be transfected.
[0593] Most of the bispecific antibodies having F.Xa
generation-promoting activity close to that of
hA69-KQ/hB26-PF/hAL-AQ had high F.Xase inhibitory action as
expected, but these bispecific antibodies (Q1-G4k/J268-G4h/L45-k,
Q1-G4k/J321-G4h/L45-k, Q31-z7/J326-z107/L2-k,
Q64-z55/J344-z107/L45-k) were found to have higher F.Xa
generation-promoting activity and lower F.Xase inhibitory action
than hA69-KQ/hB26-PF/hAL-AQ described in WO 2006/109592. The
present inventors conducted examinations to further increase the
F.Xa generation-promoting activity and reduce the F.Xase inhibitory
action using these four antibodies as prototype antibodies.
Screening of bispecific antibodies that increase F.Xa
generation-promoting activity and reduce F.Xase inhibitory action
is indicated in FIG. 2.
Example 2
Production of Modified Antibodies
[0594] The present inventors introduced various combinations of
amino acid mutations that affect the F.Xa generation-promoting
activities and F.Xase inhibitory actions found in Example 1 to each
of the chains of the prototype antibodies by a method known to
those skilled in the art such as PCR for introducing mutations and
evaluated the combinations of modified chains on a large scale to
screen for amino acid substitutions that will further increase the
F.Xa generation-promoting activities and reduce the F.Xase
inhibitory actions of the four prototype antibodies.
[0595] Each of the modified bispecific antibodies with amino acid
substitutions were expressed transiently and purified by methods
similar to those for the prototype antibodies. The F.Xa
generation-promoting activities of the antibodies were measured
using the following method. All reactions were performed at room
temperature.
[0596] Five .mu.L of antibody solution diluted with Tris-buffered
saline containing 0.1% bovine serum albumin (hereafter referred to
as TBSB) was mixed with 2.5 .mu.L of 27 ng/mL Human Factor IXa beta
(Enzyme Research Laboratories) and 2.5 .mu.L of 6 IU/mL of Novact
(registered trademark) M (Kaketsuken), and then incubated in a
384-well plate at room temperature for 30 minutes.
[0597] The enzyme reaction in this mixed solution was initiated by
adding 5 .mu.L of 24.7 .mu.g/mL of Human Factor X (Enzyme Research
Laboratories), and two minutes later, 5 .mu.L of 0.5 M EDTA was
added to stop the reaction. The coloring reaction was initiated by
adding 5 .mu.L of coloring substrate solution. After a 20-minute
coloring reaction, the change in absorbance at 405 nm was measured
using the SpectraMax 340PC.sup.384 (Molecular Devices). F.Xa
generation-promoting activity was indicated as the value obtained
by subtracting the absorbance of the antibody-free reaction
solution from the absorbance of the antibody-supplemented reaction
solution.
[0598] TBCP (TBSB containing 93.75 .mu.M phospholipid solution
(SYSMEX CO.), 7.5 mM CaCl.sub.2, and 1.5 mM MgCl.sub.2) was used as
the solvent for Human Factor IXa, Novact (registered trademark) M,
and Human Factor X. A coloring substrate solution S-2222.TM.
(CHROMOGENIX) was dissolved in purified water at 1.47 mg/mL, and
then used in this assay.
[0599] F.Xase inhibitory actions of the antibodies were also
evaluated by previously described methods.
[0600] The F.Xa generation-promoting activities of each of the
modified bispecific antibodies are indicated in FIG. 4, and the
F.Xase inhibitory actions of each of the bispecific antibodies are
indicated in FIG. 5.
[0601] The inventors of the present application obtained
Q85-G4k/J268-G4h/L406-k, Q85-G4k/J321-G4h/L334-k,
Q64-z7/J344-z107/L406-k, and Q64-z7/J326-z107/L334-k as bispecific
antibodies with a high F.Xa generation-promoting activity and a low
F.Xase inhibitory action. In addition, they discovered Q64-z7 (SEQ
ID NO: 10) and Q85-G4k (SEQ ID NO: 11) as the anti-human F.IXa
antibody H chain, and L334-k (SEQ ID NO: 30) and L406-k (SEQ ID NO:
33) as the commonly shared antibody L chains with increased F.Xa
generation-promoting activity. Though F.Xase inhibitory actions
increased slightly, F.Xa generation-promoting activity increased
greatly in Q85-G4k/J268-G4h/L406-k, Q85-G4k/J321-G4h/L334-k,
Q64-z7/J344-z107/L406-k, and Q64-z7/J326-z107/L334-k. Since these
modified antibodies have very large F.Xa generation-promoting
activities compared to increase in F.Xase inhibitory actions, the
F.Xa generation-promoting activity and the F.Xase inhibitory action
could further be separated compared to the prototype antibodies.
This way, combinations that suppress the F.Xase inhibitory action
and increase the F.Xa generation-promoting activity were
discovered.
[0602] While a higher F.Xa generation-promoting activity is
preferred for the discovered prototype antibodies to functionally
substitute for F.VIII by bispecific antibodies, lower F.Xase
inhibitory action was considered favorable to clinically use for
patients maintaining F.VIII functions or patients receiving
treatment with F.VIII formulations. Therefore, further
modifications were performed to produce bispecific antibodies in
which F.Xase inhibitory action is not increased while F.Xa
generation-promoting activity is further increased.
[0603] As a result, Q153-G4k/J232-G4h/L406-k,
Q354-z1063259-z107/L324-k, Q360-G4k/J232-G4h/L406-k,
Q360-z118/J300-z107/L334-k, Q405-G4k/J232-G4h/L248-k,
Q458-z1063346-z107/L408-k, Q460-z121/J327-z119/L334-k,
Q499-z118/J327-z107/L334-k, Q499-z118/J327-z107/L377-k,
Q499-z118/J346-z107/L248-k, Q499-z121/J327-z119/L404-k,
Q499-z121/J339-z119/L377-k, and Q153-G4k/J142-G4h/L180-k were
obtained as bispecific antibodies with a high F.Xa
generation-promoting activity and a low F.Xase inhibitory action.
In addition, the Inventors discovered Q153-G4k (SEQ ID NO: 12),
Q354-z106 (SEQ ID NO: 13), Q360-G4k (SEQ ID NO: 14), Q360-z118 (SEQ
ID NO: 15), Q405-G4k (SEQ ID NO: 16), Q458-z106 (SEQ ID NO: 17),
Q460-z121 (SEQ ID NO: 18), Q499-z118 (SEQ ID NO: 19), and Q499-z121
(SEQ ID NO: 20) as the anti-human F.IXa antibody H chain, J232-G4h
(SEQ ID NO: 21), J259-z107 (SEQ ID NO: 22), J300-z107 (SEQ ID NO:
23), J327-z107 (SEQ ID NO: 24), J327-z119 (SEQ ID NO: 25),
J339-z119 (SEQ ID NO: 26), J346-z107 (SEQ ID NO: 27), J142-G4h (SEQ
ID NO: 170) as the anti-human F.X antibody H chains with increased
F.Xa generation-promoting activity, and L248-k (SEQ ID NO: 28),
L324-k (SEQ ID NO: 29), L377-k (SEQ ID NO: 31), L404-k (SEQ ID NO:
32), L408-k (SEQ ID NO: 34), and L180-k (SEQ ID NO: 171) as the
commonly shared antibody L chains.
[0604] Since these antibodies have very high F.Xa
generation-promoting activities while having suppressed F.Xase
inhibitory actions, they may have very useful properties for
patients maintaining an F.VIII function and patients receiving
treatment with F.VIII formulations. Since antibodies generally have
long half-lives, and can be administered subcutaneously, these
bispecific antibodies may be of great value to hemophilia A
patients, when compared to existing replacement therapy by
intravenous administration of existing F. VIII formulations for
hemophilia A.
[0605] Sequence comparisons of the variable regions of each of the
chains used in Example 1 and Example 2 are shown in FIGS. 6A to D.
For example, to enhance the F.Xa generation-promoting activity of a
bispecific antibody, the following amino acids were found to be
important: in the anti-human F.IXa antibody H chain, isoleucine at
position 34, asparagine, glutamine, or serine at position 35,
serine at position 49, arginine at position 61, glutamic acid at
position 62, serine or threonine at position 96, lysine or arginine
at position 98, serine or glutamic acid at position 99,
phenylalanine or tyrosine at position 100, glycine at position
100b, tyrosine at position 102, and such; in the anti-human F.X
antibody H chain, aspartic acid at position 35, arginine at
position 53, lysine at position 73, glycine at position 76, lysine
or arginine at position 96, tyrosine at position 98, tyrosine at
position 100, histidine at position 100a, and such; in the commonly
shared antibody L chain, lysine or arginine at position 27,
glutamic acid at position 30, arginine at position 31, glutamine at
position 32, arginine or glutamine at position 50, serine at
position 52, arginine at position 53, lysine at position 54,
glutamic acid at position 55, serine at position 92, serine at
position 93, proline at position 94, proline at position 95, and
such (the variable region amino acids are numbered by Kabat
numbering (Kabat EA et al. 1991. Sequences of Proteins of
Immunological Interest. NIH)).
INDUSTRIAL APPLICABILITY
[0606] The present invention provides multispecific antigen-binding
molecules having a high activity of functionally substituting for
F.VIII, which are antibodies that recognize both an enzyme and its
substrate. Furthermore, the present invention provides
multispecific antigen-binding molecules with a high activity of
functionally substituting for F. VIII and a low F.Xase inhibitory
action, which are antibodies that recognize both an enzyme and its
substrate.
[0607] Since humanized antibodies may generally have high stability
in blood and low immunogenicity, multispecific antibodies of the
present invention may be very promising as pharmaceuticals.
Sequence CWU 1
1
1791448PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 1Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Tyr Tyr 20 25 30Asp Met Ala Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ser Ile Ser Pro Ser Gly Gly
Ser Thr Tyr Tyr Arg His Ser Val 50 55 60Lys Gly Arg Phe Thr Val Ser
Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg Ala
Gly His Asn Leu Gly Ala Gly Trp Tyr Phe Asp Phe 100 105 110Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120
125Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser
130 135 140Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val145 150 155 160Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe 165 170 175Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val 180 185 190Thr Val Pro Ser Ser Ser Leu
Gly Thr Lys Thr Tyr Thr Cys Asn Val 195 200 205Asp His Lys Pro Ser
Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys 210 215 220Tyr Gly Pro
Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly225 230 235
240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
Gln Glu 260 265 270Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His 275 280 285Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Phe Asn Ser Thr Tyr Arg 290 295 300Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys305 310 315 320Glu Tyr Lys Cys Lys
Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu 325 330 335Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys 340 345 350Thr
Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360
365Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val385 390 395 400Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Arg Leu Thr Val Asp 405 410 415Lys Ser Arg Trp Gln Glu Gly Asn Val
Phe Ser Cys Ser Val Met His 420 425 430Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Leu 435 440 4452448PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
2Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr
Tyr 20 25 30Asp Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ala Ser Ile Ser Pro Ser Gly Gly Ser Thr Tyr Tyr Arg
Arg Ser Val 50 55 60Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys
Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg Ala Gly His Asn Leu Gly
Ala Gly Trp Tyr Phe Asp Phe 100 105 110Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125Pro Ser Val Phe Pro
Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 130 135 140Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val145 150 155
160Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
165 170 175Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val 180 185 190Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr
Thr Cys Asn Val 195 200 205Asp His Lys Pro Ser Asn Thr Lys Val Asp
Lys Arg Val Glu Ser Lys 210 215 220Tyr Gly Pro Pro Cys Pro Pro Cys
Pro Ala Pro Glu Phe Leu Gly Gly225 230 235 240Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu 260 265 270Asp
Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280
285Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg
290 295 300Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys305 310 315 320Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
Pro Ser Ser Ile Glu 325 330 335Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr 340 345 350Thr Leu Pro Pro Ser Gln Lys
Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val385 390 395
400Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp
405 410 415Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
Met His 420 425 430Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Leu 435 440 4453448PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 3Gln Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Tyr 20 25 30Asp Met Ala Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ser Ile
Ser Pro Ser Gly Gly Ser Thr Tyr Tyr Arg Arg Ser Val 50 55 60Lys Gly
Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Arg Ala Gly His Asn Phe Gly Ala Gly Trp Tyr Phe Asp
Phe 100 105 110Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser
Thr Lys Gly 115 120 125Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg
Ser Thr Ser Glu Ser 130 135 140Thr Ala Ala Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val145 150 155 160Thr Val Ser Trp Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175Pro Ala Val Leu
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190Thr Val
Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val 195 200
205Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys
210 215 220Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu
Gly Gly225 230 235 240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile 245 250 255Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser Gln Glu 260 265 270Asp Pro Glu Val Gln Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His 275 280 285Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg 290 295 300Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys305 310 315
320Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu
325 330 335Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr 340 345 350Thr Leu Pro Pro Ser Gln Lys Glu Met Thr Lys Asn
Gln Val Ser Leu 355 360 365Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp 370 375 380Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val385 390 395 400Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp 405 410 415Lys Ser Arg
Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu 435 440
4454444PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 4Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu
Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Asp Asn 20 25 30Asn Met Asp Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Asp Ile Asn Thr Arg Ser Gly
Gly Ser Ile Tyr Asn Glu Glu Phe 50 55 60Gln Asp Arg Val Ile Met Thr
Val Asp Lys Ser Thr Gly Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr His Cys 85 90 95Ala Arg Arg Lys
Ser Arg Gly Tyr His Leu Asp Glu Trp Gly Glu Gly 100 105 110Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120
125Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu
130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro Ala Val Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr Val Pro Ser 180 185 190Ser Ser Leu Gly Thr Lys Thr
Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205Ser Asn Thr Lys Val
Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro 210 215 220Cys Pro Pro
Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe225 230 235
240Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro
Glu Val 260 265 270Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr 275 280 285Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr
Tyr Arg Val Val Ser Val 290 295 300Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys305 310 315 320Lys Val Ser Asn Lys
Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 325 330 335Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350Ser
Gln Cys Glu Met Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val 355 360
365Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser Asp385 390 395 400Gly Ser Phe Phe Leu Val Ser Arg Leu Thr Val
Asp Lys Ser Arg Trp 405 410 415Gln Glu Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu His 420 425 430Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Leu 435 4405444PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 5Gln Val Gln Leu Val Gln
Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Asn 20 25 30Asn Met His Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Asp Ile
Asn Thr Arg Ser Gly Gly Ser Ile Tyr Asn Glu Glu Phe 50 55 60Gln Asp
Arg Val Ile Met Thr Val Asp Lys Ser Thr Asp Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr His Cys
85 90 95Ala Arg Arg Lys Ser Tyr Gly Tyr His Leu Asp Glu Trp Gly Glu
Gly 100 105 110Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe 115 120 125Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
Ser Thr Ala Ala Leu 130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Ser Ser
Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200
205Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro
210 215 220Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser
Val Phe225 230 235 240Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro 245 250 255Glu Val Thr Cys Val Val Val Asp Val
Ser Gln Glu Asp Pro Glu Val 260 265 270Gln Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr 275 280 285Lys Pro Arg Glu Glu
Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys305 310 315
320Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser
325 330 335Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro 340 345 350Ser Gln Cys Glu Met Thr Lys Asn Gln Val Ser Leu
Ser Cys Ala Val 355 360 365Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly 370 375 380Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp385 390 395 400Gly Ser Phe Phe Leu
Val Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 405 410 415Gln Glu Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu 435
4406444PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 6Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu
Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Asp Asn 20 25 30Asn Met Asp Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Asp Ile Asn Thr Arg Ser Gly
Gly Ser Ile Tyr Asn Glu Glu Phe 50 55 60Gln Asp Arg Val Ile Met Thr
Val Asp Lys Ser Thr Asp Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr His Cys 85 90 95Ala Arg Arg Lys
Ser Tyr Gly Asn His Leu Asp Glu Trp Gly Glu Gly 100 105 110Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120
125Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu
130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro Ala Val Leu
165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser 180 185 190Ser Ser Leu Gly Thr Gln Thr Tyr Thr Cys Asn Val
Asp His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Arg Val Glu
Ser Lys Tyr Gly Pro Pro 210 215 220Cys Pro Pro Cys Pro Ala Pro Glu
Phe Leu Gly Gly Pro Ser Val Phe225 230 235 240Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255Glu Val Thr
Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 260 265 270Gln
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280
285Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys305 310 315 320Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile
Glu Lys Thr Ile Ser 325 330 335Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro 340 345 350Ser Gln Glu Glu Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp385 390 395
400Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
Leu His 420 425 430Asn Arg Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro
435 4407444PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 7Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu
Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Asp Asn 20 25 30Asn Met Asp Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Asp Ile Asn Thr Lys Ser Gly
Gly Ser Ile Tyr Asn Glu Glu Phe 50 55 60Gln Asp Arg Val Ile Met Thr
Val Asp Lys Ser Thr Asp Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Thr Tyr His Cys 85 90 95Ala Arg Arg Gln
Ser Tyr Gly Tyr His Leu Asp Glu Trp Gly Glu Gly 100 105 110Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120
125Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu
130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro Ala Val Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr Val Pro Ser 180 185 190Ser Ser Leu Gly Thr Gln Thr
Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205Ser Asn Thr Lys Val
Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro 210 215 220Cys Pro Pro
Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe225 230 235
240Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro
Glu Val 260 265 270Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr 275 280 285Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val 290 295 300Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys305 310 315 320Lys Val Ser Asn Lys
Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 325 330 335Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350Ser
Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360
365Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser Asp385 390 395 400Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp 405 410 415Gln Glu Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu His 420 425 430Asn Arg Tyr Thr Gln Glu Ser
Leu Ser Leu Ser Pro 435 4408213PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 8Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Lys Ala Ser Gln Asn Ile Tyr Lys Asn 20 25 30Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala
Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Ser Gly 50 55 60Ser Arg
Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Leu Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Gly Leu Thr
85 90 95Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
Pro 100 105 110Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
Ser Gly Thr 115 120 125Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
Pro Arg Glu Ala Lys 130 135 140Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser Gly Asn Ser Gln Glu145 150 155 160Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200
205Asn Arg Gly Glu Cys 2109214PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 9Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Lys Ala Ser Gln Asn Ile Tyr Lys Asn 20 25 30Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala
Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Ser Gly 50 55 60Ser Arg
Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Leu Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Pro Pro Leu
85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala
Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200
205Phe Asn Arg Gly Glu Cys 21010448PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
10Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr
Tyr 20 25 30Asp Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ala Ser Ile Ser Pro Ser Gly Gly Ser Thr Tyr Tyr Arg
Arg Ser Val 50 55 60Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys
Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg Ala Gly His Asn Phe Gly
Ala Gly Trp Tyr Phe Asp Phe 100 105 110Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125Pro Ser Val Phe Pro
Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 130 135 140Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val145 150 155
160Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
165 170 175Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val 180 185 190Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr
Thr Cys Asn Val 195 200 205Asp His Lys Pro Ser Asn Thr Lys Val Asp
Lys Arg Val Glu Ser Lys 210 215 220Tyr Gly Pro Pro Cys Pro Pro Cys
Pro Ala Pro Glu Phe Leu Gly Gly225 230 235 240Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu 260 265 270Asp
Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280
285Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg
290 295 300Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys305 310 315 320Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
Pro Ser Ser Ile Glu 325 330 335Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr 340 345 350Thr Leu Pro Pro Ser Gln Lys
Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val385 390 395
400Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp
405 410 415Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
Met His 420 425 430Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Leu 435 440 44511448PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 11Gln Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Tyr 20 25 30Asp Met Ala
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ser
Ile Ser Pro Ser Gly Gly Ser Thr Tyr Tyr Arg Arg Ser Val 50 55 60Lys
Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Arg Ala Gly His Asn Tyr Gly Ala Gly Trp Tyr Phe Asp
Tyr 100 105 110Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser
Thr Lys Gly 115 120 125Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg
Ser Thr Ser Glu Ser 130 135 140Thr Ala Ala Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val145 150 155 160Thr Val Ser Trp Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175Pro Ala Val Leu
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190Thr Val
Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val 195 200
205Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys
210 215 220Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu
Gly Gly225 230 235 240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile 245 250 255Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser Gln Glu 260 265 270Asp Pro Glu Val Gln Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His 275 280 285Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg 290 295 300Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys305 310 315
320Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu
325 330 335Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Cys 340 345 350Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn
Gln Val Ser Leu 355 360 365Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp 370 375 380Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val385 390 395 400Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp 405 410 415Lys Ser Arg
Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu 435 440
44512448PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 12Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Tyr Tyr 20 25 30Asp Ile Asn Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ser Ile Ser Pro Ser Gly Gly
Ser Thr Tyr Tyr Arg Arg Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Thr Arg Ala
Gly His Asn Tyr Gly Ala Gly Trp Tyr Phe Asp Tyr 100 105 110Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120
125Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser
130 135 140Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val145 150 155 160Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe 165 170 175Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val 180 185 190Thr Val Pro Ser Ser Ser Leu
Gly Thr Lys Thr Tyr Thr Cys Asn Val 195 200 205Asp His Lys Pro Ser
Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys 210 215 220Tyr Gly Pro
Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly225 230 235
240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
Gln Glu 260 265 270Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His 275 280 285Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Phe Asn Ser Thr Tyr Arg 290 295 300Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys305 310 315 320Glu Tyr Lys Cys Lys
Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu 325 330 335Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys 340 345 350Thr
Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp 370 375 380Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val385 390 395 400Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Arg Leu Thr Val Asp 405 410 415Lys Ser Arg Trp Gln Glu Gly
Asn Val Phe Ser Cys Ser Val Met His 420 425 430Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu 435 440
44513448PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 13Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Tyr Tyr 20 25 30Asp Ile Asn Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Ser Pro Ser Gly Gln
Ser Thr Tyr Tyr Arg Arg Glu Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg Ser
Gly His Asn Tyr Gly Gly Gly Trp Tyr Phe Asp Tyr 100 105 110Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120
125Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser
130 135 140Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val145 150 155 160Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe 165 170 175Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val 180 185 190Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Thr Cys Asn Val 195 200 205Asp His Lys Pro Ser
Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys 210 215 220Tyr Gly Pro
Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly225 230 235
240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
Gln Glu 260 265 270Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His 275 280 285Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg 290 295 300Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys305 310 315 320Glu Tyr Lys Cys Lys
Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu 325 330 335Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350Thr
Leu Pro Pro Ser Gln Lys Glu Met Thr Lys Asn Gln Val Ser Leu 355 360
365Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val385 390 395 400Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp 405 410 415Lys Ser Arg Trp Gln Glu Gly Asn Val
Phe Ser Cys Ser Val Met His 420 425 430Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Leu 435 440 44514448PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
14Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr
Tyr 20 25 30Asp Ile Gln Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ser Ser Ile Ser Pro Ser Gly Gln Ser Thr Tyr Tyr Arg
Arg Glu Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg Ser Gly His Asn Tyr Gly
Gly Gly Trp Tyr Phe Asp Tyr 100 105 110Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125Pro Ser Val Phe Pro
Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 130 135 140Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val145 150 155
160Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
165 170 175Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val 180 185 190Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr
Thr Cys Asn Val 195 200 205Asp His Lys Pro Ser Asn Thr Lys Val Asp
Lys Arg Val Glu Ser Lys 210 215 220Tyr Gly Pro Pro Cys Pro Pro Cys
Pro Ala Pro Glu Phe Leu Gly Gly225 230 235 240Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu 260 265 270Asp
Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280
285Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg
290 295 300Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys305 310 315 320Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
Pro Ser Ser Ile Glu 325 330 335Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Cys 340 345 350Thr Leu Pro Pro Ser Gln Glu
Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365Trp Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val385 390 395
400Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp
405 410 415Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
Met His 420 425 430Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Leu 435 440 44515448PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 15Gln Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Tyr 20 25 30Asp Ile Gln
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ser
Ile Ser Pro Ser Gly Gln Ser Thr Tyr Tyr Arg Arg Glu Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Arg Ser Gly His Asn Tyr Gly Gly Gly Trp Tyr Phe Asp
Tyr 100 105 110Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser
Thr Lys Gly 115 120 125Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg
Ser Thr Ser Glu Ser 130 135 140Thr Ala Ala Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val145 150 155 160Thr Val Ser Trp Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175Pro Ala Val Leu
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190Thr Val
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Thr Cys Asn Val 195 200
205Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys
210 215 220Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu
Gly Gly225 230 235 240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile 245 250 255Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser Gln Glu 260 265 270Asp Pro Glu Val Gln Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His 275 280 285Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys305 310 315
320Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu
325 330 335Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr 340 345 350Thr Leu Pro Pro Ser Gln Lys Glu Met Thr Lys Asn
Gln Val Ser Leu 355 360 365Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp 370 375 380Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val385 390 395 400Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415Lys Ser Arg
Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440
44516448PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 16Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Tyr Tyr 20 25 30Asp Ile Ser Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Ser Pro Ser Gly Gln
Ser Thr Tyr Tyr Arg Arg Glu Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg Ser
Gly His Asn Phe Gly Gly Gly Trp Tyr Phe Asp Tyr 100 105 110Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120
125Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser
130 135 140Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val145 150 155 160Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe 165 170 175Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val 180 185 190Thr Val Pro Ser Ser Ser Leu
Gly Thr Lys Thr Tyr Thr Cys Asn Val 195 200 205Asp His Lys Pro Ser
Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys 210 215 220Tyr Gly Pro
Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly225 230 235
240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
Gln Glu 260 265 270Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His 275 280 285Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Phe Asn Ser Thr Tyr Arg 290 295 300Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys305 310 315 320Glu Tyr Lys Cys Lys
Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu 325 330 335Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys 340 345 350Thr
Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360
365Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val385 390 395 400Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Arg Leu Thr Val Asp 405 410 415Lys Ser Arg Trp Gln Glu Gly Asn Val
Phe Ser Cys Ser Val Met His 420 425 430Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Leu 435 440 44517448PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
17Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr
Tyr 20 25 30Asp Ile Gln Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ser Ser Ile Ser Pro Ser Gly Gln Ser Thr Tyr Tyr Arg
Arg Glu Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg Ser Gly Lys Ser Tyr Gly
Gly Gly Trp Tyr Phe Asp Tyr 100 105 110Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125Pro Ser Val Phe Pro
Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 130 135 140Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val145 150 155
160Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
165 170 175Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val 180 185 190Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
Thr Cys Asn Val 195 200 205Asp His Lys Pro Ser Asn Thr Lys Val Asp
Lys Arg Val Glu Ser Lys 210 215 220Tyr Gly Pro Pro Cys Pro Pro Cys
Pro Ala Pro Glu Phe Leu Gly Gly225 230 235 240Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu 260 265 270Asp
Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280
285Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys305 310 315 320Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
Pro Ser Ser Ile Glu 325 330 335Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr 340 345 350Thr Leu Pro Pro Ser Gln Lys
Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val385 390 395
400Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
Met His 420 425 430Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Leu 435 440 44518448PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 18Gln Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Tyr 20 25 30Asp Ile Gln
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ser
Ile Ser Pro Ser Gly Gln Ser Thr Tyr Tyr Arg Arg Glu
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Arg Ser Gly Arg Glu Tyr Gly Gly Gly
Trp Tyr Phe Asp Tyr 100 105 110Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly 115 120 125Pro Ser Val Phe Pro Leu Ala
Pro Cys Ser Arg Ser Thr Ser Glu Ser 130 135 140Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val145 150 155 160Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170
175Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
180 185 190Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Thr Cys
Asn Val 195 200 205Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg
Val Glu Ser Lys 210 215 220Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala
Pro Glu Phe Leu Gly Gly225 230 235 240Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser Gln Glu 260 265 270Asp Pro Glu
Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295
300Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys305 310 315 320Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
Ser Ser Ile Glu 325 330 335Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr 340 345 350Thr Leu Pro Pro Ser Gln Lys Glu
Met Thr Lys Asn Gln Val Ser Leu 355 360 365Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val385 390 395 400Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410
415Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430Glu Ala Leu His Asn Arg Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro 435 440 44519448PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 19Gln Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Tyr 20 25 30Asp Ile Gln Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ser Ile
Ser Pro Ser Gly Gln Ser Thr Tyr Tyr Arg Arg Glu Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Arg Thr Gly Arg Glu Tyr Gly Gly Gly Trp Tyr Phe Asp
Tyr 100 105 110Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser
Thr Lys Gly 115 120 125Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg
Ser Thr Ser Glu Ser 130 135 140Thr Ala Ala Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val145 150 155 160Thr Val Ser Trp Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175Pro Ala Val Leu
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190Thr Val
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Thr Cys Asn Val 195 200
205Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys
210 215 220Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu
Gly Gly225 230 235 240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile 245 250 255Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser Gln Glu 260 265 270Asp Pro Glu Val Gln Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His 275 280 285Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys305 310 315
320Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu
325 330 335Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr 340 345 350Thr Leu Pro Pro Ser Gln Lys Glu Met Thr Lys Asn
Gln Val Ser Leu 355 360 365Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp 370 375 380Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val385 390 395 400Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415Lys Ser Arg
Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440
44520448PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 20Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Tyr Tyr 20 25 30Asp Ile Gln Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Ser Pro Ser Gly Gln
Ser Thr Tyr Tyr Arg Arg Glu Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg Thr
Gly Arg Glu Tyr Gly Gly Gly Trp Tyr Phe Asp Tyr 100 105 110Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120
125Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser
130 135 140Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val145 150 155 160Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe 165 170 175Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val 180 185 190Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Thr Cys Asn Val 195 200 205Asp His Lys Pro Ser
Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys 210 215 220Tyr Gly Pro
Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly225 230 235
240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
Gln Glu 260 265 270Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His 275 280 285Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg 290 295 300Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys305 310 315 320Glu Tyr Lys Cys Lys
Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu 325 330 335Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350Thr
Leu Pro Pro Ser Gln Lys Glu Met Thr Lys Asn Gln Val Ser Leu 355 360
365Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val385 390 395 400Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp 405 410 415Lys Ser Arg Trp Gln Glu Gly Asn Val
Phe Ser Cys Ser Val Met His 420 425 430Glu Ala Leu His Asn Arg Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 44521444PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
21Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
Asn 20 25 30Asn Met Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Asp Ile Asn Thr Arg Ser Gly Gly Ser Ile Tyr Asn
Glu Glu Phe 50 55 60Gln Asp Arg Val Ile Met Thr Val Asp Lys Ser Thr
Gly Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg Lys Ser Tyr Gly Tyr His
Leu Asp Glu Trp Gly Glu Gly 100 105 110Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Cys
Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155
160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser 180 185 190Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val
Asp His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Arg Val Glu
Ser Lys Tyr Gly Pro Pro 210 215 220Cys Pro Pro Cys Pro Ala Pro Glu
Phe Leu Gly Gly Pro Ser Val Phe225 230 235 240Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255Glu Val Thr
Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 260 265 270Gln
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280
285Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys305 310 315 320Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile
Glu Lys Thr Ile Ser 325 330 335Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro 340 345 350Ser Gln Cys Glu Met Thr Lys
Asn Gln Val Ser Leu Ser Cys Ala Val 355 360 365Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp385 390 395
400Gly Ser Phe Phe Leu Val Ser Arg Leu Thr Val Asp Lys Ser Arg Trp
405 410 415Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
Leu His 420 425 430Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu
435 44022444PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 22Gln Val Gln Leu Val Gln Ser Gly
Ser Glu Leu Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Asp Asn 20 25 30Asn Met Asp Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Asp Ile Asn Thr
Arg Ser Gly Gly Ser Ile Tyr Asn Glu Glu Phe 50 55 60Gln Asp Arg Val
Ile Met Thr Val Asp Lys Ser Thr Gly Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr His Cys 85 90 95Ala
Arg Arg Lys Ser Tyr Gly Tyr Tyr Leu Asp Glu Trp Gly Glu Gly 100 105
110Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala
Ala Leu 130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala Val Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Ser Ser Leu Gly Thr
Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205Ser Asn Thr
Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro 210 215 220Cys
Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe225 230
235 240Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro 245 250 255Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp
Pro Glu Val 260 265 270Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr 275 280 285Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val 290 295 300Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys305 310 315 320Lys Val Ser Asn
Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 325 330 335Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345
350Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly 370 375 380Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp385 390 395 400Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp 405 410 415Gln Glu Gly Asn Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His 420 425 430Asn Arg Tyr Thr Gln
Glu Ser Leu Ser Leu Ser Pro 435 44023444PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
23Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
Asn 20 25 30Asn Met Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Asp Ile Asn Thr Arg Ser Gly Gly Ser Ile Tyr Asn
Glu Glu Phe 50 55 60Gln Asp Arg Val Ile Met Thr Val Asp Lys Ser Thr
Asp Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr His Cys 85 90 95Ala Arg Arg Lys Ser Tyr Gly Tyr Tyr
Leu Asp Glu Trp Gly Glu Gly 100 105 110Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Cys
Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155
160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser 180 185 190Ser Ser Leu Gly Thr Gln Thr Tyr Thr Cys Asn Val
Asp His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Arg Val Glu
Ser Lys Tyr Gly Pro Pro 210 215 220Cys Pro Pro Cys Pro Ala Pro Glu
Phe Leu
Gly Gly Pro Ser Val Phe225 230 235 240Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255Glu Val Thr Cys Val
Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 260 265 270Gln Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295
300Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys305 310 315 320Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu
Lys Thr Ile Ser 325 330 335Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro 340 345 350Ser Gln Glu Glu Met Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val 355 360 365Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp385 390 395 400Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410
415Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430Asn Arg Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435
44024444PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 24Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu
Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Asp Asn 20 25 30Asn Met Asp Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Asp Ile Asn Thr Arg Ser Gly
Gly Ser Ile Tyr Asn Glu Glu Phe 50 55 60Gln Asp Arg Val Ile Met Thr
Val Asp Lys Ser Thr Asp Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Thr Tyr His Cys 85 90 95Ala Arg Arg Lys
Ser Tyr Gly Tyr Tyr Leu Asp Glu Trp Gly Glu Gly 100 105 110Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120
125Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu
130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro Ala Val Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr Val Pro Ser 180 185 190Ser Ser Leu Gly Thr Gln Thr
Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205Ser Asn Thr Lys Val
Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro 210 215 220Cys Pro Pro
Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe225 230 235
240Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro
Glu Val 260 265 270Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr 275 280 285Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val 290 295 300Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys305 310 315 320Lys Val Ser Asn Lys
Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 325 330 335Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350Ser
Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360
365Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser Asp385 390 395 400Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp 405 410 415Gln Glu Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu His 420 425 430Asn Arg Tyr Thr Gln Glu Ser
Leu Ser Leu Ser Pro 435 44025444PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 25Gln Val Gln Leu Val
Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Asn 20 25 30Asn Met Asp
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Asp
Ile Asn Thr Arg Ser Gly Gly Ser Ile Tyr Asn Glu Glu Phe 50 55 60Gln
Asp Arg Val Ile Met Thr Val Asp Lys Ser Thr Asp Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr His Cys
85 90 95Ala Arg Arg Lys Ser Tyr Gly Tyr Tyr Leu Asp Glu Trp Gly Glu
Gly 100 105 110Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe 115 120 125Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
Ser Thr Ala Ala Leu 130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Ser Ser
Leu Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200
205Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro
210 215 220Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser
Val Phe225 230 235 240Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro 245 250 255Glu Val Thr Cys Val Val Val Asp Val
Ser Gln Glu Asp Pro Glu Val 260 265 270Gln Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr 275 280 285Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys305 310 315
320Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser
325 330 335Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro 340 345 350Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val 355 360 365Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly 370 375 380Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp385 390 395 400Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415Gln Glu Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430Asn
His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435
44026444PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 26Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu
Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Asp Asn 20 25 30Asn Met Asp Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Asp Ile Asn Thr Arg Ser Gly
Gly Ser Ile Tyr Asn Glu Glu Phe 50 55 60Gln Asp Arg Val Ile Met Thr
Val Asp Thr Ser Thr Asp Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Thr Tyr His Cys 85 90 95Ala Arg Arg Lys
Ser Tyr Gly Tyr His Leu Asp Glu Trp Gly Glu Gly 100 105 110Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120
125Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu
130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro Ala Val Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr Val Pro Ser 180 185 190Ser Ser Leu Gly Thr Gln Thr
Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205Ser Asn Thr Lys Val
Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro 210 215 220Cys Pro Pro
Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe225 230 235
240Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro
Glu Val 260 265 270Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr 275 280 285Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val 290 295 300Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys305 310 315 320Lys Val Ser Asn Lys
Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 325 330 335Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350Ser
Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360
365Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser Asp385 390 395 400Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp 405 410 415Gln Glu Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu His 420 425 430Asn His Tyr Thr Gln Glu Ser
Leu Ser Leu Ser Pro 435 44027444PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 27Gln Val Gln Leu Val
Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Asn 20 25 30Asn Met Asp
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Asp
Ile Asn Thr Arg Ser Gly Gly Ser Ile Tyr Asn Glu Glu Phe 50 55 60Gln
Asp Arg Val Ile Met Thr Val Asp Lys Ser Thr Asp Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr His Cys
85 90 95Ala Arg Arg Lys Ser Tyr Gly Tyr His Leu Asp Glu Trp Gly Glu
Gly 100 105 110Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe 115 120 125Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
Ser Thr Ala Ala Leu 130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Ser Ser
Leu Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200
205Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro
210 215 220Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser
Val Phe225 230 235 240Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro 245 250 255Glu Val Thr Cys Val Val Val Asp Val
Ser Gln Glu Asp Pro Glu Val 260 265 270Gln Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr 275 280 285Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys305 310 315
320Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser
325 330 335Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro 340 345 350Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val 355 360 365Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly 370 375 380Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp385 390 395 400Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415Gln Glu Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430Asn
Arg Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435
44028214PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 28Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser
Lys Asn Ile Glu Arg Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Arg Ala Ser Arg Lys Glu Ser
Gly Val Pro Asp Arg Phe Ser Gly 50 55 60Ser Arg Tyr Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Ile Ala Thr
Tyr Tyr Cys Gln Gln Tyr Ser Ser Pro Pro Leu 85 90 95Thr Phe Gly Gly
Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120
125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu
Cys 21029214PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 29Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Lys Ala Ser Arg Asn Ile Glu Arg Asn 20 25 30Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Gln Ala Pro Glu Leu Leu Ile 35 40 45Tyr Arg Ala Asp Arg
Lys Glu Ser Gly Val Pro Asp Arg Phe Ser Gly 50 55 60Ser Arg Tyr Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp
Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser Pro Pro Leu 85 90 95Thr
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105
110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200
205Phe Asn Arg Gly Glu Cys 21030214PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
30Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Arg Asn Ile Glu Arg
Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Glu Leu
Leu Ile 35 40 45Tyr Gln Ala Ser Arg Lys Glu Ser Gly Val Pro Asp Arg
Phe Ser Gly 50 55 60Ser Arg Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln
Tyr Ser Ser Pro Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155
160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys
21031214PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 31Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser
Arg Asn Ile Glu Arg Gln 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Gln Ala Pro Glu Leu Leu Ile 35 40 45Tyr Gln Ala Ser Arg Lys Glu Ser
Gly Val Pro Asp Arg Phe Ser Gly 50 55 60Ser Arg Tyr Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Ile Ala Thr
Tyr Tyr Cys Gln Gln Tyr Ser Ser Pro Pro Leu 85 90 95Thr Phe Gly Gly
Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120
125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu
Cys 21032214PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 32Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Lys Ala Ser Arg Asn Ile Glu Arg Gln 20 25 30Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Gln Ala Pro Glu Leu Leu Ile 35 40 45Tyr Gln Ala Ser Arg
Lys Glu Ser Gly Val Pro Asp Arg Phe Ser Gly 50 55 60Ser Arg Tyr Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp
Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Asp Pro Pro Leu 85 90 95Thr
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105
110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg
Gly Glu Cys 21033214PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 33Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Lys Ala Ser Arg Asn Ile Glu Arg Gln 20 25 30Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Gln Ala Pro Glu Leu Leu Ile 35 40 45Tyr Arg Ala Ser Arg
Lys Glu Ser Gly Val Pro Asp Arg Phe Ser Gly 50 55 60Ser Arg Tyr Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp
Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Asp Pro Pro Leu 85 90 95Thr
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105
110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg
Gly Glu Cys 21034214PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 34Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Lys Ala Ser Arg Asn Ile Glu Arg Gln 20 25 30Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Gln Ala Pro Glu Leu Leu Ile 35 40 45Tyr Arg Ala Asp Arg
Lys Glu Ser Gly Val Pro Asp Arg Phe Ser Gly 50 55 60Ser Arg Tyr Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp
Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Asp Pro Pro Leu 85 90 95Thr
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105
110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg
Gly Glu Cys 21035123PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 35Gln Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Tyr Tyr 20 25 30Asp Met Ala Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ser Ile Ser Pro
Ser Gly Gly Ser Thr Tyr Tyr Arg His Ser Val 50 55 60Lys Gly Arg Phe
Thr Val Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Arg Ala Gly His Asn Leu Gly Ala Gly Trp Tyr Phe Asp Phe 100 105
110Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
12036123PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 36Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Tyr Tyr 20 25 30Asp Met Ala Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ser Ile Ser Pro Ser Gly Gly
Ser Thr Tyr Tyr Arg Arg Ser Val 50 55 60Lys Gly Arg Phe Thr Val Ser
Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg Ala
Gly His Asn Leu Gly Ala Gly Trp Tyr Phe Asp Phe 100 105 110Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 115 12037123PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
37Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr
Tyr 20 25 30Asp Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ala Ser Ile Ser Pro Ser Gly Gly Ser Thr Tyr Tyr Arg
Arg Ser Val 50 55 60Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys
Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg Ala Gly His Asn Phe Gly
Ala Gly Trp Tyr Phe Asp Phe 100 105 110Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser 115 12038123PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 38Gln Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Tyr 20 25 30Asp Met Ala Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ser Ile
Ser Pro Ser Gly Gly Ser Thr Tyr Tyr Arg Arg Ser Val 50 55 60Lys Gly
Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Arg Ala Gly His Asn Tyr Gly Ala Gly Trp Tyr Phe Asp
Tyr 100 105 110Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
12039123PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 39Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Tyr Tyr 20 25 30Asp Ile Asn Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ser Ile Ser Pro Ser Gly Gly
Ser Thr Tyr Tyr Arg Arg Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Thr Arg Ala
Gly His Asn Tyr Gly Ala Gly Trp Tyr Phe Asp Tyr 100 105 110Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 115 12040123PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
40Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr
Tyr 20 25 30Asp Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ser Ser Ile Ser Pro Ser Gly Gln Ser Thr Tyr Tyr Arg
Arg Glu Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg Ser Gly His Asn Tyr Gly
Gly Gly Trp Tyr Phe Asp Tyr 100 105 110Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser 115 12041123PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 41Gln Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Tyr 20 25 30Asp Ile Gln Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ser Ile
Ser Pro Ser Gly Gln Ser Thr Tyr Tyr Arg Arg Glu Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Arg Ser Gly His Asn Tyr Gly Gly Gly Trp Tyr Phe Asp
Tyr 100 105 110Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
12042123PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 42Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Tyr Tyr 20 25 30Asp Ile Ser Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Ser Pro Ser Gly Gln
Ser Thr Tyr Tyr Arg Arg Glu Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg Ser
Gly His Asn Phe Gly Gly Gly Trp Tyr Phe Asp Tyr 100 105 110Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 115 12043123PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
43Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr
Tyr 20 25 30Asp Ile Gln Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ser Ser Ile Ser Pro Ser Gly Gln Ser Thr Tyr Tyr Arg
Arg Glu Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg Ser Gly Lys Ser Tyr Gly
Gly Gly Trp Tyr Phe Asp Tyr 100 105 110Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser 115 12044123PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 44Gln Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Tyr 20 25 30Asp Ile Gln Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ser Ile
Ser Pro Ser Gly Gln Ser Thr Tyr Tyr Arg Arg Glu Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Arg Ser Gly Arg Glu Tyr Gly Gly Gly Trp Tyr Phe Asp
Tyr 100
105 110Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
12045123PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 45Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Tyr Tyr 20 25 30Asp Ile Gln Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Ser Pro Ser Gly Gln
Ser Thr Tyr Tyr Arg Arg Glu Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg Thr
Gly Arg Glu Tyr Gly Gly Gly Trp Tyr Phe Asp Tyr 100 105 110Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 115 12046119PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
46Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
Asn 20 25 30Asn Met Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Asp Ile Asn Thr Arg Ser Gly Gly Ser Ile Tyr Asn
Glu Glu Phe 50 55 60Gln Asp Arg Val Ile Met Thr Val Asp Lys Ser Thr
Gly Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg Lys Ser Tyr Gly Tyr His
Leu Asp Glu Trp Gly Glu Gly 100 105 110Thr Leu Val Thr Val Ser Ser
11547119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 47Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu
Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Asp Asn 20 25 30Asn Met Asp Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Asp Ile Asn Thr Arg Ser Gly
Gly Ser Ile Tyr Asn Glu Glu Phe 50 55 60Gln Asp Arg Val Ile Met Thr
Val Asp Lys Ser Thr Gly Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr His Cys 85 90 95Ala Arg Arg Lys
Ser Tyr Gly Tyr Tyr Leu Asp Glu Trp Gly Glu Gly 100 105 110Thr Leu
Val Thr Val Ser Ser 11548119PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 48Gln Val Gln Leu Val Gln
Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Asn 20 25 30Asn Met Asp Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Asp Ile
Asn Thr Arg Ser Gly Gly Ser Ile Tyr Asn Glu Glu Phe 50 55 60Gln Asp
Arg Val Ile Met Thr Val Asp Lys Ser Thr Gly Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr His Cys
85 90 95Ala Arg Arg Lys Ser Arg Gly Tyr His Leu Asp Glu Trp Gly Glu
Gly 100 105 110Thr Leu Val Thr Val Ser Ser 11549119PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
49Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
Asn 20 25 30Asn Met Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Asp Ile Asn Thr Arg Ser Gly Gly Ser Ile Tyr Asn
Glu Glu Phe 50 55 60Gln Asp Arg Val Ile Met Thr Val Asp Lys Ser Thr
Asp Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr His Cys 85 90 95Ala Arg Arg Lys Ser Tyr Gly Tyr Tyr
Leu Asp Glu Trp Gly Glu Gly 100 105 110Thr Leu Val Thr Val Ser Ser
11550119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 50Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu
Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Asp Asn 20 25 30Asn Met His Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Asp Ile Asn Thr Arg Ser Gly
Gly Ser Ile Tyr Asn Glu Glu Phe 50 55 60Gln Asp Arg Val Ile Met Thr
Val Asp Lys Ser Thr Asp Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr His Cys 85 90 95Ala Arg Arg Lys
Ser Tyr Gly Tyr His Leu Asp Glu Trp Gly Glu Gly 100 105 110Thr Leu
Val Thr Val Ser Ser 11551119PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 51Gln Val Gln Leu Val Gln
Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Asn 20 25 30Asn Met Asp Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Asp Ile
Asn Thr Arg Ser Gly Gly Ser Ile Tyr Asn Glu Glu Phe 50 55 60Gln Asp
Arg Val Ile Met Thr Val Asp Lys Ser Thr Asp Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr His Cys
85 90 95Ala Arg Arg Lys Ser Tyr Gly Asn His Leu Asp Glu Trp Gly Glu
Gly 100 105 110Thr Leu Val Thr Val Ser Ser 11552119PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
52Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
Asn 20 25 30Asn Met Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Asp Ile Asn Thr Arg Ser Gly Gly Ser Ile Tyr Asn
Glu Glu Phe 50 55 60Gln Asp Arg Val Ile Met Thr Val Asp Lys Ser Thr
Asp Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Thr Tyr His Cys 85 90 95Ala Arg Arg Lys Ser Tyr Gly Tyr Tyr
Leu Asp Glu Trp Gly Glu Gly 100 105 110Thr Leu Val Thr Val Ser Ser
11553119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 53Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu
Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Asp Asn 20 25 30Asn Met Asp Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Asp Ile Asn Thr Arg Ser Gly
Gly Ser Ile Tyr Asn Glu Glu Phe 50 55 60Gln Asp Arg Val Ile Met Thr
Val Asp Thr Ser Thr Asp Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Thr Tyr His Cys 85 90 95Ala Arg Arg Lys
Ser Tyr Gly Tyr His Leu Asp Glu Trp Gly Glu Gly 100 105 110Thr Leu
Val Thr Val Ser Ser 11554119PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 54Gln Val Gln Leu Val Gln
Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Asn 20 25 30Asn Met Asp Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Asp Ile
Asn Thr Lys Ser Gly Gly Ser Ile Tyr Asn Glu Glu Phe 50 55 60Gln Asp
Arg Val Ile Met Thr Val Asp Lys Ser Thr Asp Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr His Cys
85 90 95Ala Arg Arg Gln Ser Tyr Gly Tyr His Leu Asp Glu Trp Gly Glu
Gly 100 105 110Thr Leu Val Thr Val Ser Ser 11555119PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
55Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
Asn 20 25 30Asn Met Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Asp Ile Asn Thr Arg Ser Gly Gly Ser Ile Tyr Asn
Glu Glu Phe 50 55 60Gln Asp Arg Val Ile Met Thr Val Asp Lys Ser Thr
Asp Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Thr Tyr His Cys 85 90 95Ala Arg Arg Lys Ser Tyr Gly Tyr His
Leu Asp Glu Trp Gly Glu Gly 100 105 110Thr Leu Val Thr Val Ser Ser
11556106PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 56Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser
Gln Asn Ile Tyr Lys Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Gln Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Tyr Arg Tyr Ser
Gly Val Pro Asp Arg Phe Ser Gly 50 55 60Ser Arg Tyr Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Leu Ala Thr
Tyr Tyr Cys Gln Gln Tyr Tyr Ser Gly Leu Thr 85 90 95Phe Gly Gly Gly
Thr Lys Val Glu Ile Lys 100 10557107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
57Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Ile Tyr Lys
Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg
Phe Ser Gly 50 55 60Ser Arg Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Leu Ala Thr Tyr Tyr Cys Gln Gln
Tyr Tyr Ser Pro Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys 100 10558107PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 58Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Lys Ala Ser Lys Asn Ile Glu Arg Asn 20 25 30Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Arg Ala Ser Arg
Lys Glu Ser Gly Val Pro Asp Arg Phe Ser Gly 50 55 60Ser Arg Tyr Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp
Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser Pro Pro Leu 85 90 95Thr
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 10559107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
59Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Arg Asn Ile Glu Arg
Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Glu Leu
Leu Ile 35 40 45Tyr Arg Ala Asp Arg Lys Glu Ser Gly Val Pro Asp Arg
Phe Ser Gly 50 55 60Ser Arg Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln
Tyr Ser Ser Pro Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys 100 10560107PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 60Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Lys Ala Ser Arg Asn Ile Glu Arg Asn 20 25 30Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Gln Ala Pro Glu Leu Leu Ile 35 40 45Tyr Gln Ala Ser Arg
Lys Glu Ser Gly Val Pro Asp Arg Phe Ser Gly 50 55 60Ser Arg Tyr Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp
Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser Pro Pro Leu 85 90 95Thr
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 10561107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
61Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Arg Asn Ile Glu Arg
Gln 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Glu Leu
Leu Ile 35 40 45Tyr Gln Ala Ser Arg Lys Glu Ser Gly Val Pro Asp Arg
Phe Ser Gly 50 55 60Ser Arg Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln
Tyr Ser Ser Pro Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys 100 10562107PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 62Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Lys Ala Ser Arg Asn Ile Glu Arg Gln 20 25 30Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Gln Ala Pro Glu Leu Leu Ile 35 40 45Tyr Gln Ala Ser Arg
Lys Glu Ser Gly Val Pro Asp Arg Phe Ser Gly 50 55 60Ser Arg Tyr Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp
Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Asp Pro Pro Leu 85 90 95Thr
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 10563107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
63Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Arg Asn Ile Glu Arg
Gln 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Glu Leu
Leu Ile 35 40 45Tyr Arg Ala Ser Arg Lys Glu Ser Gly Val Pro Asp Arg
Phe Ser Gly 50 55 60Ser Arg Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln
Tyr Ser Asp Pro Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys 100 10564107PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 64Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Lys Ala Ser Arg Asn Ile Glu Arg Gln 20 25 30Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Gln Ala Pro Glu Leu Leu Ile 35 40 45Tyr Arg Ala Asp Arg
Lys Glu Ser Gly Val Pro Asp Arg Phe Ser Gly 50 55
60Ser Arg Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Asp Pro Pro
Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100
10565325PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 65Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly Thr Lys Thr65 70 75 80Tyr Thr Cys Asn Val
Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg Val Glu Ser
Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110Glu Phe
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120
125Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
130 135 140Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr
Val Asp145 150 155 160Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Phe 165 170 175Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp 180 185 190Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205Pro Ser Ser Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220Glu Pro Gln
Val Cys Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys225 230 235
240Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
245 250 255Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys 260 265 270Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser 275 280 285Arg Leu Thr Val Asp Lys Ser Arg Trp Gln
Glu Gly Asn Val Phe Ser 290 295 300Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser305 310 315 320Leu Ser Leu Ser Leu
32566325PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 66Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly Thr Lys Thr65 70 75 80Tyr Thr Cys Asn Val
Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg Val Glu Ser
Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110Glu Phe
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120
125Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
130 135 140Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr
Val Asp145 150 155 160Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Phe 165 170 175Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp 180 185 190Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205Pro Ser Ser Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Gln Lys Glu Met Thr Lys225 230 235
240Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
245 250 255Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys 260 265 270Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser 275 280 285Arg Leu Thr Val Asp Lys Ser Arg Trp Gln
Glu Gly Asn Val Phe Ser 290 295 300Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser305 310 315 320Leu Ser Leu Ser Leu
32567325PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 67Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly Thr Lys Thr65 70 75 80Tyr Thr Cys Asn Val
Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg Val Glu Ser
Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro 100 105 110Glu Phe
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120
125Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
130 135 140Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr
Val Asp145 150 155 160Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Phe 165 170 175Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp 180 185 190Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205Pro Ser Ser Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Gln Lys Glu Met Thr Lys225 230 235
240Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
245 250 255Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys 260 265 270Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser 275 280 285Arg Leu Thr Val Asp Lys Ser Arg Trp Gln
Glu Gly Asn Val Phe Ser 290 295 300Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser305 310 315 320Leu Ser Leu Ser Leu
32568325PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 68Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Thr Cys Asn Val
Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg Val Glu Ser
Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110Glu Phe
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120
125Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
130 135 140Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr
Val Asp145 150 155 160Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr 165 170 175Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp 180 185 190Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205Pro Ser Ser Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Gln Lys Glu Met Thr Lys225 230 235
240Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
245 250 255Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys 260 265 270Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser 275 280 285Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Glu Gly Asn Val Phe Ser 290 295 300Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser305 310 315 320Leu Ser Leu Ser Leu
32569325PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 69Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Thr Cys Asn Val
Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg Val Glu Ser
Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110Glu Phe
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120
125Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
130 135 140Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr
Val Asp145 150 155 160Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr 165 170 175Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp 180 185 190Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205Pro Ser Ser Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Gln Lys Glu Met Thr Lys225 230 235
240Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
245 250 255Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys 260 265 270Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser 275 280 285Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Glu Gly Asn Val Phe Ser 290 295 300Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser305 310 315 320Leu Ser Leu Ser Pro
32570325PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 70Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Thr Cys Asn Val
Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg Val Glu Ser
Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110Glu Phe
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120
125Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
130 135 140Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr
Val Asp145 150 155 160Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr 165 170 175Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp 180 185 190Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205Pro Ser Ser Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Gln Lys Glu Met Thr Lys225 230 235
240Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
245 250 255Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys 260 265 270Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser 275 280 285Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Glu Gly Asn Val Phe Ser 290 295 300Cys Ser Val Met His Glu Ala Leu
His Asn Arg Tyr Thr Gln Lys Ser305 310 315 320Leu Ser Leu Ser Pro
32571325PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 71Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly Thr Lys Thr65 70 75 80Tyr Thr Cys Asn Val
Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg Val Glu Ser
Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110Glu Phe
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120
125Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
130 135 140Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr
Val Asp145 150 155 160Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Phe 165 170 175Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp 180 185 190Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205Pro Ser Ser Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Gln Cys Glu Met Thr Lys225 230 235
240Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp
245 250 255Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys 260 265 270Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Val Ser 275 280 285Arg Leu Thr Val Asp Lys Ser Arg Trp Gln
Glu Gly Asn Val Phe Ser 290 295 300Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser305 310 315 320Leu Ser Leu Ser Leu
32572325PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 72Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75
80Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala
Pro 100 105 110Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys 115 120 125Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val 130 135 140Asp Val Ser Gln Glu Asp Pro Glu Val
Gln Phe Asn Trp Tyr Val Asp145 150 155 160Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 165 170 175Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200
205Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
210 215 220Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met
Thr Lys225 230 235 240Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp 245 250 255Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys 260 265 270Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300Cys Ser Val
Met His Glu Ala Leu His Asn Arg Tyr Thr Gln Glu Ser305 310 315
320Leu Ser Leu Ser Pro 32573325PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 73Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Glu Ser
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75
80Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala
Pro 100 105 110Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys 115 120 125Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val 130 135 140Asp Val Ser Gln Glu Asp Pro Glu Val
Gln Phe Asn Trp Tyr Val Asp145 150 155 160Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 165 170 175Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200
205Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
210 215 220Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met
Thr Lys225 230 235 240Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp 245 250 255Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys 260 265 270Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Glu Ser305 310 315
320Leu Ser Leu Ser Pro 32574107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 74Arg Thr Val Ala Ala Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu1 5 10 15Gln Leu Lys Ser Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30Tyr Pro Arg Glu
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45Ser Gly Asn
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60Thr Tyr
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu65 70 75
80Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 95Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100
105755PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 75Tyr Tyr Asp Met Ala1 57617PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 76Ser
Ile Ser Pro Ser Gly Gly Ser Thr Tyr Tyr Arg His Ser Val Lys1 5 10
15Gly7714PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 77Arg Ala Gly His Asn Leu Gly Ala Gly Trp Tyr Phe
Asp Phe1 5 10785PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 78Tyr Tyr Asp Met Ala1
57917PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 79Ser Ile Ser Pro Ser Gly Gly Ser Thr Tyr Tyr Arg
Arg Ser Val Lys1 5 10 15Gly8014PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 80Arg Ala Gly His Asn Leu Gly
Ala Gly Trp Tyr Phe Asp Phe1 5 10815PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 81Tyr
Tyr Asp Met Ala1 58217PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 82Ser Ile Ser Pro Ser Gly Gly
Ser Thr Tyr Tyr Arg Arg Ser Val Lys1 5 10 15Gly8314PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 83Arg
Ala Gly His Asn Phe Gly Ala Gly Trp Tyr Phe Asp Phe1 5
10845PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 84Tyr Tyr Asp Met Ala1 58517PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 85Ser
Ile Ser Pro Ser Gly Gly Ser Thr Tyr Tyr Arg Arg Ser Val Lys1 5 10
15Gly8614PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 86Arg Ala Gly His Asn Tyr Gly Ala Gly Trp Tyr Phe
Asp Tyr1 5 10875PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 87Tyr Tyr Asp Ile Asn1
58817PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 88Ser Ile Ser Pro Ser Gly Gly Ser Thr Tyr Tyr Arg
Arg Ser Val Lys1 5 10 15Gly8914PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 89Arg Ala Gly His Asn Tyr Gly
Ala Gly Trp Tyr Phe Asp Tyr1 5 10905PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 90Tyr
Tyr Asp Ile Asn1 59117PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 91Ser Ile Ser Pro Ser Gly Gln
Ser Thr Tyr Tyr Arg Arg Glu Val Lys1 5 10 15Gly9214PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 92Arg
Ser Gly His Asn Tyr Gly Gly Gly Trp Tyr Phe Asp Tyr1 5
10935PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 93Tyr Tyr Asp Ile Gln1 59417PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 94Ser
Ile Ser Pro Ser Gly Gln Ser Thr Tyr Tyr Arg Arg Glu Val Lys1 5 10
15Gly9514PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 95Arg Ser Gly His Asn Tyr Gly Gly Gly Trp Tyr Phe
Asp Tyr1 5 10965PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 96Tyr Tyr Asp Ile Ser1
59717PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 97Ser Ile Ser Pro Ser Gly Gln Ser Thr Tyr Tyr Arg
Arg Glu Val Lys1 5 10 15Gly9814PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 98Arg Ser Gly His Asn Phe Gly
Gly Gly Trp Tyr Phe Asp Tyr1 5 10995PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 99Tyr
Tyr Asp Ile Gln1 510017PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 100Ser Ile Ser Pro Ser Gly
Gln Ser Thr Tyr Tyr Arg Arg Glu Val Lys1 5 10
15Gly10114PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 101Arg Ser Gly Lys Ser Tyr Gly Gly Gly Trp Tyr
Phe Asp Tyr1 5 101025PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 102Tyr Tyr Asp Ile Gln1
510317PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 103Ser Ile Ser Pro Ser Gly Gln Ser Thr Tyr Tyr
Arg Arg Glu Val Lys1 5 10 15Gly10414PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 104Arg
Ser Gly Arg Glu Tyr Gly Gly Gly Trp Tyr Phe Asp Tyr1 5
101055PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 105Tyr Tyr Asp Ile Gln1 510617PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 106Ser
Ile Ser Pro Ser Gly Gln Ser Thr Tyr Tyr Arg Arg Glu Val Lys1 5 10
15Gly10714PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 107Arg Thr Gly Arg Glu Tyr Gly Gly Gly Trp Tyr
Phe Asp Tyr1 5 101085PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 108Asp Asn Asn Met Asp1
510917PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 109Asp Ile Asn Thr Arg Ser Gly Gly Ser Ile Tyr
Asn Glu Glu Phe Gln1 5 10 15Asp11010PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 110Arg
Lys Ser Tyr Gly Tyr His Leu Asp Glu1 5 101115PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 111Asp
Asn Asn Met Asp1 511217PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 112Asp Ile Asn Thr Arg Ser
Gly Gly Ser Ile Tyr Asn Glu Glu Phe Gln1 5 10
15Asp11310PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 113Arg Lys Ser Tyr Gly Tyr Tyr Leu Asp Glu1 5
101145PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 114Asp Asn Asn Met Asp1 511517PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 115Asp
Ile Asn Thr Arg Ser Gly Gly Ser Ile Tyr Asn Glu Glu Phe Gln1 5 10
15Asp11610PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 116Arg Lys Ser Arg Gly Tyr His Leu Asp Glu1 5
101175PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 117Asp Asn Asn Met Asp1 511817PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 118Asp
Ile Asn Thr Arg Ser Gly Gly Ser Ile Tyr Asn Glu Glu Phe Gln1 5 10
15Asp11910PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 119Arg Lys Ser Tyr Gly Tyr Tyr Leu Asp Glu1 5
101205PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 120Asp Asn Asn Met His1 512117PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 121Asp
Ile Asn Thr Arg Ser Gly Gly Ser Ile Tyr Asn Glu Glu Phe Gln1 5 10
15Asp12210PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 122Arg Lys Ser Tyr Gly Tyr His Leu Asp Glu1 5
101235PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 123Asp Asn Asn Met Asp1 512417PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 124Asp
Ile Asn Thr Arg Ser Gly Gly Ser Ile Tyr Asn Glu Glu Phe Gln1 5 10
15Asp12510PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 125Arg Lys Ser Tyr Gly Asn His Leu Asp Glu1 5
101265PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 126Asp Asn Asn Met Asp1 512717PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 127Asp
Ile Asn Thr Arg Ser Gly Gly Ser Ile Tyr Asn Glu Glu Phe Gln1 5 10
15Asp12810PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 128Arg Lys Ser Tyr Gly Tyr Tyr Leu Asp Glu1 5
101295PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 129Asp Asn Asn Met Asp1 513017PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 130Asp
Ile Asn Thr Arg Ser Gly Gly Ser Ile Tyr Asn Glu Glu Phe Gln1 5 10
15Asp13110PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 131Arg Lys Ser Tyr Gly Tyr His Leu Asp Glu1 5
101325PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 132Asp Asn Asn Met Asp1 513317PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 133Asp
Ile Asn Thr Lys Ser Gly Gly Ser Ile Tyr Asn Glu Glu Phe Gln1 5 10
15Asp13410PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 134Arg Gln Ser Tyr Gly Tyr His Leu Asp Glu1 5
101355PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 135Asp Asn Asn Met Asp1 513617PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 136Asp
Ile Asn Thr Arg Ser Gly Gly Ser Ile Tyr Asn Glu Glu Phe Gln1 5 10
15Asp13710PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 137Arg Lys Ser Tyr Gly Tyr His Leu Asp Glu1 5
1013811PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 138Lys Ala Ser Gln Asn Ile Tyr Lys Asn Leu Ala1 5
101397PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 139Ser Ala Ser Tyr Arg Tyr Ser1
51408PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 140Gln Gln Tyr Tyr Ser Gly Leu Thr1
514111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 141Lys Ala Ser Gln Asn Ile Tyr Lys Asn Leu Ala1 5
101427PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 142Ser Ala Ser Tyr Arg Tyr Ser1
51439PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 143Gln Gln Tyr Tyr Ser Pro Pro Leu Thr1
514411PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 144Lys Ala Ser Lys Asn Ile Glu Arg Asn Leu Ala1 5
101457PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 145Arg Ala Ser Arg Lys Glu Ser1
51469PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 146Gln Gln Tyr Ser Ser Pro Pro Leu Thr1
514711PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 147Lys Ala Ser Arg Asn Ile Glu Arg Asn Leu Ala1 5
101487PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 148Arg Ala Asp Arg Lys Glu Ser1
51499PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 149Gln Gln Tyr Ser Ser Pro Pro Leu Thr1
515011PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 150Lys Ala Ser Arg Asn Ile Glu Arg Asn Leu Ala1 5
101517PRTArtificial SequenceDescription of Artificial Sequence
Synthetic
peptide 151Gln Ala Ser Arg Lys Glu Ser1 51529PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 152Gln
Gln Tyr Ser Ser Pro Pro Leu Thr1 515311PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 153Lys
Ala Ser Arg Asn Ile Glu Arg Gln Leu Ala1 5 101547PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 154Gln
Ala Ser Arg Lys Glu Ser1 51559PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 155Gln Gln Tyr Ser Ser Pro
Pro Leu Thr1 515611PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 156Lys Ala Ser Arg Asn Ile Glu Arg Gln
Leu Ala1 5 101577PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 157Gln Ala Ser Arg Lys Glu Ser1
51589PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 158Gln Gln Tyr Ser Asp Pro Pro Leu Thr1
515911PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 159Lys Ala Ser Arg Asn Ile Glu Arg Gln Leu Ala1 5
101607PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 160Arg Ala Ser Arg Lys Glu Ser1
51619PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 161Gln Gln Tyr Ser Asp Pro Pro Leu Thr1
516211PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 162Lys Ala Ser Arg Asn Ile Glu Arg Gln Leu Ala1 5
101637PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 163Arg Ala Asp Arg Lys Glu Ser1
51649PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 164Gln Gln Tyr Ser Asp Pro Pro Leu Thr1
5165118PRTHomo sapiens 165Glu Val Gln Leu Val Gln Ser Gly Ala Glu
Val Gln Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Tyr Met His Trp Val Arg Gln Ala
Pro Gly Glu Gly Leu Glu Trp Met 35 40 45Gly Tyr Ile Asn Pro Ser Ser
Gly Tyr Thr Lys Tyr Asn Arg Lys Phe 50 55 60Arg Asp Arg Val Thr Ile
Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly
Gly Gln Gly Tyr Tyr Leu Asp Tyr Trp Gly Glu Gly Thr 100 105 110Thr
Val Thr Val Ser Ser 115166119PRTHomo sapiens 166Glu Val Gln Leu Val
Gln Ser Gly Ala Gln Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Asn 20 25 30Asn Met Asp
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Asp
Ile Asn Thr Lys Ser Gly Gly Ser Ile Tyr Asn Gln Lys Phe 50 55 60Lys
Gly Arg Val Ile Met Thr Ile Asp Lys Ser Thr Gly Thr Ala Tyr65 70 75
80Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Ile Tyr Tyr Cys
85 90 95Ala Arg Arg Arg Ser Tyr Gly Tyr Tyr Phe Asp Tyr Trp Gly Arg
Gly 100 105 110Thr Leu Val Thr Val Ser Ser 115167106PRTHomo sapiens
167Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr
Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Ser Ala Ser Tyr Arg Ala Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Arg Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Leu Ala Thr Tyr Tyr Cys Gln Gln
Tyr Ser Asn Tyr Ile Thr 85 90 95Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys 100 105168326PRTHomo sapiens 168Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Glu Ser Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr65 70 75 80Tyr Thr
Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg
Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105
110Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
115 120 125Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val 130 135 140Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn
Trp Tyr Val Asp145 150 155 160Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Phe 165 170 175Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp 180 185 190Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205Pro Ser Ser
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys225 230
235 240Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp 245 250 255Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys 260 265 270Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser 275 280 285Arg Leu Thr Val Asp Lys Ser Arg Trp
Gln Glu Gly Asn Val Phe Ser 290 295 300Cys Ser Val Met His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser305 310 315 320Leu Ser Leu Ser
Leu Gly 325169107PRTHomo sapiens 169Arg Thr Val Ala Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu1 5 10 15Gln Leu Lys Ser Gly Thr Ala
Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30Tyr Pro Arg Glu Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45Ser Gly Asn Ser Gln
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60Thr Tyr Ser Leu
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu65 70 75 80Lys His
Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95Pro
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105170444PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
170Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
Asn 20 25 30Asn Met Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Asp Ile Asn Thr Arg Ser Gly Gly Ser Ile Tyr Asn
Glu Glu Phe 50 55 60Gln Asp Arg Val Thr Met Thr Ile Asp Lys Ser Thr
Gly Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg Arg Ser Tyr Gly Tyr Tyr
His Asp Glu Trp Gly Glu Gly 100 105 110Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Cys
Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155
160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser 180 185 190Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val
Asp His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Arg Val Glu
Ser Lys Tyr Gly Pro Pro 210 215 220Cys Pro Pro Cys Pro Ala Pro Glu
Phe Leu Gly Gly Pro Ser Val Phe225 230 235 240Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255Glu Val Thr
Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 260 265 270Gln
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280
285Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys305 310 315 320Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile
Glu Lys Thr Ile Ser 325 330 335Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro 340 345 350Ser Gln Cys Glu Met Thr Lys
Asn Gln Val Ser Leu Ser Cys Ala Val 355 360 365Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp385 390 395
400Gly Ser Phe Phe Leu Val Ser Arg Leu Thr Val Asp Lys Ser Arg Trp
405 410 415Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
Leu His 420 425 430Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu
435 440171214PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 171Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Lys Ala Ser Arg Asn Ile Glu Arg Asn 20 25 30Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Gln Ala Pro Glu Leu Leu Ile 35 40 45Tyr Ser Ala Ser Arg
Lys Glu Ser Gly Val Pro Asp Arg Phe Ser Gly 50 55 60Ser Arg Tyr Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp
Leu Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Pro Pro Leu 85 90 95Thr
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105
110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg
Gly Glu Cys 210172119PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 172Gln Val Gln Leu Val
Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Asn 20 25 30Asn Met Asp
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Asp
Ile Asn Thr Arg Ser Gly Gly Ser Ile Tyr Asn Glu Glu Phe 50 55 60Gln
Asp Arg Val Thr Met Thr Ile Asp Lys Ser Thr Gly Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Arg Arg Ser Tyr Gly Tyr Tyr His Asp Glu Trp Gly Glu
Gly 100 105 110Thr Leu Val Thr Val Ser Ser 115173107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
173Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Arg Asn Ile Glu Arg
Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Glu Leu
Leu Ile 35 40 45Tyr Ser Ala Ser Arg Lys Glu Ser Gly Val Pro Asp Arg
Phe Ser Gly 50 55 60Ser Arg Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Leu Ala Thr Tyr Tyr Cys Gln Gln
Tyr Tyr Ser Pro Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys 100 1051745PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 174Asp Asn Asn Met Asp1
517517PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 175Asp Ile Asn Thr Arg Ser Gly Gly Ser Ile Tyr
Asn Glu Glu Phe Gln1 5 10 15Asp17610PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 176Arg
Arg Ser Tyr Gly Tyr Tyr His Asp Glu1 5 1017711PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 177Lys
Ala Ser Arg Asn Ile Glu Arg Asn Leu Ala1 5 101787PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 178Ser
Ala Ser Arg Lys Glu Ser1 51799PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 179Gln Gln Tyr Tyr Ser Pro
Pro Leu Thr1 5
* * * * *
References