U.S. patent application number 14/713416 was filed with the patent office on 2015-09-17 for anti-glypican 3 antibody.
The applicant listed for this patent is Chugai Seiyaku Kabushiki Kaisha. Invention is credited to Tomoyuki Igawa, Takahiro Ishiguro, Shigeto Kawai, Yasuko Kinoshita, Hiroko Konishi, Kiyotaka Nakano, Jun-Ichi Nezu, Izumi Sugo, Megumi Tanaka, Hiroyuki Tsunoda, Takeshi Yoshino.
Application Number | 20150259417 14/713416 |
Document ID | / |
Family ID | 35784028 |
Filed Date | 2015-09-17 |
United States Patent
Application |
20150259417 |
Kind Code |
A1 |
Nakano; Kiyotaka ; et
al. |
September 17, 2015 |
Anti-Glypican 3 Antibody
Abstract
An antibody capable of binding to a specific region of glypican
3, as well as a humanized antibody created based on that antibody
are disclosed. The anti-GPC3 antibody of the invention has a higher
ADCC activity and CDC activity compared with those of a
conventional antibody. The antibody of the present invention is
useful as a cell growth inhibitor, an anticancer agent and an agent
for diagnosis of cancers.
Inventors: |
Nakano; Kiyotaka; (Ibaraki,
JP) ; Yoshino; Takeshi; (Ibaraki, JP) ; Nezu;
Jun-Ichi; (Ibaraki, JP) ; Tsunoda; Hiroyuki;
(Ibaraki, JP) ; Igawa; Tomoyuki; (Shizuoka,
JP) ; Konishi; Hiroko; (Shizuoka, JP) ;
Tanaka; Megumi; (Shizuoka, JP) ; Sugo; Izumi;
(Shizuoka, JP) ; Kawai; Shigeto; (Kanagawa,
JP) ; Ishiguro; Takahiro; (Kanagawa, JP) ;
Kinoshita; Yasuko; (Kanagawa, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Chugai Seiyaku Kabushiki Kaisha |
Tokyo |
|
JP |
|
|
Family ID: |
35784028 |
Appl. No.: |
14/713416 |
Filed: |
May 15, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12797349 |
Jun 9, 2010 |
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14713416 |
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10583795 |
Jun 21, 2006 |
7919086 |
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PCT/JP2005/013103 |
Jul 8, 2005 |
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12797349 |
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Current U.S.
Class: |
530/324 ;
435/252.3; 435/252.31; 435/252.33; 435/254.11; 435/254.2;
435/254.21; 435/254.3; 435/320.1; 435/325; 435/348; 435/352;
435/357; 435/358; 435/364; 435/365; 435/367; 435/419; 530/328;
530/330; 536/23.53 |
Current CPC
Class: |
C07K 2317/565 20130101;
C07K 2317/92 20130101; C07K 2317/34 20130101; A61P 43/00 20180101;
C07K 14/47 20130101; A61P 1/16 20180101; C07K 2317/732 20130101;
C07K 16/303 20130101; C07K 16/28 20130101; C07K 2317/734 20130101;
A61P 1/00 20180101; C07K 7/06 20130101; A61P 35/00 20180101; C07K
2317/24 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; C07K 7/06 20060101 C07K007/06; C07K 14/47 20060101
C07K014/47 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 9, 2004 |
JP |
2004-203637 |
Claims
1.-32. (canceled)
33. Nucleic acid comprising a sequence that encodes a polypeptide
comprising an anti-glypican 3 antibody heavy chain variable region
comprising the amino acid sequence of SEQ ID NO: 84, 85, 86, 87,
88, 89, or 90.
34. The nucleic acid of claim 33, wherein the nucleic acid
comprises a sequence that encodes a polypeptide comprising an
anti-glypican 3 antibody light chain variable region comprising the
amino acid sequence of SEQ ID NO: 92, 191, 192, 193, 194, 195, 196,
197, 198, 199, 200, 201, 202, 203, 204, or 205.
35. An expression vector comprising the nucleic acid of claim
33.
36. A host cell comprising the nucleic acid of claim 33.
37. Nucleic acid comprising a sequence that encodes a polypeptide
comprising an anti-glypican 3 antibody light chain variable region
comprising the amino acid sequence of SEQ ID NO: 92, 191, 192, 193,
194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, or 205.
38. An expression vector comprising the nucleic acid of claim
37.
39. A host cell comprising the nucleic acid of claim 37.
40. A set of one or more nucleic acids that together encode an
anti-glypican 3 antibody that comprises: (a) an antibody heavy
chain variable region having CDRs 1, 2, and 3 comprising the amino
acid sequences of SEQ ID NOs: 123, 124, 125, respectively, or (b)
an antibody light chain variable region having a CDR2 comprising
the sequence of SEQ ID NO: 144, a CDR3 comprising the sequence of
SEQ ID NO: 158, and a CDR1 comprising the sequence of SEQ ID NO:
143, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185,
186, 187, or 188.
41. An expression vector that comprises the set of nucleic acids of
claim 40.
42. A set of expression vectors that together comprise the set of
nucleic acids of claim 40.
43. A host cell comprising the set of nucleic acids of claim
40.
44. The set of nucleic acids of claim 40, wherein the antibody is a
humanized antibody.
45. An expression vector that comprises the set of nucleic acids of
claim 44.
46. A set of expression vectors that together comprise the set of
nucleic acids of claim 44.
47. A host cell comprising the set of nucleic acids of claim
44.
48. A peptide comprising the amino acid sequence of amino acid
residues 524-563, amino acid residues 537-563, amino acid residues
544-553, or amino acids residues 546-551 of glypican 3.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to an anti-glypican 3
antibody, a cell growth inhibitor and an anticancer agent
containing the antibody as an active ingredient.
[0003] 2. Description of Related Art
[0004] Glypican 3 (GPC3) is one of the glypican family of heparan
sulfate proteoglycans that are present on cell surfaces. It is
suggested that GPC3 may be involved in cell division in development
or cancer cell growth, however, its function has not been well
elucidated yet.
[0005] It has been found that a certain type of antibody binding to
GPC3 has a cell growth-inhibiting activity via an
antibody-dependent cell-mediated cytotoxicity (ADCC) activity and a
complement-dependent cytotoxicity (CDC) activity (International
Patent Application WO 2003/000883). In addition, it has been
suggested that GPC3 is cleaved in vivo and secreted into blood as a
secreted form of GPC3, and the diagnosis of cancers may be possible
by using an antibody capable of detecting the secreted form of GPC3
(International Patent Applications WO 2004/022739, WO 03/100429 and
WO 2004/018667).
[0006] When developing an anticancer agent based on the
cytotoxicity activity of an antibody, it is preferred that the
antibody to be used has high ADCC activity or CDC activity.
Accordingly, an anti-GPC3 antibody having a high cytotoxicity
activity has been desired as an antibody recognizing GPC3.
[0007] An object of the present invention is to provide an
anti-GPC3 antibody having a higher ADCC activity and CDC activity
compared with those of a conventional antibody.
SUMMARY OF THE INVENTION
[0008] The present inventors have succeeded in obtaining an
antibody having a higher cytotoxicity activity compared with that
of a conventional anti-glypican 3 antibody. Furthermore, they
analyzed epitopes for such an antibody and succeeded in determining
the regions on GPC 3 recognized by the antibody with a high
cytotoxicity activity.
[0009] In one aspect, the present invention provides an antibody
comprising a heavy chain variable region having CDRs 1, 2 and 3 of
any one of (1)-(12):
(1) CDR1 comprising the amino acid sequence set forth in SEQ ID NO:
123, CDR2 comprising the amino acid sequence set forth in SEQ ID
NO: 124, and CDR3 comprising the amino acid sequence set forth in
SEQ ID NO: 125; (2) CDR1 comprising the amino acid sequence set
forth in SEQ ID NO: 109, CDR2 comprising the amino acid sequence
set forth in SEQ ID NO: 110, and CDR3 comprising the amino acid
sequence set forth in SEQ ID NO: 111; (3) CDR1 comprising the amino
acid sequence set forth in SEQ ID NO: 106, CDR2 comprising the
amino acid sequence set forth in SEQ ID NO: 107, and CDR3
comprising the amino acid sequence set forth in SEQ ID NO: 108; (4)
CDR1 comprising the amino acid sequence set forth in SEQ ID NO:
132, CDR2 comprising the amino acid sequence set forth in SEQ ID
NO: 133, and CDR3 comprising the amino acid sequence set forth in
SEQ ID NO: 134; (5) CDR1 comprising the amino acid sequence set
forth in SEQ ID NO: 106, CDR2 comprising the amino acid sequence
set forth in SEQ ID NO: 135, and CDR3 comprising the amino acid
sequence set forth in SEQ ID NO: 136; (6) CDR1 comprising the amino
acid sequence set forth in SEQ ID NO: 126, CDR2 comprising the
amino acid sequence set forth in SEQ ID NO: 127, and CDR3
comprising the amino acid sequence set forth in SEQ ID NO: 128; (7)
CDR1 comprising the amino acid sequence set forth in SEQ ID NO:
129, CDR2 comprising the amino acid sequence set forth in SEQ ID
NO: 130, and CDR3 comprising the amino acid sequence set forth in
SEQ ID NO: 131; (8) CDR1 comprising the amino acid sequence set
forth in SEQ ID NO: 103, CDR2 comprising the amino acid sequence
set forth in SEQ ID NO: 104, and CDR3 comprising the amino acid
sequence set forth in SEQ ID NO: 105; (9) CDR1 comprising the amino
acid sequence set forth in SEQ ID NO: 118, CDR2 comprising the
amino acid sequence set forth in SEQ ID NO: 121, and CDR3
comprising the amino acid sequence set forth in SEQ ID NO: 122;
(10) CDR1 comprising the amino acid sequence set forth in SEQ ID
NO: 115, CDR2 comprising the amino acid sequence set forth in SEQ
ID NO: 116, and CDR3 comprising the amino acid sequence set forth
in SEQ ID NO: 117; (11) CDR1 comprising the amino acid sequence set
forth in SEQ ID NO: 112, CDR2 comprising the amino acid sequence
set forth in SEQ ID NO: 113, and CDR3 comprising the amino acid
sequence set forth in SEQ ID NO: 114; or (12) CDR1 comprising the
amino acid sequence set forth in SEQ ID NO: 118, CDR2 comprising
the amino acid sequence set forth in SEQ ID NO: 119, and CDR3
comprising the amino acid sequence set forth in SEQ ID NO: 120.
[0010] In another aspect, the invention provides an antibody
comprising a light chain variable region having CDRs 1, 2 and 3 of
any one of (1)-(13):
(1) CDR1 comprising the amino acid sequence set forth in SEQ ID NO:
143, CDR2 comprising the amino acid sequence set forth in SEQ ID
NO: 144, and CDR3 comprising the amino acid sequence set forth in
SEQ ID NO: 158; (2) CDR1 comprising the amino acid sequence set
forth in SEQ ID NO: 143, CDR2 comprising the amino acid sequence
set forth in SEQ ID NO: 144, and CDR3 comprising the amino acid
sequence set forth in SEQ ID NO: 145; (3) CDR1 comprising the amino
acid sequence set forth in SEQ ID NO: 140, CDR2 comprising the
amino acid sequence set forth in SEQ ID NO: 141, and CDR3
comprising the amino acid sequence set forth in SEQ ID NO: 142; (4)
CDR1 comprising the amino acid sequence set forth in SEQ ID NO:
167, CDR2 comprising the amino acid sequence set forth in SEQ ID
NO: 168, and CDR3 comprising the amino acid sequence set forth in
SEQ ID NO: 169; (5) CDR1 comprising the amino acid sequence set
forth in SEQ ID NO: 170, CDR2 comprising the amino acid sequence
set forth in SEQ ID NO: 144, and CDR3 comprising the amino acid
sequence set forth in SEQ ID NO: 171; (6) CDR1 comprising the amino
acid sequence set forth in SEQ ID NO: 159, CDR2 comprising the
amino acid sequence set forth in SEQ ID NO: 160, and CDR3
comprising the amino acid sequence set forth in SEQ ID NO: 161; (7)
CDR1 comprising the amino acid sequence set forth in SEQ ID NO:
162, CDR2 comprising the amino acid sequence set forth in SEQ ID
NO: 147, and CDR3 comprising the amino acid sequence set forth in
SEQ ID NO: 163; (8) CDR1 comprising the amino acid sequence set
forth in SEQ ID NO: 164, CDR2 comprising the amino acid sequence
set forth in SEQ ID NO: 165, and CDR3 comprising the amino acid
sequence set forth in SEQ ID NO: 166; (9) CDR1 comprising the amino
acid sequence set forth in SEQ ID NO: 137, CDR2 comprising the
amino acid sequence set forth in SEQ ID NO: 138, and CDR3
comprising the amino acid sequence set forth in SEQ ID NO: 139;
(10) CDR1 comprising the amino acid sequence set forth in SEQ ID
NO: 155, CDR2 comprising the amino acid sequence set forth in SEQ
ID NO: 156, and CDR3 comprising the amino acid sequence set forth
in SEQ ID NO: 157; (11) CDR1 comprising the amino acid sequence set
forth in SEQ ID NO: 149, CDR2 comprising the amino acid sequence
set forth in SEQ ID NO: 150, and CDR3 comprising the amino acid
sequence set forth in SEQ ID NO: 151; (12) CDR1 comprising the
amino acid sequence set forth in SEQ ID NO: 146, CDR2 comprising
the amino acid sequence set forth in SEQ ID NO: 147, and CDR3
comprising the amino acid sequence set forth in SEQ ID NO: 148; or
(13) CDR1 comprising the amino acid sequence set forth in SEQ ID
NO: 152, CDR2 comprising the amino acid sequence set forth in SEQ
ID NO: 153, and CDR3 comprising the amino acid sequence set forth
in SEQ ID NO: 154.
[0011] Preferably, the antibody of the invention is selected from
the group consisting of any one of (1)-(13):
(1) an antibody comprising a heavy chain variable region having
CDRs 1, 2 and 3 comprising the amino acid sequence set forth in SEQ
ID NO: 123, 124 and 125, respectively, and a light chain variable
region having CDRs 1, 2 and 3 comprising the amino acid sequence
set forth in SEQ ID NO: 143, 144 and 158, respectively; (2) an
antibody comprising a heavy chain variable region having CDRs 1, 2
and 3 comprising the amino acid sequence set forth in SEQ ID NO:
109, 110 and 111, respectively, and a light chain variable region
having CDRs 1, 2 and 3 comprising the amino acid sequence set forth
in SEQ ID NO: 143, 144 and 145, respectively; (3) an antibody
comprising a heavy chain variable region having CDRs 1, 2 and 3
comprising the amino acid sequence set forth in SEQ ID NO: 106, 107
and 108, respectively, and a light chain variable region having
CDRs 1, 2 and 3 comprising the amino acid sequence set forth in SEQ
ID NO: 140, 141 and 142, respectively; (4) an antibody comprising a
heavy chain variable region having CDRs 1, 2 and 3 comprising the
amino acid sequence set forth in SEQ ID NO: 132, 133 and 134,
respectively, and a light chain variable region having CDRs 1, 2
and 3 comprising the amino acid sequence set forth in SEQ ID NO:
167, 168 and 169, respectively; (5) an antibody comprising a heavy
chain variable region having CDRs 1, 2 and 3 comprising the amino
acid sequence set forth in SEQ ID NO: 106, 135 and 136,
respectively, and a light chain variable region having CDRs 1, 2
and 3 comprising the amino acid sequence set forth in SEQ ID NO:
170, 144 and 171, respectively; (6) an antibody comprising a heavy
chain variable region having CDRs 1, 2 and 3 comprising the amino
acid sequence set forth in SEQ ID NO: 126, 127 and 128,
respectively, and a light chain variable region having CDRs 1, 2
and 3 comprising the amino acid sequence set forth in SEQ ID NO:
159, 160 and 161, respectively; (7) an antibody comprising a heavy
chain variable region having CDRs 1, 2 and 3 comprising the amino
acid sequence set forth in SEQ ID NO: 129, 130 and 131,
respectively, and a light chain variable region having CDRs 1, 2
and 3 comprising the amino acid sequence set forth in SEQ ID NO:
162, 147 and 163, respectively; (8) an antibody comprising a heavy
chain variable region having CDRs 1, 2 and 3 comprising the amino
acid sequence set forth in SEQ ID NO: 129, 130 and 131,
respectively, and a light chain variable region having CDRs 1, 2
and 3 comprising the amino acid sequence set forth in SEQ ID NO:
164, 165 and 166, respectively; (9) an antibody comprising a heavy
chain variable region having CDRs 1, 2 and 3 comprising the amino
acid sequence set forth in SEQ ID NO: 103, 104 and 105,
respectively, and a light chain variable region having CDRs 1, 2
and 3 comprising the amino acid sequence set forth in SEQ ID NO:
137, 138 and 139, respectively; (10) an antibody comprising a heavy
chain variable region having CDRs 1, 2 and 3 comprising the amino
acid sequence set forth in SEQ ID NO: 118, 121 and 122,
respectively, and a light chain variable region having CDRs 1, 2
and 3 comprising the amino acid sequence set forth in SEQ ID NO:
155, 156 and 157, respectively; (11) an antibody comprising a heavy
chain variable region having CDRs 1, 2 and 3 comprising the amino
acid sequence set forth in SEQ ID NO: 115, 116 and 117,
respectively, and a light chain variable region having CDRs 1, 2
and 3 comprising the amino acid sequence set forth in SEQ ID NO:
149, 150 and 151, respectively; (12) an antibody comprising a heavy
chain variable region having CDRs 1, 2 and 3 comprising the amino
acid sequence set forth in SEQ ID NO: 112, 113 and 114,
respectively, and a light chain variable region having CDRs 1, 2
and 3 comprising the amino acid sequence set forth in SEQ ID NO:
146, 147 and 148, respectively; and (13) an antibody comprising a
heavy chain variable region having CDRs 1, 2 and 3 comprising the
amino acid sequence set forth in SEQ ID NO: 118, 119 and 120,
respectively, and a light chain variable region having CDRs 1, 2
and 3 comprising the amino acid sequence set forth in SEQ ID NO:
152, 153 and 154, respectively.
[0012] In another aspect, the invention provides an antibody having
a heavy chain variable region of any one of (1)-(7):
(1) a heavy chain variable region comprising the amino acid
sequence set forth in SEQ ID NO: 84; (2) a heavy chain variable
region comprising the amino acid sequence set forth in SEQ ID NO:
85; (3) a heavy chain variable region comprising the amino acid
sequence set forth in SEQ ID NO: 86; (4) a heavy chain variable
region comprising the amino acid sequence set forth in SEQ ID NO:
87; (5) a heavy chain variable region comprising the amino acid
sequence set forth in SEQ ID NO: 88; (6) a heavy chain variable
region comprising the amino acid sequence set forth in SEQ ID NO:
89; or (7) a heavy chain variable region comprising the amino acid
sequence set forth in SEQ ID NO: 90.
[0013] In another aspect, the invention provides an antibody having
a light chain variable region comprising the amino acid sequence
set forth in SEQ ID NO: 92.
[0014] Preferably, the antibody of the invention is selected from
the group consisting of the antibody of any one of (1)-(7):
(1) an antibody comprising a heavy chain variable region comprising
the amino acid sequence set forth in SEQ ID NO: 84 and a light
chain variable region comprising the amino acid sequence set forth
in SEQ ID NO: 92; (2) an antibody comprising a heavy chain variable
region comprising the amino acid sequence set forth in SEQ ID NO:
85 and a light chain variable region comprising the amino acid
sequence set forth in SEQ ID NO: 92; (3) an antibody comprising a
heavy chain variable region comprising the amino acid sequence set
forth in SEQ ID NO: 86 and a light chain variable region comprising
the amino acid sequence set forth in SEQ ID NO: 92; (4) an antibody
comprising a heavy chain variable region comprising the amino acid
sequence set forth in SEQ ID NO: 87 and a light chain variable
region comprising the amino acid sequence set forth in SEQ ID NO:
92; (5) an antibody comprising a heavy chain variable region
comprising the amino acid sequence set forth in SEQ ID NO: 88 and a
light chain variable region comprising the amino acid sequence set
forth in SEQ ID NO: 92; (6) an antibody comprising a heavy chain
variable region comprising the amino acid sequence set forth in SEQ
ID NO: 89 and a light chain variable region comprising the amino
acid sequence set forth in SEQ ID NO: 92; and (7) an antibody
comprising a heavy chain variable region comprising the amino acid
sequence set forth in SEQ ID NO: 90 and a light chain variable
region comprising the amino acid sequence set forth in SEQ ID NO:
92.
[0015] In another aspect, the invention provides an antibody
comprising a light chain variable region having CDRs 1, 2 and 3 of
any one of (1)-(15):
(1) CDR1 comprising the amino acid sequence set forth in SEQ ID NO:
174, CDR2 comprising the amino acid sequence set forth in SEQ ID
NO: 144, and CDR3 comprising the amino acid sequence set forth in
SEQ ID NO: 158; (2) CDR1 comprising the amino acid sequence set
forth in SEQ ID NO: 175, CDR2 comprising the amino acid sequence
set forth in SEQ ID NO: 144, and CDR3 comprising the amino acid
sequence set forth in SEQ ID NO: 158; (3) CDR1 comprising the amino
acid sequence set forth in SEQ ID NO: 176, CDR2 comprising the
amino acid sequence set forth in SEQ ID NO: 144, and CDR3
comprising the amino acid sequence set forth in SEQ ID NO: 158; (4)
CDR1 comprising the amino acid sequence set forth in SEQ ID NO:
177, CDR2 comprising the amino acid sequence set forth in SEQ ID
NO: 144, and CDR3 comprising the amino acid sequence set forth in
SEQ ID NO: 158; (5) CDR1 comprising the amino acid sequence set
forth in SEQ ID NO: 178, CDR2 comprising the amino acid sequence
set forth in SEQ ID NO: 144, and CDR3 comprising the amino acid
sequence set forth in SEQ ID NO: 158; (6) CDR1 comprising the amino
acid sequence set forth in SEQ ID NO: 179, CDR2 comprising the
amino acid sequence set forth in SEQ ID NO: 144, and CDR3
comprising the amino acid sequence set forth in SEQ ID NO: 158; (7)
CDR1 comprising the amino acid sequence set forth in SEQ ID NO:
180, CDR2 comprising the amino acid sequence set forth in SEQ ID
NO: 144, and CDR3 comprising the amino acid sequence set forth in
SEQ ID NO: 158; (8) CDR1 comprising the amino acid sequence set
forth in SEQ ID NO: 181, CDR2 comprising the amino acid sequence
set forth in SEQ ID NO: 144, and CDR3 comprising the amino acid
sequence set forth in SEQ ID NO: 158; (9) CDR1 comprising the amino
acid sequence set forth in SEQ ID NO: 182, CDR2 comprising the
amino acid sequence set forth in SEQ ID NO: 144, and CDR3
comprising the amino acid sequence set forth in SEQ ID NO: 158;
(10) CDR1 comprising the amino acid sequence set forth in SEQ ID
NO: 183, CDR2 comprising the amino acid sequence set forth in SEQ
ID NO: 144, and CDR3 comprising the amino acid sequence set forth
in SEQ ID NO: 158; (11) CDR1 comprising the amino acid sequence set
forth in SEQ ID NO: 184, CDR2 comprising the amino acid sequence
set forth in SEQ ID NO: 144, and CDR3 comprising the amino acid
sequence set forth in SEQ ID NO: 158; (12) CDR1 comprising the
amino acid sequence set forth in SEQ ID NO: 185, CDR2 comprising
the amino acid sequence set forth in SEQ ID NO: 144, and CDR3
comprising the amino acid sequence set forth in SEQ ID NO: 158;
(13) CDR1 comprising the amino acid sequence set forth in SEQ ID
NO: 186, CDR2 comprising the amino acid sequence set forth in SEQ
ID NO: 144, and CDR3 comprising the amino acid sequence set forth
in SEQ ID NO: 158; (14) CDR1 comprising the amino acid sequence set
forth in SEQ ID NO: 187, CDR2 comprising the amino acid sequence
set forth in SEQ ID NO: 144, and CDR3 comprising the amino acid
sequence set forth in SEQ ID NO: 158; or (15) CDR1 comprising the
amino acid sequence set forth in SEQ ID NO: 188, CDR2 comprising
the amino acid sequence set forth in SEQ ID NO: 144, and CDR3
comprising the amino acid sequence set forth in SEQ ID NO: 158.
[0016] In another aspect, the invention provides an antibody
selected from the group consisting of the antibody of (1)-(15):
(1) an antibody comprising a heavy chain variable region having
CDRs 1, 2 and 3 comprising the amino acid sequence set forth in SEQ
ID NO: 123, 124 and 125, respectively, and a light chain variable
region having CDRs 1, 2 and 3 comprising the amino acid sequence
set forth in SEQ ID NO: 174, 144 and 158, respectively; (2) an
antibody comprising a heavy chain variable region having CDRs 1, 2
and 3 comprising the amino acid sequence set forth in SEQ ID NO:
123, 124 and 125, respectively, and a light chain variable region
having CDRs 1, 2 and 3 comprising the amino acid sequence set forth
in SEQ ID NO: 175, 144 and 158, respectively; (3) an antibody
comprising a heavy chain variable region having CDRs 1, 2 and 3
comprising the amino acid sequence set forth in SEQ ID NO: 123, 124
and 125, respectively, and a light chain variable region having
CDRs 1, 2 and 3 comprising the amino acid sequence set forth in SEQ
ID NO: 176, 144 and 158, respectively; (4) an antibody comprising a
heavy chain variable region having CDRs 1, 2 and 3 comprising the
amino acid sequence set forth in SEQ ID NO: 123, 124 and 125,
respectively, and a light chain variable region having CDRs 1, 2
and 3 comprising the amino acid sequence set forth in SEQ ID NO:
177, 144 and 158, respectively; (5) an antibody comprising a heavy
chain variable region having CDRs 1, 2 and 3 comprising the amino
acid sequence set forth in SEQ ID NO: 123, 124 and 125,
respectively, and a light chain variable region having CDRs 1, 2
and 3 comprising the amino acid sequence set forth in SEQ ID NO:
178, 144 and 158, respectively; (6) an antibody comprising a heavy
chain variable region having CDRs 1, 2 and 3 comprising the amino
acid sequence set forth in SEQ ID NO: 123, 124 and 125,
respectively, and a light chain variable region having CDRs 1, 2
and 3 comprising the amino acid sequence set forth in SEQ ID NO:
179, 144 and 158, respectively; (7) an antibody comprising a heavy
chain variable region having CDRs 1, 2 and 3 comprising the amino
acid sequence set forth in SEQ ID NO: 123, 124 and 125,
respectively, and a light chain variable region having CDRs 1, 2
and 3 comprising the amino acid sequence set forth in SEQ ID NO:
180, 144 and 158, respectively; (8) an antibody comprising a heavy
chain variable region having CDRs 1, 2 and 3 comprising the amino
acid sequence set forth in SEQ ID NO: 123, 124 and 125,
respectively, and a light chain variable region having CDRs 1, 2
and 3 comprising the amino acid sequence set forth in SEQ ID NO:
181, 144 and 158, respectively; (9) an antibody comprising a heavy
chain variable region having CDRs 1, 2 and 3 comprising the amino
acid sequence set forth in SEQ ID NO: 123, 124 and 125,
respectively, and a light chain variable region having CDRs 1, 2
and 3 comprising the amino acid sequence set forth in SEQ ID NO:
182, 144 and 158, respectively; (10) an antibody comprising a heavy
chain variable region having CDRs 1, 2 and 3 comprising the amino
acid sequence set forth in SEQ ID NO: 123, 124 and 125,
respectively, and a light chain variable region having CDRs 1, 2
and 3 comprising the amino acid sequence set forth in SEQ ID NO:
183, 144 and 158, respectively; (11) an antibody comprising a heavy
chain variable region having CDRs 1, 2 and 3 comprising the amino
acid sequence set forth in SEQ ID NO: 123, 124 and 125,
respectively, and a light chain variable region having CDRs 1, 2
and 3 comprising the amino acid sequence set forth in SEQ ID NO:
184, 144 and 158, respectively; (12) an antibody comprising a heavy
chain variable region having CDRs 1, 2 and 3 comprising the amino
acid sequence set forth in SEQ ID NO: 123, 124, and 125,
respectively, and a light chain variable region having CDRs 1, 2
and 3 comprising the amino acid sequence set forth in SEQ ID NO:
185, 144, and 158, respectively; (13) an antibody comprising a
heavy chain variable region having CDRs 1, 2 and 3 comprising the
amino acid sequence set forth in SEQ ID NO: 123, 124, and 125,
respectively, and a light chain variable region having CDRs 1, 2
and 3 comprising the amino acid sequence set forth in SEQ ID NO:
186, 144 and 158, respectively; (14) an antibody comprising a heavy
chain variable region having CDRs 1, 2 and 3 comprising the amino
acid sequence set forth in SEQ ID NO: 123, 124 and 125,
respectively, and a light chain variable region having CDRs 1, 2
and 3 comprising the amino acid sequence set forth in SEQ ID NO:
187, 144 and 158, respectively; and (15) an antibody comprising a
heavy chain variable region having CDRs 1, 2 and 3 comprising the
amino acid sequence set forth in SEQ ID NO: 123, 124 and 125,
respectively, and a light chain variable region having CDRs 1, 2
and 3 comprising the amino acid sequence set forth in SEQ ID NO:
188, 144 and 158, respectively.
[0017] In further aspect, the invention provides an antibody having
a light chain variable region selected from (1)-(15):
(1) a light chain variable region comprising the amino acid
sequence set forth in SEQ ID NO: 191; (2) a light chain variable
region comprising the amino acid sequence set forth in SEQ ID NO:
192; (3) a light chain variable region comprising the amino acid
sequence set forth in SEQ ID NO: 193; (4) a light chain variable
region comprising the amino acid sequence set forth in SEQ ID NO:
194; (5) a light chain variable region comprising the amino acid
sequence set forth in SEQ ID NO: 195; (6) a light chain variable
region comprising the amino acid sequence set forth in SEQ ID NO:
196; (7) a light chain variable region comprising the amino acid
sequence set forth in SEQ ID NO: 197; (8) a light chain variable
region comprising the amino acid sequence set forth in SEQ ID NO:
198; (9) a light chain variable region comprising the amino acid
sequence set forth in SEQ ID NO: 199; (10) a light chain variable
region comprising the amino acid sequence set forth in SEQ ID NO:
200; (11) a light chain variable region comprising the amino acid
sequence set forth in SEQ ID NO: 201; (12) a light chain variable
region comprising the amino acid sequence set forth in SEQ ID NO:
202; (13) a light chain variable region comprising the amino acid
sequence set forth in SEQ ID NO: 203; (14) a light chain variable
region comprising the amino acid sequence set forth in SEQ ID NO:
204; and (15) a light chain variable region comprising the amino
acid sequence set forth in SEQ ID NO: 205.
[0018] In another aspect, the invention provides an antibody having
a light chain variable region selected from the group consisting of
(1)-(15):
(1) a light chain variable region comprising the amino acid
sequence set forth in SEQ ID NO: 191; (2) a light chain variable
region comprising the amino acid sequence set forth in SEQ ID NO:
192; (3) a light chain variable region comprising the amino acid
sequence set forth in SEQ ID NO: 193; (4) a light chain variable
region comprising the amino acid sequence set forth in SEQ ID NO:
194; (5) a light chain variable region comprising the amino acid
sequence set forth in SEQ ID NO: 195; (6) a light chain variable
region comprising the amino acid sequence set forth in SEQ ID NO:
196; (7) a light chain variable region comprising the amino acid
sequence set forth in SEQ ID NO: 197; (8) a light chain variable
region comprising the amino acid sequence set forth in SEQ ID NO:
198; (9) a light chain variable region comprising the amino acid
sequence set forth in SEQ ID NO: 199; (10) a light chain variable
region comprising the amino acid sequence set forth in SEQ ID NO:
200; (11) a light chain variable region comprising the amino acid
sequence set forth in SEQ ID NO: 201; (12) a light chain variable
region comprising the amino acid sequence set forth in SEQ ID NO:
202; (13) a light chain variable region comprising the amino acid
sequence set forth in SEQ ID NO: 203; (14) a light chain variable
region comprising the amino acid sequence set forth in SEQ ID NO:
204; and (15) a light chain variable region comprising the amino
acid sequence set forth in SEQ ID NO: 205; and a heavy chain
variable region selected from the group consisting of (1)-(7): (1)
a heavy chain variable region comprising the amino acid sequence
set forth in SEQ ID NO: 84; (2) a heavy chain variable region
comprising; the amino acid sequence set forth in SEQ ID NO: 85; (3)
a heavy chain variable region comprising the amino acid sequence
set forth in SEQ ID NO: 86; (4) a heavy chain variable region
comprising the amino acid sequence set forth in SEQ ID NO: 87; (5)
a heavy chain variable region comprising the amino acid sequence
set forth in SEQ ID NO: 88; (6) a heavy chain variable region
comprising the amino acid sequence set forth in SEQ ID NO: 89; and
(7) a heavy chain variable region comprising the amino acid
sequence set forth in SEQ ID NO: 90.
[0019] The heavy chain variable region, a light chain variable
region, and the amino acid sequence of the CDRs 1, 2 and 3, as well
as the SEQ ID NOs are summarized in the table below.
TABLE-US-00001 Antibody and variable regions SEQ ID NO M3C11 H 22
M13B3 H 23 M1E7 H 24 M3B8 H 25 M11F1 H 26 M19B11 H 27 M6B1 H 28
M18D4 H 29 M5B9 H 30 M10D2 H 31 L9G11 H 32 M3C11 L 44 M13B3 L 45
M1E7 L 46 M3B8 L 47 M11F1 L 48 M19B11 L 49 M6B1 L 50 M18D4 L 51
M5B9 L 52 M10D2 L 53 L9G11 L 54 GC199 H 60 GC202 H 61 GC33 H 62
GC179 H 63 GC194 H 64 GC199 L 71 GC202 L 72 GC33 L 73 GC179 L 74
GC194(1) L 75 GC194(2) L 76 GC33.ver.a H 84 GC33.ver.c H 85
GC33.ver.f H 86 GC33.ver.h H 87 GC33.ver.i H 88 GC33.ver.j H 89
GC33.ver.k H 90 GC33.ver.a L 92 M13B3(H) CDR1 103 CDR2 104 CDR3 105
M3B8(H) CDR1 106 CDR2 107 CDR3 108 M11F1(H) CDR1 109 CDR2 110 CDR3
111 M5B9(H) CDR1 112 CDR2 113 CDR3 114 M6B1(H) CDR1 115 CDR2 116
CDR3 117 M10D2(H) CDR1 118 CDR2 119 CDR3 120 L9G11(H) CDR1 118 CDR2
121 CDR3 122 GC33(H) CDR1 123 CDR2 124 CDR3 125 GC179(H) CDR1 126
CDR2 127 CDR3 128 GC194(H) CDR1 129 CDR2 130 CDR3 131 GC199(H) CDR1
132 CDR2 133 CDR3 134 GC202(H) CDR1 106 CDR2 135 CDR3 136 M13B3(L)
CDR1 137 CDR2 138 CDR3 139 M3B8(L) CDR1 140 CDR2 141 CDR3 142
M11F1(L) CDR1 143 CDR2 144 CDR3 145 M5B9(L) CDR1 146 CDR2 147 CDR3
148 M6B1(L) CDR1 149 CDR2 150 CDR3 151 M10D2(L) CDR1 152 CDR2 153
CDR3 154 L9G11(L) CDR1 155 CDR2 156 CDR3 157 GC33(L) CDR1 143 CDR2
144 CDR3 158 GC179(L) CDR1 159 CDR2 160 CDR3 161 GC194(L)1 CDR1 162
CDR2 147 CDR3 163 GC194(L)2 CDR1 164 CDR2 165 CDR3 166 GC199(L)
CDR1 167 CDR2 168 CDR3 169 GC202(L) CDR1 170 CDR2 144 CDR3 171
GC33(L) G34A 174 GC33(L) G34D 175 GC33(L) G34E 176 GC33(L) G34F 177
GC33(L) G34H 178 GC33(L) G34N 179 GC33(L) G34P 180 GC33(L) G34Q 181
GC33(L) G34I 182 GC33(L) G34K 183 GC33(L) G34L 184 GC33(L) G34V 185
GC33(L) G34W 186 GC33(L) G34Y 187 GC33(L) G34R 188
[0020] Also the invention features an antibody having an activity
equivalent to the activity of the antibody described above, wherein
one or more amino acid residues are substituted, deleted or added
and/or inserted from the amino acid sequences described above.
[0021] Preferably, the antibody of the invention is a humanized
antibody.
[0022] Thus, in another aspect, the invention provides a humanized
antibody capable of binding to glypican 3.
[0023] In further aspect, the invention provides an antibody
capable of binding to a peptide consisting of the sequence of the
amino acid residues 524-563 of glypican 3.
[0024] Preferably, the antibody of the invention is capable of
binding to a peptide consisting of the sequence of the amino acid
residues 537-563 of glypican 3. More preferably, the antibody of
the invention does not bind to a peptide consisting of the sequence
of the amino acid residues 550-563 of glypican 3.
[0025] Preferably, the antibody is capable of binding to a peptide
consisting of the sequence of the amino acid residues 544-553 of
glypican 3 or a peptide consisting of the sequence of the amino
acid residues 546-551 of glypican 3.
[0026] In still another aspect, the invention provides an antibody
capable of binding to an epitope to which a second antibody is
capable of binding, wherein said second antibody comprises a heavy
chain variable region having CDRs 1, 2 and 3 comprising the amino
acid sequence set forth in SEQ ID NO: 123, 124 and 125,
respectively, and a light chain variable region having CDRs 1, 2
and 3 comprising the amino acid sequence set forth in SEQ ID NO:
143, 144 and 158, respectively. Namely, the antibody of the
invention is capable of competing in binding to GPC3 with the
second antibody.
[0027] In a preferred embodiment, the antibody of the invention is
capable of binding to glypican 3 and has a high CDC activity
against a cell expressing glypican 3 and/or has a high ADCC
activity against a cell expressing glypican 3.
[0028] In another aspect, the invention provides a polynucleotide
coding for a heavy chain variable region or a light chain variable
region of the antibody of the invention.
[0029] Preferably, the polynucleotide of the invention has the
sequence set forth in SEQ ID NOs: 11-21, 33-43, 55-59, 65-70 and
77-83.
[0030] In still another aspect, the invention provides a
cell-growth inhibitor and an anticancer agent comprising as an
active ingredient the antibody of the invention. Preferably, the
anticancer agent of the invention is used for treatment of
hepatoma.
[0031] In further aspect, the invention provides a peptide
comprising the sequence of the amino acid residues 524-563 of
glypican 3, the sequence of the amino acid residues 537-563 of
glypican 3, the sequence of the amino acid residues 544-553 of
glypican 3 or the amino acid sequence of the amino acid residues
546-551 of glypican 3.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] FIG. 1 shows the binding activity of the anti-GPC3 antibody
to a CHO cell, a CHO cell expressing full-length GPC3, HepG2 and
HuH-7, which was evaluated by flow cytometry. M1E7 (solid line) and
M11F1 (dashed line) were used at a concentration of 5 .mu.g/mL,
respectively.
[0033] FIG. 2 is a table showing the results of epitope
classification by a competitive ELISA. The degrees of competitive
inhibition against the binding of the biotinylated anti-GPC3
antibody are indicated by percentage. The epitopes were classified
into 5 groups, a to e, according to the competitive inhibition
pattern.
[0034] FIG. 3 shows the results of evaluating by Western blotting
whether an anti-GPC3 antibody binds to the N-terminal fragment of
40 kDa of the soluble form of GPC3 core protein or to the
C-terminal fragment of 30 kDa thereof. It was found that L9G11
binds to the N-terminal fragment and M3C11 binds to the C-terminal
fragment.
[0035] FIG. 4 shows the results of detecting a secreted form of
GPC3 is present in the culture supernatant of HepG2 by a sandwich
ELISA. It was strongly detected with the combination of antibodies
that bind to the N-terminal fragment such as M6B1, M18D4 or M19B11,
and it was not strongly detected with an antibody that binds to the
C-terminal fragment such as M3C11, M13B3 or M3B8.
[0036] FIG. 5 shows the results of immunoprecipitation of the
culture supernatant of HepG2 with the use of an anti-GPC3 antibody
and detecton of a secreted form of GPC3. The medium as a control
(lanes 1 and 3) and the culture supernatant of HepG2 (lanes 2 and
4) were immunoprecipitated using M1E7 (lanes 1 and 2) and M10D2
(lanes 3 and 4). Secretory GPC3 was detected by M10D2 that binds to
the N-terminal fragment.
[0037] FIG. 6 shows the results of analyzing the epitope of the
antibodies that bind to the C-terminal fragment of GPC3 by Western
blotting with the use of a fusion protein of the C-terminal peptide
of GPC3 and GST. The soluble form of GPC3 core protein (lane 1),
GST (lane 2), GC-1 (lane 3), GC-2 (lane 4), GC-3 (lane 5), GC-4
(lane 6) and GC-5 (lane 7) were subjected to SDS electrophoresis
under reducing conditions, and detected by Western blotting using
M3C11 and M11F1.
[0038] FIG. 7 shows the results of evaluating the CDC activity of
the anti-GPC3 mouse-human chimeric antibody to a CHO cell that
expresses GPC3.
[0039] FIG. 8 shows the results of evaluating the ADCC activity of
the anti-GPC3 mouse-human chimeric antibody to a CHO cell that
expresses GPC3 and HepG2.
[0040] FIG. 9 shows the results of evaluating the ADCC activity of
GC33 to a human hepatoma cell line, HuH-7, using a mouse bone
marrow-derived effector cell.
[0041] FIG. 10 shows the results of evaluating the antitumor
activity of GC33 antibody to a mouse model transplanted with human
hepatoma.
[0042] FIG. 11 shows the results of evaluating the CDC activity of
the mouse-human chimeric antibody GC33 to a CHO cell that expresses
GPC3.
[0043] FIG. 12 shows the results of evaluating the ADCC activity of
the mouse-human chimeric antibody GC33 to HepG2.
[0044] FIG. 13 shows GPC3-derived sequences contained in GST-fusion
proteins (GC-4, 5, 6, 7, 8, 9, 11, 12, 13 and 14) prepared for
analyzing the epitope of GC33.
[0045] FIG. 14 shows the results of Western blotting with the use
of GC33 after separating GST, GC-7, 8, 9, 11, 12, 13 and 14 by
SDS-PAGE under reducing conditions.
[0046] FIG. 15 shows the results of evaluating the binding activity
of humanized GC33 to GPC3 by an ELISA.
[0047] FIG. 16 shows an antibody panel which summarizes isotypes
and the results of an ELISA, BIAcore, FACS, an epitope analysis and
an immunoprecipitation test for clones derived from a mouse
immunized with a soluble form of GPC3.
[0048] FIG. 17 shows an antibody panel in which isotypes and the
results of an ELISA, FACS and an epitope analysis for clones
derived from a mouse immunized with GC-3 are summarized.
[0049] FIG. 18 shows the results of evaluating the binding activity
of the modified antibodies to the soluble form of GPC3 core protein
by an ELISA. Gly34 located at CDR1 in a humanized GC33 L chain
variable region was replaced with any of 17 amino acids other than
Cys and Met.
[0050] FIG. 19 shows the results of evaluating the CDC activity of
the mouse-human chimeric antibodies GC33, M3C11, and M1E7 to a CHO
cell that expresses full-length GPC3.
[0051] FIG. 20 shows the results of evaluating the ADCC activity of
the mouse-human chimeric antibodies GC33, M3C11, and M1E7 to a
human hepatoma cell line SK-03 that expresses full-length GPC3.
DETAILED DESCRIPTION OF THE INVENTION
Antibody
[0052] The present invention provides antibodies described in the
following (I) to (XI).
(I) An antibody containing heavy chain variable regions having CDRs
1, 2 and 3 consisting of the amino acid sequences set forth in SEQ
ID NOs: in any of the following (1) to (12):
(1) SEQ ID NOs: 123, 124 and 125 (GC33),
(2) SEQ ID NOs: 109, 110 and 111 (M11F1),
(3) SEQ ID NOs: 106, 107 and 108 (M3B8),
(4) SEQ ID NOs: 132, 133 and 134 (GC199),
(5) SEQ ID NOs: 106, 135 and 136 (GC202),
(6) SEQ ID NOs: 126, 127 and 128 (GC179),
(7) SEQ ID NOs: 129, 130 and 131 (GC194),
(8) SEQ ID NOs: 103, 104 and 105 (M13B3),
(9) SEQ ID NOs: 118, 121 and 122 (L9G11),
(10) SEQ ID NOs: 115, 116 and 117 (M6B1),
(11) SEQ ID NOs: 112, 113 and 114 (M5B9), and
(12) SEQ ID NOs: 118, 119 and 120 (M10D2).
[0053] Among the antibodies described in (1) to (12), preferred are
the antibodies described in (1) to (8), more preferred are the
antibodies described in (1) to (5), and particularly preferred is
the antibody described in (1). The antibodies described in (1) to
(8) recognize the C-terminal peptide of glypican 3 (a peptide
comprising the 374th amino acid to the 580th amino acid of glypican
3); and are useful as a therapeutic antibody. In addition, the
antibodies described in (9) to (12) recognize the N-terminal
peptide of glypican 3 (a peptide comprising from the 1st amino acid
to the 373rd amino acid of glypican 3); and are useful as a
diagnostic antibody.
(II) An antibody containing light chain variable regions having
CDRs 1, 2 and 3 consisting of the amino acid sequences set forth in
SEQ ID NOs: in any of the following (1) to (13):
(1) SEQ ID NOs: 143, 144 and 158 (GC33),
(2) SEQ ID NOs: 143, 144 and 145 (M11F1),
(3) SEQ ID NOs: 140, 141 and 142 (M3B8),
(4) SEQ ID NOs: 167, 168 and 169 (GC199),
(5) SEQ ID NOs: 170, 144 and 171 (GC202),
(6) SEQ ID NOs: 159, 160 and 161 (GC179),
(7) SEQ ID NOs: 162, 147 and 163 (GC194 (1)),
(8) SEQ ID NOs: 164, 165 and 166 (GC194 (2)),
(9) SEQ ID NOs: 137, 138 and 139 (M13B3),
(10) SEQ ID NOs: 155, 156 and 157 (L9G11),
(11) SEQ ID NOs: 149, 150 and 151 (M6B1),
(12) SEQ ID NOs: 146, 147 and 148 (M5B9), and
(13) SEQ ID NOs: 152, 153 and 154 (M10D2).
[0054] Among the antibodies described in (1) to (13), preferred are
the antibodies described in (1) to (8), more preferred are the
antibodies described in (1) to (5), and particularly preferred is
the antibody described in (1). The antibodies described in (1) to
(8) recognize the C-terminal peptide of glypican 3 (a peptide
comprising from the 374th amino acid to the 580th amino acid of
glypican 3); and are useful as a therapeutic antibody. In addition,
the antibodies described in (9) to (13) recognize the N-terminal
peptide of glypican 3 (a peptide comprising from the 1st amino acid
to the 373rd amino acid of glypican 3); and are useful as a
diagnostic antibody.
(III) An antibody selected from the group consisting of the
antibodies described in the following (1) to (13): (1) an antibody
containing heavy chain variable regions having CDRs 1, 2 and 3
consisting of the amino acid sequences set forth in SEQ ID NOs:
123, 124 and 125, and light chain variable regions having CDRs 1, 2
and 3 consisting of the amino acid sequences set forth in SEQ ID
NOs: 143, 144 and 158 (GC33), (2) an antibody containing heavy
chain variable regions having CDRs 1, 2 and 3 consisting of the
amino acid sequences set forth in SEQ ID NOs: 109, 110 and 111, and
light chain variable regions having CDRs 1, 2 and 3 consisting of
the amino acid sequences set forth in SEQ ID NOs: 143, 144 and 145
(M11F1), (3) an antibody containing heavy chain variable regions
having CDRs 1, 2 and 3 consisting of the amino acid sequences set
forth in SEQ ID NOs: 106, 107 and 108, and light chain variable
regions having CDRs 1, 2 and 3 consisting of the amino acid
sequences set forth in SEQ ID NOs: 140, 141 and 142 (M3B8), (4) an
antibody containing heavy chain variable regions having CDRs 1, 2
and 3 consisting of the amino acid sequences set forth in SEQ ID
NOs: 132, 133 and 134, and light chain variable regions having CDRs
1, 2 and 3 consisting of the amino acid sequences set forth in SEQ
ID NOs: 167, 168 and 169 (GC199), (5) an antibody containing heavy
chain variable regions having CDRs 1, 2 and 3 consisting of the
amino acid sequences set forth in SEQ ID NOs: 106, 135 and 136, and
light chain variable regions having CDRs 1, 2 and 3 consisting of
the amino acid sequences set forth in SEQ ID NOs: 170, 144 and 171
(GC202), (6) an antibody containing heavy chain variable regions
having CDRs 1, 2 and 3 consisting of the amino acid sequences set
forth in SEQ ID NOs: 126, 127 and 128, and light chain variable
regions having CDRs 1, 2 and 3 consisting of the amino acid
sequences set forth in SEQ ID NOs: 159, 160 and 161 (GC179), (7) an
antibody containing heavy chain variable regions having CDRs 1, 2
and 3 consisting of the amino acid sequences set forth in SEQ ID
NOs: 129, 130 and 131, and light chain variable regions having CDRs
1, 2 and 3 consisting of the amino acid sequences set forth in SEQ
ID NOs: 162, 147 and 163 (GC194 (1)), (8) an antibody containing
heavy chain variable regions having CDRs 1, 2 and 3 consisting of
the amino acid sequences set forth in SEQ ID NOs: 129, 130 and 131,
and light chain variable regions having CDRs 1, 2 and 3 consisting
of the amino acid sequences set forth in SEQ ID NOs: 164, 165 and
166 (GC194 (2)), (9) an antibody containing heavy chain variable
regions having CDRs 1, 2 and 3 consisting of the amino acid
sequences set forth in SEQ ID NOs: 103, 104 and 105, and light
chain variable regions having CDRs 1, 2 and 3 consisting of the
amino acid sequences set forth in SEQ ID NOs: 137, 138 and 139
(M13B3), (10) an antibody containing heavy chain variable regions
having CDRs 1, 2 and 3 consisting of the amino acid sequences set
forth in SEQ ID NOs: 118, 121 and 122, and light chain variable
regions having CDRs 1, 2 and 3 consisting of the amino acid
sequences set forth in SEQ ID NOs: 155, 156 and 157 (L9G11), (11)
an antibody containing heavy chain variable regions having CDRs 1,
2 and 3 consisting of the amino acid sequences set forth in SEQ ID
NOs: 115, 116 and 117, and light chain variable regions having CDRs
1, 2 and 3 consisting of the amino acid sequences set forth in SEQ
ID NOs: 149, 150 and 151 (M6B1), (12) an antibody containing heavy
chain variable regions having CDRs 1, 2 and 3 consisting of the
amino acid sequences set forth in SEQ ID NOs: 112, 113 and 114, and
light chain variable regions having CDRs 1, 2 and 3 consisting of
the amino acid sequences set forth in SEQ ID NOs: 146, 147 and 148
(M5B9), (13) an antibody containing heavy chain variable regions
having CDRs 1, 2 and 3 consisting of the amino acid sequences set
forth in SEQ ID NOs: 118, 119 and 120, and light chain variable
regions having CDRs 1, 2 and 3 consisting of the amino acid
sequences set forth in SEQ ID NOs: 152, 153 and 154 (M10D2).
[0055] Among the antibodies described in (1) to (13), preferred are
the antibodies described in (1) to (8), more preferred are the
antibodies described in (1) to (5), and particularly preferred is
the antibody described in (1). The antibodies described in (1) to
(8) recognize the C-terminal peptide of glypican 3 (a peptide
comprising from the 374th amino acid to the 580th amino acid of
glypican 3); and are useful as a therapeutic antibody. In addition,
the antibodies described in (9) to (13) recognize the N-terminal
peptide of glypican 3 (a peptide comprising from the 1st amino acid
to the 373rd amino acid of glypican 3); and are useful as a
diagnostic antibody.
(IV) An antibody having a heavy chain variable region described in
any of the following (1) to (7): (1) a heavy chain variable region
containing the amino acid sequence set forth in SEQ ID NO: 84 (GC33
VH ver.a), (2) a heavy chain variable region containing the amino
acid sequence set forth in SEQ ID NO: 85 (GC33 VH ver.c), (3) a
heavy chain variable region containing the amino acid sequence set
forth in SEQ ID NO: 86 (GC33 VH ver.f), (4) a heavy chain variable
region containing the amino acid sequence set forth in SEQ ID NO:
87 (GC33 VH ver.h), (5) a heavy chain variable region containing
the amino acid sequence set forth in SEQ ID NO: 88 (GC33 VH ver.i),
(6) a heavy chain variable region containing the amino acid
sequence set forth in SEQ ID NO: 89 (GC33 VH ver.j), and (7) a
heavy chain variable region containing the amino acid sequence set
forth in SEQ ID NO: 90 (GC33 VH ver.k).
[0056] Among the antibodies described in (1) to (7), particularly
preferred are the antibodies described in (2) to (7).
(V) An antibody having a light chain variable region containing the
amino acid sequence set forth in SEQ ID NO: 92 (GC33 VL ver.a).
(VI) An antibody selected from the group consisting of the
antibodies described in the following (1) to (7): (1) an antibody
having a heavy chain variable region containing the amino acid
sequence set forth in SEQ ID NO: 84 (GC33 VH ver.a) and a light
chain variable region containing the amino acid sequence set forth
in SEQ ID NO: 92 (GC33 VL ver.a), (2) an antibody having a heavy
chain variable region containing the amino acid sequence set forth
in SEQ ID NO: 85 (GC33 VH ver. c) and a light chain variable region
containing the amino acid sequence set forth in SEQ ID NO: 92 (GC33
VL ver.a), (3) an antibody having a heavy chain variable region
containing the amino acid sequence set forth in SEQ ID NO: 86 (GC33
VH ver. f) and a light chain variable region containing the amino
acid sequence set forth in SEQ ID NO: 92 (GC33 VL ver.a), (4) an
antibody having a heavy chain variable region containing the amino
acid sequence set forth in SEQ ID NO: 87 (GC33 VH ver.h) and a
light chain variable region containing the amino acid sequence set
forth in SEQ ID NO: 92 (GC33 VL ver.a), (5) an antibody having a
heavy chain variable region containing the amino acid sequence set
forth in SEQ ID NO: 88 (GC33 VH ver. i) and a light chain variable
region containing the amino acid sequence set forth in SEQ ID NO:
92 (GC33 VL ver.a), (6) an antibody having a heavy chain variable
region containing the amino acid sequence set forth in SEQ ID NO:
89 (GC33 VH ver. j) and a light chain variable region containing
the amino acid sequence set forth in SEQ ID NO: 92 (GC33 VL ver.a),
and (7) an antibody having a heavy chain variable region containing
the amino acid sequence set forth in SEQ ID NO: 90 (GC33 VH ver. k)
and a light chain variable region containing the amino acid
sequence set forth in SEQ ID NO: 92 (GC33 VL ver.a).
[0057] Among the antibodies described in (1) to (7), particularly
preferred are the antibodies described in (2) to (7).
(VII) An antibody described in any of the following (1) to (15):
(1) an antibody containing light chain variable regions having CDRs
1, 2 and 3 consisting of the amino acid sequences set forth in SEQ
ID NOs: 174, 144 and 158, (2) an antibody containing light chain
variable regions having CDRs 1, 2 and 3 consisting of the amino
acid sequences set forth in SEQ ID NOs: 175, 144 and 158, (3) an
antibody containing light chain variable regions having CDRs 1, 2
and 3 consisting of the amino acid sequences set forth in SEQ ID
NOs: 176, 144 and 158, (4) an antibody containing light chain
variable regions having CDRs 1, 2 and 3 consisting of the amino
acid sequences set forth in SEQ ID NOs: 177, 144 and 158, (5) an
antibody containing light chain variable regions having CDRs 1, 2
and 3 consisting of the amino acid sequences set forth in SEQ ID
NOs: 178, 144 and 158, (6) an antibody containing light chain
variable regions having CDRs 1, 2 and 3 consisting of the amino
acid sequences set forth in SEQ ID NOs: 179, 144 and 158, (7) an
antibody containing light chain variable regions having CDRs 1, 2
and 3 consisting of the amino acid sequences set forth in SEQ ID
NOs: 180, 144 and 158, (8) an antibody containing light chain
variable regions having CDRs 1, 2 and 3 consisting of the amino
acid sequences set forth in SEQ ID NOs: 181, 144 and 158, (9) an
antibody containing light chain variable regions having CDRs 1, 2
and 3 consisting of the amino acid sequences set forth in SEQ ID
NOs: 182, 144 and 158, (10) an antibody containing light chain
variable regions having CDRs 1, 2 and 3 consisting of the amino
acid sequences set forth in SEQ ID NOs: 183, 144 and 158, (11) an
antibody containing light chain variable regions having CDRs 1, 2
and 3 consisting of the amino acid sequences set forth in SEQ ID
NOs: 184, 144 and 158, (12) an antibody containing light chain
variable regions having CDRs 1, 2 and 3 consisting of the amino
acid sequences set forth in SEQ ID NOs: 185, 144 and 158, (13) an
antibody containing light chain variable regions having CDRs 1, 2
and 3 consisting of the amino acid sequences set forth in SEQ ID
NOs: 186, 144 and 158, (14) an antibody containing light chain
variable regions having CDRs 1, 2 and 3 consisting of the amino
acid sequences set forth in SEQ ID NOs: 187, 144 and 158, and (15)
an antibody containing light chain variable regions having CDRs 1,
2 and 3 consisting of the amino acid sequences set forth in SEQ ID
NOs: 188, 144 and 158.
[0058] Among the antibodies described in (1) to (15), preferred is
the antibody described in (15).
(VIII) An antibody described in any of the following (1) to (15):
(1) an antibody containing heavy chain variable regions having CDRs
1, 2 and 3 consisting of the amino acid sequences set forth in SEQ
ID NOs: 123, 124 and 125 and light chain variable regions having
CDRs 1, 2 and 3 consisting of the amino acid sequences set forth in
SEQ ID NOs: 174, 144 and 158, (2) an antibody containing heavy
chain variable regions having CDRs 1, 2 and 3 consisting of the
amino acid sequences set forth in SEQ ID NOs: 123, 124 and 125 and
light chain variable regions having CDRs 1, 2 and 3 consisting of
the amino acid sequences set forth in SEQ ID NOs: 175, 144 and 158,
(3) an antibody containing heavy chain variable regions having CDRs
1, 2 and 3 consisting of the amino acid sequences set forth in SEQ
ID NOs: 123, 124 and 125 and light chain variable regions having
CDRs 1, 2 and 3 consisting of the amino acid sequences set forth in
SEQ ID NOs: 176, 144 and 158, (4) an antibody containing heavy
chain variable regions having CDRs 1, 2 and 3 consisting of the
amino acid sequences set forth in SEQ ID NOs: 123, 124 and 125 and
light chain variable regions having CDRs 1, 2 and 3 consisting of
the amino acid sequences set forth in SEQ ID NOs: 177, 144 and 158,
(5) an antibody containing heavy chain variable regions having CDRs
1, 2 and 3 consisting of the amino acid sequences set forth in SEQ
ID NOs: 123, 124 and 125 and light chain variable regions having
CDRs 1, 2 and 3 consisting of the amino acid sequences set forth in
SEQ ID NOs: 178, 144 and 158, (6) an antibody containing heavy
chain variable regions having CDRs 1, 2 and 3 consisting of the
amino acid sequences set forth in SEQ ID NOs: 123, 124 and 125 and
light chain variable regions having CDRs 1, 2 and 3 consisting of
the amino acid sequences set forth in SEQ ID NOs: 179, 144 and 158,
(7) an antibody containing heavy chain variable regions having CDRs
1, 2 and 3 consisting of the amino acid sequences set forth in SEQ
ID NOs: 123, 124 and 125 and light chain variable regions having
CDRs 1, 2 and 3 consisting of the amino acid sequences set forth in
SEQ ID NOs: 180, 144 and 158, (8) an antibody containing heavy
chain variable regions having CDRs 1, 2 and 3 consisting of the
amino acid sequences set forth in SEQ ID NOs: 123, 124 and 125 and
light chain variable regions having CDRs 1, 2 and 3 consisting of
the amino acid sequences set forth in SEQ ID NOs: 181, 144 and 158,
(9) an antibody containing heavy chain variable regions having CDRs
1, 2 and 3 consisting of the amino acid sequences set forth in SEQ
ID NOs: 123, 124 and 125 and light chain variable regions having
CDRs 1, 2 and 3 consisting of the amino acid sequences set forth in
SEQ ID NOs: 182, 144 and 158, (10) an antibody containing heavy
chain variable regions having CDRs 1, 2 and 3 consisting of the
amino acid sequences set forth in SEQ ID NOs: 123, 124 and 125 and
light chain variable regions having CDRs 1, 2 and 3 consisting of
the amino acid sequences set forth in SEQ ID NOs: 183, 144 and 158,
(11) an antibody containing heavy chain variable regions having
CDRs 1, 2 and 3 consisting of the amino acid sequences set forth in
SEQ ID NOs: 123, 124 and 125 and light chain variable regions
having CDRs 1, 2 and 3 consisting of the amino acid sequences set
forth in SEQ ID NOs: 184, 144 and 158, (12) an antibody containing
heavy chain variable regions having CDRs 1, 2 and 3 consisting of
the amino acid sequences set forth in SEQ ID NOs: 123, 124 and 125
and light chain variable regions having CDRs 1, 2 and 3 consisting
of the amino acid sequences set forth in SEQ ID NOs: 185, 144 and
158, (13) an antibody containing heavy chain variable regions
having CDRs 1, 2 and 3 consisting of the amino acid sequences set
forth in SEQ ID NOs: 123, 124 and 125 and light chain variable
regions having CDRs 1, 2 and 3 consisting of the amino acid
sequences set forth in SEQ ID NOs: 186, 144 and 158, (14) an
antibody containing heavy chain variable regions having CDRs 1, 2
and 3 consisting of the amino acid sequences set forth in SEQ ID
NOs: 123, 124 and 125 and light chain variable regions having CDRs
1, 2 and 3 consisting of the amino acid sequences set forth in SEQ
ID NOs: 187, 144 and 158, and (15) an antibody containing heavy
chain variable regions having CDRs 1, 2 and 3 consisting of the
amino acid sequences set forth in SEQ ID NOs: 123, 124 and 125 and
light chain variable regions having CDRs 1, 2 and 3 consisting of
the amino acid sequences set forth in SEQ ID NOs: 188, 144 and
158.
[0059] Among the antibodies described in (1) to (15), preferred is
the antibody described in (15).
(IX) An antibody described in any of the following (1) to (15): (1)
an antibody having a light chain variable region containing the
amino acid sequence set forth in SEQ ID NO: 191, (2) an antibody
having a light chain variable region containing the amino acid
sequence set forth in SEQ ID NO: 192, (3) an antibody having a
light chain variable region containing the amino acid sequence set
forth in SEQ ID NO: 193, (4) an antibody having a light chain
variable region containing the amino acid sequence set forth in SEQ
ID NO: 194, (5) an antibody having a light chain variable region
containing the amino acid sequence set forth in SEQ ID NO: 195, (6)
an antibody having a light chain variable region containing the
amino acid sequence set forth in SEQ ID NO: 196, (7) an antibody
having a light chain variable region containing the amino acid
sequence set forth in SEQ ID NO: 197, (8) an antibody having a
light chain variable region containing the amino acid sequence set
forth in SEQ ID NO: 198, (9) an antibody having a light chain
variable region containing the amino acid sequence set forth in SEQ
ID NO: 199, (10) an antibody having a light chain variable region
containing the amino acid sequence set forth in SEQ ID NO: 200,
(11) an antibody having a light chain variable region containing
the amino acid sequence set forth in SEQ ID NO: 201, (12) an
antibody having a light chain variable region containing the amino
acid sequence set forth in SEQ ID NO: 202, (13) an antibody having
a light chain variable region containing the amino acid sequence
set forth in SEQ ID NO: 203, (14) an antibody having a light chain
variable region containing the amino acid sequence set forth in SEQ
ID NO: 204, and (15) an antibody having a light chain variable
region containing the amino acid sequence set forth in SEQ ID NO:
205.
[0060] Among the antibodies described in (1) to (15), preferred is
the antibody described in (15).
(X) An antibody having a light chain variable region selected from
the group consisting of the light chain variable regions described
in the following (1) to (15): (1) a light chain variable region
containing the amino acid sequence set forth in SEQ ID NO: 191, (2)
a light chain variable region containing the amino acid sequence
set forth in SEQ ID NO: 192, (3) a light chain variable region
containing the amino acid sequence set forth in SEQ ID NO: 193, (4)
a light chain variable region containing the amino acid sequence
set forth in SEQ ID NO: 194, (5) a light chain variable region
containing the amino acid sequence set forth in SEQ ID NO: 195, (6)
a light chain variable region containing the amino acid sequence
set forth in SEQ ID NO: 196, (7) a light chain variable region
containing the amino acid sequence set forth in SEQ ID NO: 197, (8)
a light chain variable region containing the amino acid sequence
set forth in SEQ ID NO: 198, (9) a light chain variable region
containing the amino acid sequence set forth in SEQ ID NO: 199,
(10) a light chain variable region containing the amino acid
sequence set forth in SEQ ID NO: 200, (11) a light chain variable
region containing the amino acid sequence set forth in SEQ ID NO:
201, (12) a light chain variable region containing the amino acid
sequence set forth in SEQ ID NO: 202, (13) a light chain variable
region containing the amino acid sequence set forth in SEQ ID NO:
203, (14) a light chain variable region containing the amino acid
sequence set forth in SEQ ID NO: 204, and (15) a light chain
variable region containing the amino acid sequence set forth in SEQ
ID NO: 205, and a heavy chain variable region selected from the
group consisting of the heavy chain variable regions described in
the following (1) to (7): (1) a heavy chain variable region
containing the amino acid sequence set forth in SEQ ID NO: 84, (2)
a heavy chain variable region containing the amino acid sequence
set forth in SEQ ID NO: 85, (3) a heavy chain variable region
containing the amino acid sequence set forth in SEQ ID NO: 86, (4)
a heavy chain variable region containing the amino acid sequence
set forth in SEQ ID NO: 87, (5) a heavy chain variable region
containing the amino acid sequence set forth in SEQ ID NO: 88, (6)
a heavy chain variable region containing the amino acid sequence
set forth in SEQ ID NO: 89, and
[0061] Among the antibodies described above, preferred is the
antibody having a light chain variable region containing the amino
acid sequence set forth in SEQ ID NO: 205 and a heavy chain
variable region containing the amino acid sequence set forth in SEQ
ID NO: 90.
(XI) An antibody, in which one or more amino acids have been
replaced, deleted, added and/or inserted in the amino acid sequence
described in any one of the above-mentioned (I) to (X), and which
has an activity equivalent to that of the antibody described in any
of (I) to (X).
[0062] In the present invention, the activity equivalent to that of
the antibody described in any of (I) to (X) means that the binding
activity to a human glypican 3 antibody or the cytotoxicity
activity on a cell that expresses human glypican 3 (e.g., HepG2 or
a recombinant CHO cells expressing human glypican 3, etc.) is
equivalent.
Humanized Antibody
[0063] One preferred embodiment of the antibody according to the
present invention is a humanized antibody that binds to glypican 3.
The humanized antibody can be prepared by using a known method.
[0064] The humanized antibody is also referred to as a reshaped
human antibody, which is made by transplanting the complementarity
determining region (CDR) of an antibody of a non-human mammal, for
example a mouse antibody, into the CDR of a human antibody. The
general recombinant DNA technology for preparation of such
antibodies is also known (see European Patent Application EP 125023
and International Patent Application WO 96/02576).
[0065] Specifically, for example, in the case where a CDR is
derived from a mouse antibody, a DNA sequence which has been
designed to link the CDRs of the mouse antibody with the framework
region (FR) of a human antibody is synthesized by the PCR method
using several oligonucleotides as primers, which have been prepared
so as to have portions overlapping with one another at both ends of
the CDR and the FR (see the method described in International
Patent Application WO 98/13388).
[0066] As for the framework region of a human antibody to be linked
with the CDR, the one which allows a complementarity determining
region to form a favorable antigen-binding site is selected. If
necessary, an amino acid in the framework region of a variable
region of the antibody may be replaced so that the complementarity
determining region of a reshaped human antibody may form an
appropriate antigen-binding site (Sato, K. et al., Cancer Res.
(1993) 53, 851-856).
[0067] The C region of a human antibody may be used as the C region
of a chimeric antibody or a humanized antibody, for example,
C.gamma.1, C.gamma.2, C.gamma.3, and C.gamma.4 may be used in the H
chain, and C.kappa. and C.lamda., may be used in the L chain. The C
region of a human antibody may also be modified in order to improve
the stability of the antibody or the production thereof. The human
antibody to be used in the humanization may be any isotype of human
antibody, for example, IgG, IgM, IgA, IgE and IgD, preferably, IgG,
more preferably IgG1 or IgG3, and particularly preferably IgG1. the
present invention IgG1 is effective when an antibody is used as an
anticancer agent in terms of having a high cytotoxicity activity
(Chemical immunology, 65: 88 (1997)).
[0068] In addition, after the humanized antibody is prepared, an
amino acid in a variable region (e.g., FR) or a constant region may
be replaced with another amino acid.
[0069] The origin of the CDR in a humanized antibody is not
particularly limited, and the CDR may be derived from any animals.
For example, it is possible to use a sequence derived from a mouse
antibody, a rat antibody, a rabbit antibody, a camel antibody or
the like. Preferred is a CDR sequence of a mouse antibody.
[0070] With regard to the humanization of an antibody, it is
generally difficult to humanize it while maintaining the agonist
activity of the original antibody. In the present invention,
however, a humanized antibody having an agonist activity equivalent
to that of the original mouse antibody was successful acquired.
Since the antigenicity of the humanized antibody in the human body
is reduced, it is useful in administering it into the human for a
therapeutic purpose.
[0071] Preferred examples of the humanized anti-glypican 3 antibody
in the present invention include, for example, an antibody having a
heavy chain variable region set forth in SEQ ID NO: 84 (GC33 VH
ver.a), SEQ ID NO: 85 (GC33 VH ver.c), SEQ ID NO: 86 (GC33 VH
ver.f), SEQ ID NO: 87 (GC33 VH ver.h), SEQ ID NO: 88 (GC33 VH
ver.i), SEQ ID NO: 89 (GC33 VH ver.j) or SEQ ID NO: 90 (GC33 VH
ver.k) or an antibody having a light chain variable region set
forth in SEQ ID NO: 92 (GC33 VL ver.a). Particularly preferred
examples thereof include an antibody having a heavy chain variable
region set forth in SEQ ID NO: 84 (GC33 VH ver.a), SEQ ID NO: 85
(GC33 VH ver.c), SEQ ID NO: 86 (GC33 VH ver.f), SEQ ID NO: 87 (GC33
VH ver.h), SEQ ID NO: 88 (GC33 VH ver.i), SEQ ID NO: 89 (GC33 VH
ver.j) or SEQ ID NO: 90 (GC33 VH ver.k) and a light chain variable
region set forth in SEQ ID NO: 92 (GC33 VL ver.a).
[0072] In addition, a preferred example of the humanized
anti-glypican 3 antibody includes an antibody having a heavy chain
variable region containing the amino acid sequence set forth in SEQ
ID NO: 90 and a light chain variable region containing the amino
acid sequence set forth in SEQ ID NO: 205.
[0073] A preferred embodiment of the antibody according to the
present invention is an antibody that binds to the epitope to which
the antibody set forth in any of the following (1) to (8)
binds:
(1) an antibody containing a heavy chain variable region having the
amino acid sequence set forth in SEQ ID NO: 62 and a light chain
variable region having the amino acid sequence set forth in SEQ ID
NO: 73 (GC33), (2) an antibody containing a heavy chain variable
region having the amino acid sequence set forth in SEQ ID NO: 26
and a light chain variable region the amino acid sequence set forth
in SEQ ID NO: 48 (M11F1), (3) an antibody containing a heavy chain
variable region having the amino acid sequence set forth in SEQ ID
NO: 25 and a light chain variable region the amino acid sequence
set forth in SEQ ID NO: 47 (M3B8), (4) an antibody containing a
heavy chain variable region having the amino acid sequence set
forth in SEQ ID NO: 60 and a light chain variable region the amino
acid sequence set forth in SEQ ID NO: 71 (GC199), (5) an antibody
containing a heavy chain variable region having the amino acid
sequence set forth in SEQ ID NO: 61 and a light chain variable
region the amino acid sequence set forth in SEQ ID NO: 72 (GC202),
(6) an antibody containing a heavy chain variable region having the
amino acid sequence set forth in SEQ ID NO: 63 and a light chain
variable region the amino acid sequence set forth in SEQ ID NO: 74
(GC179), (7) an antibody containing a heavy chain variable region
having the amino acid sequence set forth in SEQ ID NO: 64 and a
light chain variable region the amino acid sequence set forth in
SEQ ID NO: 75 (GC194 (1)), and (8) an antibody containing a heavy
chain variable region having the amino acid sequence set forth in
SEQ ID NO: 64 and a light chain variable region the amino acid
sequence set forth in SEQ ID NO: 76 (GC194 (2)). More preferred is
an antibody that binds to the epitope to which the antibody
described in any of (1) to (5) binds, and particularly preferred is
an antibody that binds to the epitope to which the antibody
described in (1) binds.
[0074] The antibody that binds to the epitope to which any of the
above-mentioned antibodies bind is useful because it has a
particularly high cytotoxicity.
[0075] The antibody described in any of (1) to (7) binds to a
region from the 524th amino acid to the 580th amino acid of human
glypican 3. In particular, it binds to a region from the 524th
amino acid to the 563rd amino acid. The antibody described in any
of (1) to (5) binds to a region from the 537th amino acid to the
563rd amino acid of human glypican 3. The antibody described in (1)
binds to a region from the 544th amino acid to the 553rd amino acid
of human glypican 3. In particular, it binds to a region from the
546th amino acid to the 551st amino acid.
[0076] The antibodies recognizing the above-mentioned epitopes have
a high cytotoxicity, therefore they are useful in the treatment of
a disease such as cancer. In particular, the antibody which binds
to a region from the 546th amino acid to the 551st amino acid is
useful as it has a particularly high cytotoxicity
[0077] Accordingly, the present invention includes the antibodies
which binds to an epitope in a region from the 524th amino acid to
the 580th amino acid of human glypican 3, preferably a region from
the 524th amino acid to the 563rd amino acid, more preferably a
region from the 537th amino acid to the 563rd amino acid, further
more preferably a region from the 544th amino acid to the 553rd
amino acid, particularly preferably a region from the 546th amino
acid to the 551st amino acid.
[0078] Another preferred embodiment according to the present
invention is an antibody that recognizes a region from the 524th
amino acid to the 563rd amino acid of human glypican 3 and does not
recognize a region from the 537th amino acid to the 563rd amino
acid.
[0079] A further preferred embodiment according to the present
invention is an antibody that recognizes a region from the 537th
amino acid to the 563rd amino acid of human glypican 3 and does not
recognize a region from the 550th amino acid to the 563rd amino
acid.
[0080] The analysis of an epitope recognized by an antibody can be
carried out by a method known to those skilled in the art, for
example, by Western blotting described in Examples below.
[0081] The antibody that recognizes the above-mentioned regions as
an epitope can be obtained by a method known to those skilled in
the art. For example, it can be obtained by preparing a peptide
containing an amino acid sequence of a target region based on an
amino acid sequence of human glypican 3 and preparing an antibody
with the use of the peptide as an immunogene, or by preparing an
antibody by a usual method and determining an epitope that the
obtained antibody recognizes, and then selecting an antibody that
recognizes the target epitope.
[0082] A preferred example of the anti-glypican 3 antibody of the
present invention is an antibody having a high ADCC activity or an
antibody having a high CDC activity to a cell that expresses
glypican 3.
[0083] The phrase "a high ADCC activity" or "a high CDC activity"
as used herein means that the antibody of the invention has a
higher ADCC activity or a higher CDC activity than that of a known
anti-glypican 3 antibody. Known glypican 3 antibodies include, for
example, M3C11 and M1E07 described in International Patent
Application WO 2004/22739.
[0084] The ADCC activity or the CDC activity can be measured by a
method known to those skilled in the art. For example, it can be
measured by the chromium release test. Specific conditions of the
chromium release test for measuring the ADCC activity are not
particularly limited, however, for example, it can be measured
using the conditions described in the Examples below.
[0085] Examples of the cells that express glypican 3 include, for
example, a hepatoma cell line such as HepG2, a CHO cell line having
a gene encoding glypican 3 incorporated therein and the like. To
measure the ADCC activity, it is preferred to use a HepG2 cell
line, and to measure the CDC activity, it is preferred to use a
recombinant CHO cell line that expresses GPC3. The recombinant CHO
cell line that expresses GPC3 may be prepared by any method,
however, it can be prepared by, for example, the method described
in the Examples below.
[0086] In the case where the anti-glypican 3 antibody is used as an
anticancer agent, it is preferred that it has an ADCC activity at
the same level as that of an antibody containing a heavy chain
variable region having the amino acid sequence set forth in SEQ ID
NO: 62 and a light chain variable region having the amino acid
sequence set forth in SEQ ID NO: 73 (GC33). In the case where the
anti-glypican 3 antibody is used as an anticancer agent, it is
preferred that it has a CDC activity at the same level as that of
an antibody containing a heavy chain variable region having the
amino acid sequence set forth in SEQ ID NO: 62 and a light chain
variable region having the amino acid sequence set forth in SEQ ID
NO: 73 (GC33).
[0087] Further, the present invention includes an antibody having a
high binding activity to glypican 3.
[0088] In the present invention, the binding activity of the
antibody to glypican 3 can be measured by using a method known to
those skilled in the art. For example, it can be measured by
utilizing the surface plasmon resonance with BIAcore. Specifically,
a glypican 3 protein is immobilized on a sensor chip to react with
an antibody, and the interaction between the antibody and glypican
3 can be calculated as a reaction rate constant from the
measurement value. In addition, with regard to the evaluation of
the binding activity, an enzyme linked immunosorbent assay (ELISA),
an enzyme immunoassay (EIA), a radioimmunoassay (RIA) or a
fluorescent antibody technique can be used. For example, in the
case where an enzyme immunoassay is used, a sample containing an
antibody to be tested, for example, a culture supernatant of a cell
producing an antibody to be tested or a purified antibody is added
to a plate which has been coated with an antigen to which the
antibody to be tested binds. Then, a secondary antibody labeled
with an enzyme such as alkaline phosphatase is added, and the
plated is incubated and washed. Then, an enzyme substrate such as
p-nitrophenyl phosphate is added and the absorbance is measured,
whereby an antigen binding activity can be evaluated. The upper
limit of the binding activity is not particularly limited. However,
for example, the upper limit can be defined within the range which
is technically possible by those skilled in the art. It will be
appreciated that the range which is technically possible will be
expanded by the advancement of technology.
[0089] Further, in the present invention, an amino acid to be
deamidated or an amino acid adjacent to an amino acid to be
deamidated may be replaced with another amino acid for the purpose
of, for example, suppressing deamidation to increase the stability
of the antibody. The amino acid to be deamidated includes,
asparagine and glutamine, preferably asparagine. An amino acid
adjacent to asparagine is not particularly limited and may be any
amino acid. It is known that an asparagine-glycine sequence is
particularly susceptible to deamidation, thus, glycine is preferred
as the amino acid adjacent to asparagine. An amino acid used for
replacement is not particularly limited and may be any amino acid
other than asparagine and glutamine. Preferred is an amino acid
other than valine and proline. Therefore, in the present invention,
in the case where the antibody is deamidated, it is preferred to
replace the amino acid with an amino acid other than asparagine,
glutamine, valine and proline. Suppression of deamidation by amino
acid replacement can be carried out with reference to, for example,
International Patent Application WO 03/057881. In the case where
amino acid replacement is carried out for the purpose of
suppression of deamidation, it is preferred that the antigen
binding activity before replacement is maintained.
[0090] Another embodiment of stabilization of the antibody includes
replacement of glutamic acid with another amino acid. In addition,
in the present invention, it was found that, in the case where the
6th amino acid of the heavy chain of an antibody is glutamic acid,
the antibody can be significantly stabilized by replacing the
glutamic acid with glutamine. Accordingly, the present invention
also relates to a method of stabilizing an antibody by replacing
the glutamic acid at the 6th position of the heavy chain of the
antibody with glutamine. The amino acid numbering of the antibody
is known to those skilled in the art (e.g., Kabat, E. A. et al.,
"Sequences of Proteins of Immunological Interest", US Dept. Health
and Human Services 1983).
[0091] The antibody of the invention may be a conjugated antibody
in which the antibody is conjugated with various molecules, such as
polyethyleneglycol (PEG), radioactive materials and toxin. Such a
conjugated antibody may be prepared by chemically modifying the
antibody obtained as above. Methods for modifying antibodies have
already been established in the art. The antibody of the invention
encompasses such a conjugated antibody.
[0092] The antibody of the invention may also be a bispecific
antibody (see, for example, Journal of Immunology, 1994, 152,
5368-5374). The bispecific antibody may recognize glypican 3 and
another antigen, or may recognize different epitopes on a GPC3
molecule.
[0093] Further, the antibody of the invention may carry a certain
protein fused to the N- or C-terminus of the antibody (Clinical
Cancer Research, 2004, 10, 1274-1281). The protein to be fused to
the antibody may be conveniently selected by those skilled in the
art.
[0094] In addition, the antibody of the invention includes an
antibody with an enhanced cytotoxicity. Examples of the antibody
with an enhanced cytotoxicity include an antibody lacking fucose,
an antibody having bisecting N-acetyl glucosamine (GlcNAc) attached
to its sugar chain, and an antibody having altered binding activity
for Fc.gamma. receptor obtained by substituting one or more amino
acids in the Fc region. Such antibodies with an enhanced
cytotoxicity can be prepared by a method known in the art.
Method of Preparing Antibody
[0095] The antibody that binds to glypican 3 can be prepared by a
method known to those skilled in the art. For example, a monoclonal
antibody-producing hybridoma can be prepared as follows basically
using a known technique. That is, the hybridoma can be prepared by
immunizing a mammal in accordance with a usual immunization method
using a glypican 3 protein or a cell that expresses glypican 3 as a
sensitizing antigen. The thus obtained immunocyte is fused with a
known parent cell by a usual cell fusion method, and then selecting
a monoclonal antibody-producing cell by a usual screening
method.
[0096] Specifically, a monoclonal antibody can be prepared as
follows. First, a glypican 3 protein is obtained based on the
glypican 3 gene/amino acid sequence shown in SEQ ID NOs: 3 and 4,
which is used as a sensitizing antigen to obtain an antibody. More
specifically, the gene sequence encoding glypican 3 is inserted
into a known expression vector system, and an appropriate host cell
is transformed with the vector, and then a target human glypican 3
protein is purified by a known method from the host cell or the
culture supernatant.
[0097] Subsequently, this purified glypican 3 protein is used as a
sensitizing antigen. Alternatively, a partial peptide of glypican 3
can be used as a sensitizing antigen. In this case, the partial
peptide can also be obtained by chemical synthesis according to the
amino acid sequence of human glypican 3.
[0098] The epitope on a glypican 3 molecule which is recognized by
the anti-glypican 3 antibody of the present invention is not
limited to a particular epitope. The anti-glypican 3 antibody may
recognize any epitope, as long as the epitope is present on a
glypican 3 molecule. Accordingly, any fragment can also be used as
an antigen for producing the anti-glypican 3 antibody of the
present invention, as long as it contains an epitope that is
present on a glypican 3 molecule.
[0099] A mammal to be immunized with a sensitizing antigen is not
particularly limited, but it is preferably selected in view of
compatibility with a parent cell to be used for cell fusion. For
example, rodents such as mice, rats and hamsters, rabbits or
monkeys are generally used.
[0100] Immunization of an animal with a sensitizing antigen is
carried out in accordance with a known method. For example,
immunization is carried out by a general method in which a mammal
is injected intraperitoneally or subcutaneously with a sensitizing
antigen. Specifically, a sensitizing antigen is diluted with or
suspended in an appropriate amount of PBS (Phosphate-Buffered
Saline), physiological saline or the like, an appropriate amount of
a standard adjuvant such as a Freund's complete adjuvant is mixed
with the product if necessary, and then the solution is emulsified
and is administered to a mammal several times every 4 to 21 days.
In addition, an appropriate carrier can also be used upon
immunization with a sensitizing antigen.
[0101] A mammal is immunized as described above, and then an
increased level of a target antibody in the serum is confirmed.
Subsequently, immunocytes are collected from the mammal, and then
subjected to cell fusion. A particularly preferred immunocyte is a
splenocyte.
[0102] As a parent partner cell to be fused with the
above-mentioned immunocyte, a mammalian myeloma cell is used.
Examples of a cell line of the myeloma cell that is preferably used
herein include various known cell lines such as P3 (P3x63 Ag8.653)
(J. Immnol. (1979) 123, 1548-1550), P3x63 Ag8U.1 (Current Topics in
Microbiology and Immunology (1978) 81, 1-7), NS-1 (Kohler. G. and
Milstein, C. Eur. J. Immunol. (1976) 6, 511-519), MPC-11
(Margulies. D. H. et al., Cell (1976) 8, 405-415), SP2/0 (Shulman,
M. et al., Nature (1978) 276, 269-270), FO (de St. Groth, S. F. et
al., J. Immunol. Methods (1980) 35, 1-21), 5194 (Trowbridge, I. S.
J. Exp. Med. (1978) 148, 313-323) and R210 (Galfre, G. et al.,
Nature (1979) 277, 131-133).
[0103] Cell fusion of the above-mentioned immunocytes with myeloma
cells can be basically carried out in accordance with a known
method, for example, the method of Kohler and Milstein et al.
(Kohler. G. and Milstein, C., Methods Enzymol. (1981) 73,
3-46).
[0104] More specifically, the above-mentioned cell fusion is
carried out in a normal nutrition culture solution in the presence
of, for example, a cell-fusion accelerator. As the cell-fusion
accelerator, for example, polyethylene glycol (PEG), a
hemagglutinating virus of Japan (HVJ) is used. If desired, an
adjuvant such as dimethylsulfoxide can be added to further enhance
the fusion efficiency.
[0105] The ratio of immunocytes to myeloma cells may be
appropriately selected. For example, it is preferred that the
number of immunocytes is 1 to 10 times greater than that of myeloma
cells. The culture solution to be used for the above-mentioned cell
fusion include, for example, a RPMI1640 culture solution or a MEM
culture solution which is suitable for the growth of the
above-mentioned myeloma cell line, or another normal culture
solution which is used for this type of cell culture. Moreover, a
serum supplement such as fetal calf serum (FCS) can be used in
combination therewith.
[0106] Cell fusion is carried out as follows. Predetermined amounts
of the above-mentioned immunocytes and myeloma cells are mixed well
in the above-mentioned culture solution, a PEG (e.g., with an
average molecular weight of approximately 1000 to 6000) solution (a
general concentration of 30 to 60% (w/v)), which had been
pre-heated at approximately 37.degree. C., is added, and then the
solution is mixed, whereby a target fusion cell (hybridoma) is
formed. Subsequently, an appropriate culture solution is added
successively, and then a step of removing the supernatant by
centrifugation is repeated to remove a reagent for cell fusion or
the like that is unfavorable for the growth of the hybridoma.
[0107] The thus obtained hybridoma is selected by culturing the
hybridoma in a standard selective culture solution such as a HAT
culture solution (a culture solution containing hypoxanthine,
aminopterin and thymidine). Cultivation in the above-mentioned HAT
culture solution is continued for a time period sufficient for the
cells (unfused cells) other than the target hybridoma to die
(normally, several days to several weeks). Subsequently, a standard
limiting dilution method is conducted to screen for and monoclone
of hybridoma that produces a target antibody.
[0108] In addition to the method of immunizing a non-human animal
with an antigen to obtain hybridoma, a desired human antibody
having a binding activity to glypican 3 can also be obtained by
sensitizing a human lymphocyte with glypican 3 in vitro, and
allowing the sensitized lymphocyte to fuse with a human-derived
myeloma cell having a permanent division potential (see
JP-B-1-59878). In another method, glypican 3 antigen is
administered to a transgenic animal having all the repertories of
human antibody genes to obtain anti-glypican 3 antibody-producing
cells, which are then immortalized, and a human antibody for
glypican 3 may be obtained from the immortalized anti-glypican 3
antibody-producing cells (see International Patent Applications WO
94/25585, WO 93/12227, WO 92/03918 and WO 94/02602).
[0109] The thus prepared hybridoma that produce a monoclonal
antibody can be passage-cultured in a standard culture solution, or
can be stored for a long period in liquid nitrogen.
[0110] One example of a method employed to obtain a monoclonal
antibody from the hybridoma involves culturing the hybridoma and
obtaining a monoclonal antibody from the culture supernatant in
accordance with a standard method. Another method involves
administering the hybridoma to a mammal that is compatible with the
hybridoma to allow it to proliferate, and obtaining a monoclonal
antibody from the ascites. The former method is suitable to obtain
an antibody of high purity, while the latter method is suitable for
the mass production of antibodies.
[0111] It is also possible to prepare a recombinant antibody by
cloning the antibody gene from the hybridoma, incorporating the
gene into an appropriate vector, introducing the vector into a
host, and then allowing the host to produce the recombinant
antibody by a genetic engineering technique (e.g., see Vandamme, A.
M. et al., Eur. J. Biochem. (1990) 192, 767-775, 1990).
[0112] Specifically, mRNA encoding the variable (V) region of an
anti-glypican 3 antibody is isolated from a hybridoma producing the
anti-glypican 3 antibody. mRNA is isolated by a known method such
as a guanidine ultracentrifugal method (Chirgwin, J. M. et al.,
Biochemistry (1979) 18, 5294-5299) or an AGPC method (Chomczynski,
P. et al., Anal. Biochem. (1987) 162, 156-159), and total RNA is
prepared, and then target mRNA is prepared using an mRNA
Purification Kit (Pharmacia) or the like. In addition, mRNA can
also be directly prepared using a QuickPrep mRNA Purification Kit
(Pharmacia).
[0113] The cDNA of the antibody V region is synthesized using a
reverse transcriptase from the thus obtained mRNA. cDNA may be
synthesized using an AMV Reverse Transcriptase First-strand cDNA
Synthesis Kit (SEIKAGAKU CORPORATION) or the like. In addition, for
example, a 5'-Ampli FINDER RACE Kit (Clontech), the 5'-RACE method
using PCR (Frohman, M. A. et al., Proc. Natl. Acad. Sci. USA (1988)
85, 8998-9002, Belyaysky, A. et al., Nucleic Acids Res. (1989) 17,
2919-2932) can be employed for synthesizing and amplifying
cDNA.
[0114] A target DNA fragment is purified from the thus obtained PCR
product, and then ligated to a vector DNA. A recombinant vector is
prepared from the product, and then the vector is introduced into
E. coli or the like, and a colony is selected, thereby preparing a
desired recombinant vector. The nucleotide sequence of the target
DNA is then determined by a known method such as a
dideoxynucleotide chain termination method.
[0115] After DNA encoding the V region of the target anti-glypican
3 antibody is obtained, this DNA is incorporated into an expression
vector containing DNA encoding the constant region (C region) of
the target antibody.
[0116] To produce the anti-glypican 3 antibody used in the present
invention, the antibody gene is incorporated into an expression
vector so that the gene is expressed under the regulation of the
gene expression control region including, for example, an enhancer
and a promoter. Next, a host cell is transformed with the
expression vector, thereby allowing the host to express the
antibody.
[0117] An antibody gene can be expressed by incorporating a
polynucleotide encoding the H chain or a polynucleotide encoding
the L chain separately into an expression vector, and then
simultaneously transforming a host cell with the vectors, or by
incorporating polynucleotides encoding the H chain and the L chain
into a single expression vector, and then transforming a host cell
with the vector (see International Patent Application WO
94/11523).
Polynucleotide
[0118] In another aspect, the present invention provides a
polynucleotide encoding a heavy chain variable region or a light
chain variable region of the antibody of the present invention.
Preferably, the polynucleotide of the present invention has a
nucleotide sequence described in any of SEQ ID NOs: 11-21, 33-43,
55-59, 65-70 and 77-83. In addition, a polynucleotide that is
hybridized to the above-mentioned polynucleotide under stringent
conditions and encodes an antibody having an activity equivalent to
that of the antibody of the present invention is also within the
scope of the present invention.
[0119] The polynucleotide of the present invention is not
particularly limited as long as it encodes the antibody of the
present invention. It is a polymer composed of a plurality of
nucleotides, such as deoxyribonucleic acids (DNA) or ribonucleic
acids (RNA). It may contain a base other than a naturally occurring
base. The polynucleotide of the present invention can be used for
producing an antibody by a genetic engineering technique. In
addition, the polynucleotide of the present invention can be used
as a probe to screen for an antibody having a function equivalent
to that of the antibody of the present invention. That is, a
polynucleotide encoding the antibody of the present invention or a
partial fragment thereof may be used as a probe to obtain DNA that
is hybridized to the polynucleotide under stringent conditions and
encodes an antibody having an activity equivalent to that of the
antibody of the present invention by techniques such as a
hybridization technique, a gene amplification technique (e.g.,
PCR). Such DNA is also included in the polynucleotide of the
present invention.
[0120] The hybridization technique (Sambrook, J. et al., Molecular
Cloning 2nd ed., 9.47-9.58, Cold Spring Harbor Lab. Press, 1989) is
well known to those skilled in the art. Examples of the
hybridization conditions include, for example, low stringent
conditions. The low stringent conditions are, for example, the
conditions of 42.degree. C., 0.1.times.SSC and 0.1% SDS, preferably
the conditions of 50.degree. C., 0.1.times.SSC and 0.1% SDS when
washing is performed after hybridization. More preferred examples
of the hybridization conditions include, for example, high
stringent conditions. The high stringent conditions are, for
example, the conditions of 65.degree. C., 5.times.SSC and 0.1% SDS.
Under these conditions, it can be expected that a polynucleotide
having a higher homology can be efficiently obtained under higher
temperature. Incidentally, there are plural factors that affect the
stringency of hybridization, such as temperature and the
concentration of salt, and those skilled in the art can achieve a
similar stringency by appropriately selecting these factors.
[0121] An antibody functionally equivalent to the antibody of the
present invention encoded by a polynucleotide obtained by such a
hybridization technique and a gene amplification technique usually
has a high homology with the antibody in terms of the amino acid
sequence. The antibody of the present invention also includes an
antibody that is functionally equivalent to the antibody of the
present invention and has a high homology with the amino acid
sequence of the antibody. A high homology means generally at least
50% or higher identity, preferably 75% or higher identity, more
preferably 85% or higher identity, and further more preferably 95%
or higher identity at the amino acid level. To determine the
homology of polypeptides, the algorithm described in the literature
(Wilbur, W. J. and Lipman, D. J., Proc. Natl. Acad. Sci. USA (1983)
80, 726-730) may be employed.
[0122] The present invention also provides a vector containing the
polynucleotide of the present invention. Such a vector can be used
for preparing the antibody of the present invention. As for the
vector of the present invention, in the case where E. coli is used
as a host, for example, it is not particularly limited as long as
it has "ori" for use in amplification in E. coli to produce and
amplify the vector in a large amount in E. coli (e.g., JM109,
DH5.alpha., HB101 or XL1Blue), and has a marker gene for selecting
a transformed E. coli (e.g., a drug resistance gene that can be
identified by a drug such as ampicillin, tetracycline, kanamycin or
chloramphenicol). Examples of the vector include M13-series
vectors, pUC-series vectors, pBR322, pBluescript, pCR-Script and
the like. In addition, pGEM-T, pDIRECT, and pT7 can also be used
for subcloning and extracting cDNA as well as the vectors described
above.
[0123] As the vector of the present invention, an expression vector
is particularly useful. For example, an expression vector to be
expressed in E. coli should have the above characteristics to be
amplified in E. coli. In addition, in the case where E. coli, such
as JM109, DH5.alpha., HB101, or XL1-Blue is used as a host cell, it
is indispensable that the vector should have a promoter, 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 the like, that can
efficiently express the desired product in E. coli. Examples of
such a vector include pGEX-5X-1 (Pharmacia), "QIAexpress system"
(Qiagen), pEGFP, pET (in this case, the host is preferably BL21
which expresses T7 RNA polymerase) and the like, as well as the
vectors described above.
[0124] In addition, the vector may also contain a signal sequence
for polypeptide secretion. As for the signal sequence for protein
secretion, in the case where a polypeptide is produced in the
periplasm of E. coli, the pelB signal sequence (Lei S. P. et al.,
J. Bacteriol (1987) 169, 4379) can be used. Introduction of the
vector into a host cell can be carried out by using, for example,
the calcium chloride method and the electroporation method.
[0125] In addition to E. coli, for example, expression vectors
derived from mammals (e.g., pcDNA3 (Invitrogen) and pEGF-BOS
(Nucleic Acids Res. (1990) 18(17), p 5322), pEF and pCDM8),
expression vectors derived from insect cells (e.g., "Bac-to-BAC
baculovirus expression system" (GIBCO BRL) and pBacPAK8),
expression vectors derived from plants (e.g., pMH1 and pMH2),
expression vectors derived from animal viruses (e.g., pHSV, pMV and
pAdexLcw), expression vectors derived from retroviruses (e.g.,
pZIPneo), expression vectors derived from yeast (e.g., "Pichia
Expression Kit" (Invitrogen), pNV11 and SP-Q01), and expression
vectors derived from Bacillus subtilis (e.g., pPL608 and pKTH50)
can be used as the vector of the present invention.
[0126] For the purpose of expressing the vector in an animal cell
such as a CHO cell, a COS cell, an NIH3T3 cell or the like, it is
indispensable for the vector to have a promoter required for
expression in a cell such as SV40 promoter (Mulligan et al., Nature
(1979) 277, 108), MMTV-LTRpromoter, EF1.alpha. promoter (Mizushima
et al., Nucleic Acids Res. (1990) 18, 5322), CMV promoter or the
like, and more preferably to have a marker gene (such as a drug
resistance gene that can be identified by a drug such as neomycin
or G418) for selecting transformation into the cell. Examples of
the vector having such characteristics include, for example, pMAM,
pDR2, pBK-RSV, pBK-CMV, pOPRSV and pOP13.
[0127] Further, for the purpose of stably expressing a gene and, at
the same time, amplifying the gene copy numbers in the cell, a
vector (e.g., pCHOI, etc.) having the DHFR gene is introduced into
the CHO cell deficient in the nucleic acid synthetic pathway to
complement the deficiency and is amplified with methotrexate (MTX).
In addition, for the purpose of transient expression of a gene,
transformation is effected with a vector (such as pcD) having the
origin of replication for SV40 using a COS cell having on the
chromosome a gene that expresses the SV40 T antigen. As the origin
of replication, the one derived from a polyoma virus, an
adenovirus, a bovine papilloma virus (BPV) and the like can also be
used. Further, for the amplification of gene copy numbers in the
host cell system, the expression vector can include, as a
selectable marker, the aminoglycoside transferase (APH) gene, the
thymidine kinase (TK) gene, E. coli xanthine guaninephosphoribosyl
transferase (Ecogpt) gene, the dihydrofolate reductase (dhfr) gene
and the like.
[0128] To prepare the antibody of the present invention, the vector
is introduced into a host cell. The host cell into which the vector
is introduced is not particularly limited, but includes, for
example, E. coli or any of various animal cells. For example, the
host cell can be used as a production system for the production or
expression of the antibody of the present invention. As for the
production system of polypeptide preparation, there are an in vitro
production system and an in vivo production system. In vitro
production system include a production system which employs
eukaryotic cells and a production system which employs prokaryotic
cells.
[0129] In the case where the eukaryotic cell is used, for example,
an animal cell, a plant cell or a fungal cell can be used. Known
animal cells include a mammalian cell such as a CHO cell (J. Exp.
Med. (1995) 108, 945), a COS cell, a 3T3 cell, a myeloma cell, a
baby hamster kidney (BHK) cell, a HeLa cell and a Vero cell, an
amphibian cell such as a Xenopus oocyte (Valle, et al., Nature
(1981) 291, 358-340), or an insect cell such as Sf9, Sf21, and Tn5.
In the present invention, CHO-DG44, CHO-DXB11, a COST cell, a BHK
cell are preferably used. Among the animal cells, for the purpose
of performing a large amount of expression, a CHO cell is
particularly preferred. Introduction of the vector into the host
cell can be carried out by, for example, the calcium phosphate
method, the DEAE-dextran method, the cationic ribozome DOTAP
(Boehringer Mannheim), the electroporation method, the lipofection
method or the like.
[0130] As for the plant cell, for example, a cell derived from
Nicotiana tabacum is known as a protein production system, which
may be subj ected to callus culture. Examples of the fungal cells
include yeast such as the genus Saccharomyces, more specifically
Saccharomyces cerevistae and Saccharomyces pombe, and filamentous
fungi such as the genus Aspergillus, more specifically Aspergillus
niger.
[0131] In the case where the prokaryotic cell is used, production
system using a bacterial cell may be employed. Examples of the
bacterial cells include Escherichia coli (E. coli) such as JM109,
DH5.alpha. and HB101, and Bacillus subtilis.
Preparation of Recombinant Antibody
[0132] The antibody of the present invention can be prepared by
culturing the above-mentioned host cells. The antibody can be
obtained by culturing in vitro a cell transformed with a desired
polynucleotide. Cultivation can be carried out in accordance with a
known method. Culture media for animal cells include, for example,
DMEM, MEM, RPMI 1640, and IMDM. A serum supplement such as FBS or
fetal calf serum (FCS) may be used in combination, or serum-free
medium can be used. The pH during the cultivation is preferably
about 6 to 8. Cultivation is usually carried out at about 30 to
40.degree. C. for about 15 to 200 hours with, as needed, medium
change, aeration, and agitation.
[0133] On the other hand, systems for producing a polypeptide in
vivo include, for example, a production system which employs an
animal and a production system which employs a plant. The target
polynucleotide is introduced into such an animal or a plant, and
the polypeptide is produced in the body of the animal or the plant
and recovered. The term "host cell" as used herein encompasses such
an animal and a plant.
[0134] When the animal is used, production systems employing a
mammal or an insect are available. As the mammal, goats, pigs,
sheep, mice and cattle can be used (Vicki Glaser, SPECTRUM
Biotechnology Applications, 1993). A transgenic animal can also be
used as a mammal.
[0135] For example, the target polynucleotide is prepared as a
fusion gene with a gene encoding a polypeptide which is inherently
produced in the milk such as goat .beta. casein. Then, the DNA
fragment containing this fusion gene is injected into a goat
embryo, and the embryo is transplanted into a female goat. The
target antibody can be obtained from the milk produced by the
transgenic goat borne to the goat which received the embryo or the
offspring thereof. To increase the amount of milk containing the
antibody produced by the transgenic goat, hormone may be given to
the transgenic goat as needed. (Ebert, K. M. et al., Bio/Technology
(1994) 12, 699-702).
[0136] In addition, as an insect, for example, a silkworm can be
used. In the case where a silkworm is used, a silkworm is infected
with a baculovirus into which the polynucleotide encoding the
target antibody has been inserted. The target antibody can be
obtained from the body fluid of the silkworm (Susumu, M. et al.,
Nature (1985) 315, 592-594).
[0137] In the case where a plant is used, for example, tobacco can
be used. In the case where tobacco is used, a polynucleotide
encoding the target antibody is inserted into an expression vector
for a plant, for example pMON 530, and then the vector is
introduced into a bacterium such as Agrobacterium tumefaciens.
Then, tobacco such as Nicotiana tabacum is infected with the
bacterium, whereby the target antibody can be obtained from the
leaves of the tobacco (Julian, K. -C. Ma et al., Eur. J. Immunol.
(1994) 24, 131-138).
[0138] The thus obtained antibody can be isolated from the inside
or the outside (culture medium, etc.) of the host cell and then can
be purified to a substantially pure and uniform antibody.
Separation and purification of the antibody may be carried out by a
separation and a purification method usually used in purification
of polypeptides. For example, polypeptides can be separated and
purified by any methods including chromatography columns,
filtration, ultrafiltration, salting-out, solvent precipitation,
solvent extraction, distillation, immunoprecipitation,
SDS-polyacrylamide gel electrophoresis, isoelectric focusing,
dialysis, recrystallization, and a combination thereof.
[0139] Examples of the chromatography include, for example,
affinity chromatography, ion exchange chromatography, hydrophobic
chromatography, gel-filtration, reverse phase chromatography,
adsorption chromatography (Strategies for Protein Purification and
Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak
et al., Cold Spring Harbor Laboratory Press, 1996). These
chromatographies can be carried out using a liquid phase
chromatography such as HPLC and FPLC. Examples of a column to be
used for affinity chromatography include a protein A column or a
protein G column. One example of the protein A column is Hyper D,
POROS, Sepharose F. F. (Pharmacia).
[0140] Further, before or after purification of the antibody, the
antibody can be modified or peptides can be partially removed as
needed by allowing a suitable protein-modifying enzyme to act on.
The protein-modifying enzyme for this purpose include, for example,
trypsin, chymotrypsin, lysyl endopeptidase, protein kinase,
glucosidase.
Diagnostic Method
[0141] In another aspect, the present invention provides a method
of diagnosing a disease such as cancer by detecting GPC3 protein in
a test sample with the use of the antibody of the present
invention.
[0142] The detection used herein includes quantitative detection
and non-quantitative detection. The non-quantitative detection
include, for example, determination of merely whether or not GPC3
protein is present, determination of whether or not a specific
amount or more of GPC3 protein is present, determination for
comparison of the amount of GPC3 protein with that of another
sample (e.g., a control sample). The quantitative detection
includes determination of the concentration of GPC3 protein,
determination of the amount of GPC3 protein.
[0143] The test sample is not particularly limited as long as it is
a sample that may possible contain GPC3 protein, however, preferred
is a sample collected from the body of a living organism such as a
mammal, and more preferred is a sample collected from human.
Specific examples of the test sample may include, for example,
blood, interstitial fluid, plasma, extravascular fluid, cerebral
fluid, joint fluid, pleural fluid, serum, lymph fluid, saliva,
preferably blood, serum and plasma. In addition, a sample obtained
from the test sample such as culture solution of cells collected
from the body of the living organism is also included in the test
sample of the present invention.
[0144] The cancer to be diagnosed is not particularly limited, and
specific examples may include liver cancer, pancreatic cancer, lung
cancer, colon cancer, mammary cancer, prostate cancer, leukemia and
lymphoma, preferably liver cancer. GPC3 to be detected is not
particularly limited, and may be either full-length GPC3 or a
fragment thereof. In the case where a fragment of GPC3 is detected,
it may be either the N-terminal fragment or the C-terminal
fragment, however, the N-terminal fragment is preferred. In
addition, the GPC3 protein may also be a heparan sulfate-added GPC3
or a GPC3 core protein.
[0145] The method of detecting GPC3 protein contained in a test
sample is not particularly limited, however, detection is
preferably performed by an immunological method with the use of an
anti-GPC3 antibody. Examples of the immunological method include,
for example, a radioimmunoassay, an enzyme immunoassay, a
fluorescence immunoassay, a luminescence immunoassay,
immunoprecipitation, a turbidimetric immunoassay. Preferred is an
enzyme immunoassay, and particularly preferred is an enzyme-linked
immunosorbent assay (ELISA) (e.g., a sandwich ELISA). The
above-mentioned immunological method such as an ELISA can be
carried out by a method known to those skilled in the art.
[0146] A general detection method with the use of an anti-GPC3
antibody comprises immobilizing an anti-GPC3 antibody on a support,
adding a test sample thereto, incubating the support to allow the
anti-GPC3 antibody and GPC3 protein to bind to each other, washing
the support, and detecting the GPC3 protein binding to the support
via the anti-GPC3 antibody to detect GPC3 protein in a test
sample.
[0147] The binding between the anti-GPC3 antibody and the GPC3
protein is generally carried out in a buffer. Buffers used in the
invention include, for example, a phosphate buffer, a Tris buffer.
Incubation is carried out under the conditions generally employed,
for example, at 4.degree. C. to room temperature for 1 hour to 24
hours. The washing after incubation can be carried out by any
method as long as it does not inhibit the binding between the GPC3
protein and the anti-GPC3 antibody, using for example a buffer
containing a surfactant such as Tween 20.
[0148] In the method of detecting GPC3 protein of the present
invention, a control sample may be provided in addition to a test
sample to be tested for GPC3 protein. The control samples include a
negative control sample that does not contain GPC3 protein and a
positive control sample that contains GPC3 protein. In this case,
it is possible to detect GPC3 protein in the test sample by
comparing the result obtained with the negative control sample that
does not contain GPC3 protein with the result obtained with the
positive control sample that contains GPC3 protein. It is also
possible to quantitatively detect GPC3 protein contained in the
test sample by obtaining the detection results of the control
samples and the test sample as numerical values, and comparing
these numerical values.
[0149] One preferred embodiment of detecting GPC3 protein binding
to the support via an anti-GPC3 antibody is a method using an
anti-GPC3 antibody labeled with a detectable label. For example,
GPC3 protein may be detected by contacting the test sample with an
anti-GPC3 antibody immobilized on the support, washing the support,
and then detecting GPC3 with the use of the labeled antibody that
specifically binds to GPC3 protein.
[0150] The labeling of an anti-GPC3 antibody can be carried out by
a generally known method. Examples of the detectable label known to
those skilled in the art include a fluorescent dye, an enzyme, a
coenzyme, a chemiluminescent substance or a radioactive substance.
Specific examples may include radioisotopes (.sup.32P, .sup.14C,
.sup.125I, .sup.3H, .sup.131I and the like), fluorescein,
rhodamine, dansyl chloride, umbelliferone, luciferase, peroxidase,
alkaline phosphatase, .beta.-galactosidase, .beta.-glucosidase,
horseradish peroxidase, glucoamylase, lysozyme, saccharide oxidase,
microperoxidase, biotin and the like. In the case where biotin is
used as a detectable label, it is preferred that a biotin-labeled
antibody is added, and then avidin conjugated to an enzyme such as
alkaline phosphatase is further added.
[0151] Specifically, a solution containing an anti-GPC3 antibody is
added to a support such as a plate to allow the anti-GPC3 antibody
to be immobilized. After washing, the plate is blocked with, for
example, BSA in order to prevent the nonspecific binding of a
protein. The plate is washed again, and then the test sample is
added to the plate. After being incubated, the plate is washed, and
then the labeled anti-GPC3 antibody is added. After being incubated
appropriately, the plate is washed, and then the labeled anti-GPC3
antibody remaining on the plate is detected. The detection of the
protein can be carried out by a method known to those skilled in
the art. For example, in the case where the antibody is labeled
with a radioactive substance, the protein may be detected by liquid
scintillation or the RIA method. In the case where the antibody is
labeled with an enzyme, the protein may be detected by adding a
substrate and detecting an enzymatic change of the substrate such
as color development with an absorbance reader. In the case where
the antibody is labeled with a fluorescent substance, the protein
may be detected with the use of a fluorometer.
[0152] A particularly preferred embodiment of the method of
detecting GPC3 protein of the present invention is a method using
an anti-GPC3 antibody labeled with biotin and avidin. Specifically,
a solution containing an anti-GPC3 antibody is added to a support
such as a plate to allow the anti-GPC3 antibody to be immobilized
thereon. After washing, the plate is blocked with, for example, BSA
in order to prevent the nonspecific binding of a protein, The plate
is washed again, and then the test sample is added to the plate.
After being incubated, the plate is washed, and then the
biotin-labeled anti-GPC3 antibody is added. After being incubated
appropriately, the plate is washed, and then avidin conjugated to
an enzyme such as alkaline phosphatase or peroxidase is added.
After being incubated, the plate is washed, and then a substrate of
the enzyme conjugated to avidin is added. Then, GPC3 protein is
detected by means of the enzymatic change of the substrate as an
indicator.
[0153] Another embodiment of the method of detecting GPC3 protein
of the present invention is a method using a primary antibody that
specifically binds to GPC3 protein and a secondary antibody that
specifically binds to the primary antibody. For example, the test
sample is brought into contact with an anti-GPC3 antibody
immobilized on the support, the support is incubated and washed,
and the bound GPC3 protein after washing is detected with a primary
anti-GPC3 antibody and a secondary antibody that specifically binds
to the primary antibody. In this case, the secondary antibody is
preferably labeled with a detectable label.
[0154] Specifically, a solution containing an anti-GPC3 antibody is
added to a support such as a plate to allow the anti-GPC3 antibody
to be immobilized thereon. After washing, the plate is blocked
with, for example, BSA in order to prevent the nonspecific binding
of a protein. The plate is washed again, and then the test sample
is added to the plate. After being incubated, the plate is washed,
and then a primary anti-GPC3 antibody is added. After being
incubated appropriately, the plate is washed, and then a secondary
antibody that specifically binds to the primary antibody is added.
After being incubated appropriately, the plate is washed, and then
the secondary antibody remaining on the plate is detected. The
detection of the secondary antibody can be carried out by the
above-mentioned method.
Pharmaceutical Composition
[0155] In another aspect, the present invention provides a
pharmaceutical composition containing the antibody of the present
invention. The pharmaceutical composition containing the antibody
of the present invention is useful in the treatment and/or
prevention of a disease associated with cell proliferation such as
cancer, and particularly it is useful in the treatment and/or
prevention of liver cancer. In the case where the antibody of the
present invention is used as a pharmaceutical composition, the
antibody can be formulated into a dosage form by a method known to
those skilled in the art. For example, the pharmaceutical
composition can be used parenterally in the form of an injection of
a sterile solution or a suspension with water or another
pharmaceutically acceptable solution. For example, the antibody can
be formulated into a dosage form by appropriately mixing it with a
pharmaceutically acceptable carrier or solvent, such as sterile
water, physiological saline, a plant-oil, an emulsifier, a
suspension, a surfactant, a stabilizer, a flavor, an excipient, a
vehicle, a preservative, a binder to prepare a unit dosage form
required for generally accepted Drug Implementation. The amount of
active ingredients in these preparations is selected to allow for
administration of a suitable dosage within the indicated range.
[0156] A sterile composition for injection can be formulated by
using a vehicle such as distilled water for injection in accordance
with the general Drug Implementation.
[0157] Examples of the aqueous solution for injection include, for
example, physiological saline, glucose, and other isotonic liquids
including adjuvants, such as D-sorbitol, D-mannnose, D-mannitol and
sodium chloride. They can be used in combination with a suitable
solubilizer, such as an alcohol, specifically ethanol, a
polyalcohol such as propylene glycol and polyethylene glycol, and a
non-ionic surfactant such as Polysorbate 80 (TM) and HCO-50.
[0158] Sesame oil or soybean oil can be used as a oleaginous liquid
and may be used in combination with benzyl benzoate or benzyl
alcohol as a solubilizer. It may be formulated with a buffer such
as a phosphate buffer or a sodium acetate buffer, a pain-killer
such as procaine hydrochloride, a stabilizer such as benzyl alcohol
or phenol, or an antioxidant. The prepared injection is generally
filled into a suitable ampule.
[0159] The method of administration is preferably parenteral, and
specific examples thereof include injection, transnasal
administration, transpulmonary administration, transdermal
administration and the like. The injection formulation may be
administered systemically or topically by intravenous injection,
intramuscular injection, intraperitoneal injection, subcutaneous
injection or the like.
[0160] The method of administration can be appropriately selected
according to the age and the symptoms of a patient. For example,
one dose of the pharmaceutical composition containing the antibody
or the polynucleotide encoding the antibody can be selected from
the range of 0.0001 mg to 1,000 mg per kg of body weight.
Alternatively, for example, the dose can be selected from the range
of 0.001 mg to 100,000 mg/body per patient, although it is not
always limited to these numerical values. The dose and the method
of administration vary according to the body weight, the age and
the symptoms of a patient, and are appropriately selected by those
skilled in the art.
[0161] All patents and references cited in this specification are
incorporated by reference. All the contents disclosed in the
specifications and drawings of Japanese Patent Application No.
2004-203637, on which the application claims priority, are
incorporated herein by reference.
EXAMPLE
[0162] The present invention will be described in more detail with
reference to Examples below. However, the present invention is not
limited to these Examples.
Example 1
cDNA Cloning of Human Glypican 3 (GPC3)
[0163] A full-length cDNA encoding human GPC3 was amplified by PCR
reaction with an Advantage 2 kit (CLONTECH) using 1st stranded cDNA
prepared by a usual method from a colon cancer cell line, Caco2, as
a template. More specifically, 50 .mu.L of a reaction mixture
containing 2 .mu.l of cDNA derived from Caco2, 1 .mu.L of a sense
primer (GATATC-ATGGCCGGGACCGTGCGCACCGCGT: SEQ ID NO: 1), 1 .mu.L of
an antisense primer (GCTAGC-TCAGTGCACCAGGAAGAAGAAGCAC: SEQ ID NO:
2), 5 .mu.L of Advantage 2 10.times.PCR buffer, 8 .mu.L of dNTP mix
(1.25 mM) and 1.0 .mu.L of Advantage polymerase Mix was subjected
to 35 cycles consisting of 94.degree. C. for 1 minute, 63.degree.
C. for 30 seconds and 68.degree. C. for 3 minutes. The amplified
product from the PCR reaction was inserted into a TA vector, pGEM-T
Easy, using pGEM-T Easy Vector System I (Promega). The sequence was
confirmed by using an ABI 3100 DNA sequencer. In this way, a cDNA
encoding full-length human GPC3 was isolated. The sequence shown in
SEQ ID NO: 3 indicates the nucleotide sequence of human GPC3 gene
and the sequence shown in SEQ ID NO: 4 indicates the amino acid
sequence of human GPC3 protein.
Example 2
Preparation of Soluble Form of GPC3
[0164] As immunoprotein for the generation of an anti-GPC3
antibody, a soluble form of GPC protein was prepared, in which a
hydrophobic region at the C-terminal side (564-580 amino acids) was
deleted.
[0165] By using the full-length human GPC3 cDNA as a template, a
PCR reaction was carried out using an antisense primer (ATA GAA TTC
CAC CAT GGC CGG GAC CGT GCG C: SEQ ID NO: 5) and a sense primer, to
which an EcoRI recognition sequence and a Kozak sequence were
added, (ATA GGA TCC CTT CAG CGG GGA ATG AAC GTT C: SEQ ID NO: 6).
The obtained PCR fragment (1711 bp) was cloned into pCXND2-Flag.
The pCXND2-Flag was designed to express a Flag-tagged protein by
inserting the region for DHFR gene expression of pCHOI (Hirata et
al., FEBS letter 1994; 356; 244-248) into the HindIII site of pCXN2
(Nivea et al., Gene 1991; 108; 193-199) and adding a Flag tag
sequence to the downstream of the multicloning site. The
constructed expression plasmid DNA was introduced into a CHO cell
line, DXB11, and a CHO cell line highly expressing the soluble form
of GPC3 was obtained by selection with 500 .mu.g/mL Geneticin. The
large-scale cultivation of the CHO cell line highly expressing the
soluble form of GPC3 was carried out using a 1700-cm.sup.2 roller
bottle, and the culture supernatant was recovered for the antibody
purification. The culture supernatant was applied to a DEAE
sepharose Fast Flow column (Amersham) and, after washing, the
antibody was eluted with a buffer containing 500 mM NaCl, and
affinity purified using Anti-Flag M2 agarose affinity gel (SIGMA).
The elution was carried out with 200 .mu.g/mL FLAG peptide. After
the eluate was concentrated with Centriprep-10 (Millipore), FLAG
peptide was removed by gel filtration using Superdex 200 HR 10/30
(Amersham). Lastly, the filtrate was concentrated using a DEAE
sepharose Fast Flow column and eluted with PBS (containing 500 mM
NaCl) without Tween 20 to effect buffer exchange.
Example 3
Preparation of Soluble Form of GPC3 Core Protein
[0166] GPC3 is modified by heparan sulfate to become a
macromolecule. To eliminate an antibody against heparan sulfate in
a screening for an anti-GPC3 antibody, a soluble form of GPC3 core
protein that had a point mutation in the heparan sulfate-binding
site was prepared and used in the screening.
[0167] Using the above-mentioned soluble form of GPC3 (1-563) as a
template, a cDNA in which Ser residues at the 495th and 509th
positions were replaced with Ala was prepared by the assembly PCR
method, in which primers were designed to add His tag to the
C-terminus. The obtained cDNA was cloned into pCXND3 vector. The
pCXND3 was constructed by inserting the DHFR gene expressing region
of pCHOI in the HindIII site of pCXN2. The constructed expression
plasmid DNA was introduced into DXB11 cell line and a CHO cell line
highly expressing a soluble form of GPC3 core protein was obtained
by selection with 500 .mu.g/mL Geneticin.
[0168] The large-scale cultivation was carried out using a
1700-cm.sup.2 roller bottle and the culture supernatant was
recovered for antibody purification. The culture supernatant was
applied to a Q sepharose Fast Flow column (Amersham). After
washing, the antibody was eluted with a phosphate buffer containing
500 mM NaCl, and affinity purified using a Chelating sepharose Fast
Flow column (Amersham). The antibody was eluted with a gradient of
10 to 150 mM imidazole. Lastly, the eluate was concentrated using a
Q sepharose Fast Flow column and, eluted with a phosphate buffer
containing 500 mM NaCl.
[0169] SDS polyacrylamide gel electrophoresis under reducing
conditions showed three bands of 70 kDa, 40 kDa and 30 kDa. The
result of amino acid sequencing using an ABI492 protein sequencer
(Applied Biosystems) indicated that the 30 kDa band corresponded to
the amino acid sequence of the 359th and its downstream or the
375th and its downstream. of GPC3, suggesting that GPC3 was cleaved
between Arg358 and Ser359 or between Lys374 and Va1375, hence, it
was separated into 40 kDa of the N-terminal fragment and 30 kDa of
the C-terminal fragment.
Example 4
Preparation of CHO Cell Line Expressing Full-Length Human GPC3
[0170] To obtain a cell line for evaluating a binding activity
using flow cytometry, a CHO cell line expressing full-length GPC3
was established.
[0171] Ten microgram of a full-length human GPC3 gene expression
vector and 60 .mu.L of SuperFect (QIAGEN) were mixed. After a
complex was formed, gene introduction was carried out by adding it
to a CHO cell line, DXB11. After a 24-hour cultivation in a
CO.sub.2 incubator, selection was started using .alpha.MEM (GIBCO
BRL) containing Geneticin at a final concentration of 0.5 mg/mL and
10% FBS. The resulting Geneticin-resistant colonies were collected
and cell cloning was carried out by the limiting dilution method.
Each cell clone was solubilized and the expression of full-length
human GPC3 was confirmed by Western blotting using an anti-GPC3
antibody. In this way, a stably expressing cell line was
obtained.
Example 5
Evaluation of Binding Activity by ELISA
[0172] The soluble form of GPC3 core protein was diluted to 1
.mu.g/mL with a coating buffer (0.1 mol/L NaHCO.sub.3 (pH 9.6),
0.02% (w/v) NaN.sub.3) and added to an immunoplate and left at
4.degree. C. overnight to coat the plate. After the plate was
blocked with a dilution buffer (50 mmol/L Tris-HCl (pH 8.1), 1
mmol/L MgCl.sub.2, 150 mmol/L NaCl, 0.05% (v/v) Tween 20, 0.02%
(w/v) NaN.sub.3, 1% (w/v) BSA), an anti-GPC3 antibody was added and
left at room temperature for 1 hour. After washing with a rinse
buffer (0.05% (v/v) Tween 20, PBS), an anti-mouse IgG antibody
(ZYMED) labeled with alkaline phosphatase was added and left at
room temperature for 1 hour. After washing with the rinse buffer,
SIGMA 104 (SIGMA) diluted to 1 mg/mL with a substrate buffer (50
mmol/L NaHCO.sub.3 (pH 9.8), 10 mmol/L MgCl.sub.2) was added and
left at room temperature for 1 hour for color development. Then the
absorbance (at 405 nm, reference wavelength of 655 nm) was measured
using a Benchmark Plus (BIO-RAD).
Example 6
Immunization with Soluble Form of GPC3 and Selection of
Hybridoma
[0173] Since human GPC3 and mouse GPC3 show a high homology of 94%
at the amino acid level, it was considered difficult to obtain an
anti-GPC3 antibody if a normal mouse was immunized.
[0174] Therefore, an autoimmune disease mouse,
MRL/MpJUmmCrj-lpr/lpr mouse, (hereinafter referred to as MRL/lpr
mouse, purchased from Charles River Japan, Inc.) was used as an
immunized animal. Immunization was started at the age of 7 weeks or
8 weeks. For the first immunization, a soluble form of GPC3 was
prepared at 100 .mu.g/head and emulsified using Freund's complete
adjuvant (FCA, Becton Dickinson) and subcutaneously administered.
Two weeks later, a soluble form of GPC3 was prepared at 50
.mu.g/head and emulsified using Freund's incomplete adjuvant (FIA,
Becton Dickinson) and subcutaneously administered. After that, an
additional immunization was carried out every other week for 5
times in total. To two of the immunized mice, a soluble form of
GPC3 was diluted with PBS to 50 .mu.g/head, and then administered
intravenously via the tail as the final immunization. On the forth
day after the final immunization, the spleen was excised to obtain
a spleen cell, which was mixed with a mouse myeloma cell,
P3-X63Ag8U1 (P3U1, purchased from ATCC), at a ratio of 2:1. Cell
fusion was carried out by gradually adding PEG 1500 (Roche
Diagnostic). RPMI 1640 medium (GIBCO BRL) was carefully added to
dilute PEG 1500, and after PEG 1500 was removed by centrifugation,
the cells were suspended in RPMI 1640 medium containing 10% FBS and
inoculated into a 96-well culture plate at 100 .mu.L/well. On the
next day, RPMI 1640 medium containing 10% FBS, lx HAT media
supplement (SIGMA) and 0.5.times. BM-Condimed H1 Hybridoma cloning
supplement (Roche Diagnostic) (hereinafter referred to as HAT
medium) was added at 100 .mu.L/well. After 2, 3 and 5 days, half of
the culture solution was replaced with the HAT medium. After 7
days, screening was carried out using the culture supernatant. The
screening was carried out by an ELISA using an immunoplate coated
with the soluble form. of GPC3 core protein. A positive clone was
monocloned by the limiting dilution method. As a result, 11 clones
of antibodies (M3C11, M13B3, M1E7, M3B8, M11F1, L9G11, M19B11,
M6B1, M18D4, M5B9 and M10D2) that have a strong binding activity
against GPC3 were obtained.
Example 7
Isotype Determination and Purification of Anti-GPC3 Antibody
[0175] Isotype was determined by an antigen-dependent ELISA using
an Immunopure Monoclonal Antibody Isotyping Kit I (PIERCE). The
purification of antibodies was carried out as follows. The culture
supernatant of hybridoma cultured with the HAT medium supplemented
with FBS (Ultra low IgG) (GIBCO BRL) was adsorbed to Hi Trap
ProteinG HP (Amersham), and washed with a binding buffer (20 mM
sodium phosphate (pH 7.0)). The antibody was eluted with an elution
buffer (0.1 M glycine-HCl (pH 2.7)). The eluate was immediately
neutralized with a neutralization buffer (1 M Tris-HCl (pH 9.0)),
and dialyzed against PBS for day and night for buffer exchange.
Example 8
Evaluation of Binding Activity by ELISA
[0176] In order to conveniently evaluate the binding activity of
the anti-GPC3 antibody thus obtained, concentration-dependent
binding of the antibody was detected against an immunoplate
containing the soluble form of GPC3 core protein immobilized
thereon. A 3-fold dilution series (12 dilutions in total) of the
purified antibody at a concentration of 10 .mu.g/mL was added, and
an anti-mouse IgG antibody was added as the secondary antibody.
Color development was carried out using SIGMA 104. Since the degree
of color development varies depending on the color development
time, data measured precisely after 1 hour was analyzed. Every
antibody showed a concentration-dependent color development. The
correlation between the concentration of antibody and the degree of
color development was plotted and an approximate curve was obtained
by using an analyzing software, GraphPad Prism. Its EC50 value was
determined as the index of the binding activity. EC50 values for
all clones are shown in FIG. 16.
Example 9
Evaluation of Binding Activity by Flow Cytometry
[0177] Cells were dissociated with 1 mM EDTA pH 8.0 (GIBCO)/PBS and
suspended in FACS buffer (1% FBS/PBS) at 1.times.10.sup.6 cells/mL.
The suspension was dispensed to a Multiscreen-HV Filter Plate
(Millopre) at 100 .mu.L/well and the supernatant was removed by
centrifugation. An anti-GPC3 antibody diluted to an appropriate
concentration was added and reacted on ice for 30 minutes. The
cells were washed once with FACS buffer and an FITC-labeled
anti-mouse IgG antibody was added and reacted on ice for 30
minutes. After the reaction, the cells were centrifuged at 500 rpm
for 1 minute, and the supernatant was removed. The cells were
suspended in 400 .mu.L of FACS buffer and subjected to flow
cytometry. EPICS ELITE ESP (Beckman Coulter) was used as a flow
cytometer. A gate was set on the living cell population with the
histogram of forward scatter and side scatter. As shown in FIG. 1,
an anti-GPC3 antibody (M3C11, M11F1) bound strongly to the CHO cell
expressing GPC3 and did not bind to the parent CHO cell, indicating
that the antibody specifically binds to GPC3 presented on the cell
membrane. In addition, the antibody showed the binding activity to
a hepatoma cell line, HepG2 (purchased from ATCC) and HuH-7
(purchased from Health Science Research Resources Bank), suggesting
that the antibody may specifically recognize hepatoma. The binding
activity of the clones derived from the mouse immunized with a
soluble form of GPC3 measured by flow cytometry is shown in FIG.
16, where the X-mode values of histogram at the concentration of
antibody of 5 .mu.g/mL are indicated.
Example 10
Epitope Classification by Competitive ELISA
[0178] The obtained antibodies were classified according to the
epitopes by a competitive ELISA. The antibodies were biotinylated
using a Biotin Labeling Kit (Roche). The soluble form of GPC3 core
protein was diluted to 1 .mu.g/mL with the coating buffer and added
to a plate at 100 .mu.L/well and stored at 4.degree. C. overnight
to coat the plate. On the next day, 200 .mu.L of the substrate
buffer was added for blocking. The plate was left at 4.degree. C.
overnight or longer and an anti-GPC3 antibody was added to the
plate at 100 .mu.L/well and reacted at room temperature for 1 hour.
After that, without washing of the plate, 10 .mu.L of 10 .mu.g/mL
biotin-labeled anti-GPC3 antibody was added and further reacted for
1 hour. The plate was washed with 300 .mu.L/well of the rinse
buffer for 3 times. AP-streptavidin conjugate (ZYMED) was diluted
to 1000-fold with the dilution buffer and added at 100 .mu.L/well
and reacted at room temperature for 1 hour. The plate was washed
with 300 .mu.L/well of the rinse buffer for 5 times. SIGMA 104 was
diluted to 1 mg/mL with the substrate buffer and added at 100
.mu.L/well. After incubating for 1 hour at room temperature, the
absorbance (at 405 nm, reference wavelength of 655 nm) was
measured.
[0179] The results of the competitive ELISA are shown in FIG. 2. As
for the antibody that competitively inhibited the binding of the
biotinylated antibody by 50% or more, it was considered that its
epitopes are located close together in the three-dimensional
conformation. As a result of classification according to the
competitive inhibition pattern of color development against the
binding of the 8 types of biotinylated antibodies, the 11 clones
derived from the mouse immunized with a soluble form of GPC3 were
classified into 5 groups (a, b, c, d and e) (FIG. 16).
Example 11
Epitope Classification by Western Blotting
[0180] The soluble form of GPC3 core protein was applied to a 10%
SDS-PAGE mini (TEFCO) and electrophoresed under reducing
conditions. It was transferred to Immobilon-P (Millipore) using
Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell (BIO-RAD).
After the membrane was briefly washed with TBS-T (0.05% Tween 20,
TBS), it was shaken in TBS-T containing 5% skim milk for 1 hour.
The membrane was shaken in TBS-T for about 10 minutes, then each
anti-GPC3 antibody diluted with TBS-T containing 1% skim milk was
added and the membrane was shaken for 1 hour. The membrane was
washed with TBS-T and shaken in a solution of HRP-anti-mouse IgG
antibody (Amersham) diluted with TBS-T containing 1% skim milk for
1 hour, and then washed with TBS-T. Color development was carried
out using ECL-Plus (Amersham) and detected using Hyperfilm ECL
(Amersham).
[0181] As shown in FIG. 3, L9G11 was determined to be an antibody
binding to the N-terminal side because it bound to the band of
about 40 kDa. M3C11 was determined to be an antibody binding to the
C-terminal side because it bound to the band of about 30 kDa. All
the antibodies belonging to c, d or e group based on the
competitive ELISA bound to the N-terminal side, and all those
belonging to a or b groups bound to the C-terminal side (FIG. 16).
L9G11 had higher detection sensitivity in Western blotting than the
other antibodies that bind to the N-terminal side, suggesting that
this antibody is a useful for detecting the N-terminal fragment by
Western blotting.
Example 12
Detection of Secreted Form of GPC3
[0182] Since it was found that GPC3 is cleaved at the 358th amino
acid residue or the 374th amino acid residue, the inventors
hypothesized that a secreted form of GPC3 is secreted into the
blood of a patient with liver cancer. Therefore, a GPC3 sandwich
ELISA system was constructed in order to detect a secretory form of
GPC3.
[0183] An immunoplate was coated with an anti-GPC3 antibody at 10
.mu.g/mL and blocked by the substrate buffer. After the immunoplate
was stored for several hours at room temperature or overnight at
4.degree. C., the culture supernatant of HepG2 was added and
incubated for 1 hour at room. temperature. The immunoplate was
washed with 300 .mu.L/well of the rinse buffer for 3 times, and a
biotin-labeled anti-GPC3 antibody diluted to 10 .mu.g/mL was added
and incubated for 1 hour at room temperature. The immunoplate was
washed with 300 .mu.L/well of the rinse buffer for 3 times, and
AP-streptavidin was added and incubated for 1 hour at room
temperature. The immunoplate was washed with 300 .mu.L/well of the
rinse buffer for 5 times. Color development was carried out using
AMPAK (DAKO) in accordance with the attached protocol and the
absorbance was measured using a microplate reader. The antibodies
binding to the N-terminal side (M6B1, M19B11 and M18D4) and those
binding to the C-terminal side (M3C11, M13B3 and M3B8) were
combined to construct five sandwich ELISA systems. Each of these
combinations showed an equivalent sensitivity in the standard curve
using the secreted form of GPC3. These systems were evaluated using
the culture supernatant of HepG2. The secreted form of GPC3 was
detected at a high concentration of about 1 .mu.g/mL with a
combination of the antibodies binding to the N-terminal side (FIG.
4). The concentration detected with a combination of the antibodies
binding to the C-terminal side was low, suggesting that the
N-terminal fragment was dominantly present in the secreted form of
GPC3.
[0184] Subsequently, the culture supernatant of HepG2 was
immunoprecipitated using an anti-GPC3 antibody to detect the
secreted form of GPC3. In the case where M10D2 that binds to the
N-terminal fragment was used, the secreted form of GPC3 of 40 kDa
was detected (FIG. 5). On the other hand, in the case where M1E7
that binds to the C-terminal fragment was used, the secreted form
of GPC3 was not detected. The immunoprecipitation test was carried
out for all the obtained GPC3 antibodies. Every antibody binding to
the N-terminal fragment strongly detected the secreted form of
GPC3, while the secreted form of GPC3 was not detected or was
weakly detected with the use of the antibodies binding to the
C-terminal fragment (FIG. 16). The antibody that can detect the
secreted form of GPC3 by immunoprecipitation is expected to be
useful as an antibody for diagnosing hepatoma. In addition, the
antibody that can hardly detect the secreted form of GPC3 is
expected to be useful in the development of a therapeutic antibody
having an ADCC activity and a CDC activity, because such an
antibody may migrate to the hepatoma lesion without being trapped
in the secreted form of GPC3 present in the blood.
Example 13
Cloning of Variable Region of Anti-GPC3 Antibody
[0185] A variable region of the anti-GPC3 antibody was amplified by
the RT-PCR method using the total RNA extracted from an anti-GPC3
antibody-producing hybridoma. The total RNA was extracted from
1.times.10.sup.7 cells of the hybridoma with the use of RNeasy
Plant Mini Kits (QIAGEN). By using 1 .mu.g of the total RNA, the
5'-terminal gene fragment was amplified with the use of a SMART
RACE cDNA Amplification Kit (CLONTECH) and any of the following
synthetic oligonucleotides:
a synthetic oligonucleotide MHC-IgG1 complementary to the sequence
of a mouse IgG1 constant region: GGG CCA GTG GAT AGACAG ATG (SEQ ID
NO: 7); a synthetic oligonucleotide MHC-IgG2a complementary to the
sequence of a mouse IgG2a constant region: CAG GGG CCA GTG GAT AGA
CCG ATG (SEQ ID NO: 8); a synthetic oligonucleotide MHC-IgG2b
complementary to the sequence of a mouse IgG2b constant region: CAG
GGG CCA GTG GAT AGA CTG ATG (SEQ ID NO: 9); and a synthetic
oligonucleotide kappa complementary to the sequence of a mouse
kappa chain constant region: GCT CAC TGG ATG GTG GGA AGA TG (SEQ ID
NO: 10).
[0186] A reverse transcription reaction was carried out at
42.degree. C. for 1 hour and 30 minutes. The PCR mixture (50 .mu.L)
contained 5 .mu.L of 10.times. Advantage 2 PCR buffer, 5 .mu.L of
10.times. Universal Primer AMix, 0.2 mM dNTPs (dATP, dGTP, dCTP and
dTTP), 1 .mu.L of Advantage 2 Polymerase Mix (all from CLONTECH),
2.5 .mu.L of the reverse transcription reaction product and 10 pmol
of the synthetic oligonucleotide MHC-IgG1, MHC-IgG2a, MHC-IgG2b or
kappa. PCR was carried out with 5 cycles consisting of 94.degree.
C. for 30 seconds, 94.degree. C. for 5 seconds and 72.degree. C.
for 3 minutes, 5 cycles consisting of 94.degree. C. for 5 seconds,
70.degree. C. for 10 seconds and 72.degree. C. for 3 minutes, and
25 cycles consisting of 94.degree. C. for 5 seconds, 68.degree. C.
for 10 seconds and 72.degree. C. for 3 minutes. Lastly, the
reaction product was heated at 72.degree. C. for 7 minutes. Each
PCR product was purified from the agarose gel using a QIAquick Gel
Extraction Kit (QIAGEN), cloned into pGEM-T Easy vector (Promega),
and the nucleotide sequence was determined.
[0187] The nucleotide sequences of the H chain variable regions of
M3C11, M13B3, M1E7, M3B8, M11F1, M19B11, M6B1, M18D4, M5B9, M10D2
and L9G11 are shown in SEQ ID NOs: 11, 12, 13, 14, 15, 16, 17, 18,
19, 20 and 21, respectively, the amino acid sequences thereof are
shown in SEQ ID NOs: 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 and 32,
respectively. The nucleotide sequences of the L chain variable
regions thereof are shown in SEQ ID NOs: 33, 34, 35, 36, 37, 38,
39, 40, 41, 42 and 43, respectively, and the amino acid sequences
thereof are shown in SEQ ID NOs: 44, 45, 46, 47, 48, 49, 50, 51,
52, 53 and 54, respectively.
Example 14
Epitope Classification Using GST-Fusion Protein
[0188] To carry out a detail analysis of the epitopes for the
antibodies binding to the C-terminal fragment, fusion proteins of
successively shortened C-terminal peptides of GPC3 with GST, namely
GC-1 (from Ser495 to Lys563), GC-2 (from Gly510 to Lys563), GC-3
(from A1a524 to Lys563), GC-4 (from Gly537 to Lys563) and GC-5
(from Ser550 to Lys563) were prepared. The C-terminal region of
GPC3 was cloned into pGEX-4T-3 (Amersham) to construct a plasmid
DNA in which the C-terminal region of GPC3 is ligated to the
C-terminal side of GST. The plasmid DNA was introduced into
DH5.alpha., whereby a transformant was obtained. Then, IPTG was
added at 1 mM to a culture of the transformant in the logarithmic
growth phase to induce the expression of a GST-fusion protein. The
bacterial cells were collected after 2 hours cultivation. The cells
were homogenized by sonication, and centrifuged at 35,000 rpm for
30 minutes with XL-80 ultracentrifuge (Beckman, 70.1 Ti rotor).
Then, the culture supernatant was recovered and purified with GST
Purification Modules (Amersham). The thus purified GST-fusion
proteins were separated by SDS-PAGE under reducing conditions, and
Western blotting was carried out with the anti-GPC3 antibodies
(FIG. 6). M3C11 and M1E7 detected GC-1 and GC-2, while they did not
detect GC-3, GC-4 and GC-5, indicating that the epitopes of these
antibodies are contained in the region of GC-2, and that the region
of GC-3 is not sufficient. M3B8 and M11F1 detected GC-1 GC-2, GC-3
and GC-4, while they did not detect GC-5, indicating that the
epitopes of these antibodies are contained in the region of GC-4,
and that the region of GC-5 is not sufficient. The minimum region
of the GST-fusion protein to which each antibody can bind is listed
in the column headed "Western blotting" of FIG. 16.
Example 15
Preparation of Anti-GPC3 Mouse-Human Chimeric Antibody
[0189] The sequences of the H chain and the L chain variable
regions of the anti-GPC3 antibodies were ligated to the sequences
of a human IgG1 and a kappa chain constant regions. PCR was carried
out by using a synthetic oligonucleotide, which is complementary to
the 5'-terminal nucleotide sequence of the H chain variable region
of each antibody and has a Kozak sequence, and a synthetic
oligonucleotide, which is complementary to the 3'-terminal
nucleotide sequence and has a NheI site. The obtained PCR product
was cloned into pB-CH vector in which a human IgG1 constant region
was inserted into pBluescript KS (+) vector (Toyobo). The mouse H
chain variable region and the human H chain (.gamma.1 chain)
constant region are ligated via the NheI site. The prepared H chain
gene fragment was cloned into an expression vector, pCXND3. On the
other hand, PCR was carried out by using a synthetic
oligonucleotide, which is complementary to the 5'-terminal
nucleotide sequence of the L chain variable region of each antibody
and has a Kozak sequence, and a synthetic oligonucleotide, which is
complementary to the 3'-terminal nucleotide sequence and has a
BsiWI site. The obtained PCR product was cloned into pB-CL vector
in which the human kappa chain constant region was inserted into
pBluescript KS(+) vector (Toyobo). The human L chain variable
region and the constant region are ligated via the BsiWI site. The
prepared L chain gene fragment was cloned into an expression
vector, pUCAG. This pUCAG vector was obtained by cloning a 2.6 kbp
fragment obtained by digesting pCXN (Niwa et al., Gene 1991; 108:
193-200) with a restriction enzyme BamHI into the BamHI site of
pUC19 vector (Toyobo).
[0190] To prepare an expression vector for an anti-GPC3 mouse-human
chimeric antibody, a gene fragment was obtained by digesting the
pUCAG vector containing the L chain gene fragment with a
restriction enzyme HindIII (Takara Shuzo), and cloned into the
HindIII site of the pCXND3 containing the H chain gene. This
plasmid will express a neomycin-resistance gene, DHFR gene and an
anti-GPC3 mouse-human chimeric antibody in an animal cell.
[0191] A CHO cell line (DG44 cell line) stably expressing the
antibody was prepared as follows. The gene was introduced into the
cells by the electroporation method using Gene Pulser II (Bio-Rad).
A mixture obtained by mixing 25 .mu.g of the expression vector for
each anti-GPC3 mouse-human chimeric antibody and 0.75 mL of a
solution of CHO cells suspended in PBS (1.times.10.sup.7 cell/mL)
was cooled on ice for 10 minutes, and transferred to a cuvette.
Then, a pulse was applied at 1.5 kV and a capacitance of 25 .mu.FD.
After a 10-minute recovery period at room temperature, the
electroporated cells were suspended in 40 mL of CHO-S-SFM II medium
(Invitrogen) containing 1.times. HT supplement (Invitrogen). The
suspension was diluted to 50-fold with the same medium, and
dispensed to a 96-well culture plate at 100 .mu.L/well. After a
24-hour culture in a CO.sub.2 incubator (5% CO.sub.2), Geneticin
(Invitrogen) was added at 0.5 mg/mL and the cells were cultured for
2 weeks. The culture supernatant was taken from the well having a
Geneticin resistant transformed cell colony and the amount of IgG
was measured by the concentration determination method described
below. A high-producing cell line was successively expanded to
obtain a cell line that stably expresses an anti-GPC3 mouse-human
chimeric antibody. The cell line was cultured at a large-scale and
the culture supernatant was collected. The purification of each
anti-GPC3 mouse-human chimeric antibody was carried out using Hi
Trap ProteinG HP (Amersham).
Example 16
Measurement of Complement-Dependent Cytotoxicity Activity (CDC
Activity)
16.1 Preparation of Human Albumin Veronal Buffer (HAVB)
[0192] In milli-Q water, 12.75 g of NaCl (highest grade, Wako Pure
Chemicals), 0.5625 g of Na-Barbital (highest grade, Wako Pure
Chemicals) and 0.8625 g of Barbital (highest grade, Wako Pure
Chemicals) were dissolved to a final volume of 200 mL and
autoclaved at 121.degree. C. for 20 minutes. Then, 100 mL of
autoclaved hot milli-Q water was added. The pH was 7.43
(recommended pH: 7.5). The solution was used as a 5.times. veronal
buffer. In 50 mL of milli-Q water, 0.2205 g of CaCl.sub.2.2H.sub.2O
(highest grade, Junsei Chemical) was dissolved to a final
concentration of 0.03 mol/L, which was used as a CaCl.sub.2
solution. In 50 mL of milli-Q water, 1.0165 g of
MgCl.sub.2.6H.sub.2O (highest grade, Junsei Chemical) was dissolved
to a final concentration of 0.1 mol/L, which was used as a
MgCl.sub.2 solution. In milli-Q water, 100 mL of the 5.times.
veronal buffer, 4 mL of human serum albumin (25% Buminate
(registered trademark), the concentration of human serum albumin:
250 mg/mL, Baxter Healthcare), 2.5 mL of the CaCl.sub.2 solution,
2.5 mL of the MgCl.sub.2 solution, 0.1 g of KCl (highest grade,
Junsei Chemical) 0.5 g of glucose (D(+)-glucose, anhydrous glucose,
highest grade, Wako Pure Chemicals) were dissolved to a final
volume of 500 mL, which was used as HAVB. After filter
sterilization, the HAVB was stored at a preset temperature of
5.degree. C.
16.2 Preparation of Target Cell
[0193] The CHO cell expressing full-length human GPC3 prepared in
Example 4 was cultured in .alpha.-MEM medium containing nucleic
acid (+) (GIBCO) supplemented with 10% FBS and 0.5 mg/mL Geneticin
(GIBCO). The cells were dissociated from the dish using a cell
dissociation buffer (Invitrogen Corp), dispensed to each well of a
96-well flat-bottomed plate (Falcon) at 1.times.10.sup.4
cells/well, and cultured for 3 days. After the cultivation, 5.55
MBq of chromium-51 was added, and the cells were cultured in a 5%
carbon dioxide gas incubator at 37.degree. C. for 1 hour. These
cells were washed with HAVB twice, and 50 .mu.L of HAVB was added
and used as a target cell.
16.3 Chromium Release Test (CDC Activity)
[0194] Each chimeric antibody was diluted with HAVB to make a 40
.mu.g/mL antibody solution. To the target cell, 50 .mu.L of each
antibody solution was added, and left on ice for 15 minutes.
Subsequently, to each well, 100 .mu.L of the human serum from the
peripheral blood of a healthy volunteer, which had been diluted
with HAVB, was added to a final concentration of 25% (the final
concentration of antibody: 10 .mu.g/mL), and left in a 5% carbon
dioxide gas incubator at 37.degree. C. for 90 minutes. After the
plate was centrifuged, 100 .mu.L of the supernatant was collected
from each well, the radioactivity was measured using a gamma
counter. The specific chromium release rate was obtained by the
following formula.
Specific chromium release rate (%)=(A-C).times.100/(B-C)
"A" represents the radioactivity (cpm) in each well, "B" represents
the mean value of the radioactivities (cpm) in the wells in which
100 .mu.L of 2% NP-40 aqueous solution (Nonidet P-40, Code No.
252-23, Nacalai Tesque) and 50 .mu.L of HAVB were added to the
target cell, and "C" represents the mean value of the
radioactivities (cpm) in the wells in which 150 .mu.L of HAVB was
added to the target cell. The test was carried out in triplicate
and the mean value and the standard deviation were calculated for
CDC activity (%).
[0195] The results are shown in FIG. 7. Among 9 types of the
anti-GPC3 chimeric antibodies, M3B8 and M11F1, which are an
antibody recognizing the C-terminal side, showed a strong CDC
activity against the CHO cell expressing GPC3, however, the CDC
activity was not observed in the other antibodies. M3B8 and M11F1
belong to the group called "b" based on the competitive ELISA, and
an important epitope for showing a strong CDC activity could be
found.
Example 17
Measurement of ADCC Activity Using PBMC Derived from Human
Peripheral Blood
17.1 Preparation of Human PBMC Solution
[0196] The heparinized peripheral blood obtained from a healthy
volunteer was diluted to 2-fold with PBS(-), and overlayered on
Ficoll-Paque.TM. PLUS (Amersham). After centrifugation at
500.times.g for 30 minutes at 20.degree. C., the middle layer,
which is the mononuclear leukocyte fraction, was collected. The
cells were washed 3 times, suspended in 10% FBS/RPMI and used as a
human PBMC solution.
17.2 Preparation of Target Cell
[0197] The HepG2 cells cultured in 10% FBS/RPMI 1640 medium were
dissociated from the dish using Trypsin-EDTA (Invitrogen),
dispensed to each well of a 96-well U-bottomed plate (Falcon) at
1.times.10.sup.4 cells/well, and cultured for 2 days. The CHO cell
expressing full-length human GPC3 prepared in Example 4 was
cultured in .alpha.-MEMnucleic acids (+) medium (GIBCO)
supplemented with 10% FBS and 0.5 mg/mL Geneticin (GIBCO). The
cells were dissociated from the dish using a cell dissociation
buffer (Invitrogen Corp), dispensed to each well of a 96-well
flat-bottomed plate (Falcon) at 1.times.10.sup.4 cells/well, and
cultured for 3 days. Chromium-51 (5.55 MBq) was added to each cell
and the cells were cultured in a 5% carbon dioxide gas incubator at
37.degree. C. for 1 hour. These cells were washed with the medium
once, and 50 .mu.L of 10% FBS/RPMI 1640 medium was added and used
as a target cell.
17.3 Chromium Release Test (ADCC Activity)
[0198] To the target cell, 50 .mu.L of an antibody solution
prepared at different concentrations was added, and reacted on ice
for 15 minutes. Subsequently, 100 .mu.L of the human PBMC solution
was added at 5.times.10.sup.5 cells/well, and cells were cultured
in a 5% carbon dioxide gas incubator at 37.degree. C. for 4 hours.
After the cultivation, the plate was centrifuged, and the
radioactivity in 100 .mu.L of the culture supernatant was measured
using a gamma counter. The specific chromium release rate was
obtained by the following formula.
specific chromium release rate (%)=(A-C).times.100/(B-C)
"A" represents the mean value of the radioactivities (cpm) in each
well, "B" represents the mean value of the radioactivities (cpm) in
the wells in which 100 .mu.L of 2% NP-40 aqueous solution (Nonidet
P-40, Code No. 252-23, Nacalai Tesque) and 50 .mu.L of 10% FBS/RPMI
medium were added to the target cell, and "C" represents the mean
value of the radioactivities (cpm) in the wells in which 150 .mu.L
of 10% FBS/RPMI medium was added to the target cell. The test was
carried out in triplicate and the mean value and the standard
deviation were calculated for ADCC activity (%). The results are
shown in FIG. 8. Among 9 types of the anti-GPC3 chimeric
antibodies, the antibodies recognizing the C-terminal side had a
tendency of showing a strong ADCC activity.
Example 18
Immunization with GC-3 and Selection of Hybridoma
[0199] Among the obtained anti-GPC3 antibodies, only M11F1 and M3B8
showed a strong CDC activity, indicating that the CDC activity is
epitope dependent. To obtain an antibody having both ADCC activity
and CDC activity, a GST-fusion protein containing the epitope for
M11F1 and M3B8, referred to as GC-3, was used for immunization. A
large mount of GC-3 was purified by the above-mentioned method. The
buffer was changed to PBS by gel filtration using Superdex 75
(Amersham). The obtained product was used as immunoprotein. using
three Balb/c mice (purchased from Charles River Japan, Inc.) and
three MRL/lpr mice were immunized with GC-3 in accordance with the
above-mentioned method. For the first immunization, GC-3 was
prepared at 100 .mu.g/head and emulsified using FCA, which was
subcutaneously administered. Two weeks later, GC-3 was prepared at
50 .mu.g/head and emulsified using FIA, which was subcutaneously
administered. After the fifth immunization, the final immunization
(50 .mu.g/head) was carried out for all mice by intravenously
administering the immunoprotein via the tail. After cell fusion,
hybridoma were screened by an ELISA using an immunoplate coated
with the soluble form. of GPC3 core protein. A positive clone was
monocloned by the limiting dilution method. As a result, 5 clones
of antibodies (GC199, GC202, GC33, GC179 and GC194) that have a
strong binding activity against GPC3 were obtained.
[0200] The antibody was purified from the culture supernatant of
the hybridoma using Hi Trap proteinG HP, and analyzed in accordance
with the above-mentioned method. The EC50 value was calculated by
an ELISA using an immunoplate coated with the soluble form of GPC3
core protein, and the X-mode value of histogram at 5 .mu.g/mL was
measured by flow cytometry (FIG. 17). According to the epitope
classification by a competitive ELISA, the antibodies were
classified into the group b (GC199, GC202 and GC33) and a new
epitope group f (GC179 and GC194). The epitope classification using
the GST-fusion proteins indicated that GC199, GC202 and GC33
detected GC-1, GC-2, GC-3 and GC-4, but did not detect GC-5,
suggesting that the epitopes for these antibodies are contained in
the region of GC-4 in the same manner as the epitopes for M11F1 and
M3B8, and that the region of GC-5 is not sufficient. On the other
hand, GC179 and GC194 detected GC-1, GC-2 and GC-3, but did not
detect GC-4 and GC-5, suggesting that the epitopes for these
antibodies are contained in the region of GC-3, and that the region
of GC-4 is not sufficient. The minimum region of the GST-fusion
protein to which each antibody can bind is listed in the column
headed "Western blotting" of FIG. 17.
[0201] The H chain and the L chain variable regions of GC199,
GC202, GC33, GC179 and GC194 were cloned in accordance with the
above-mentioned method, and their sequences were determined. As for
the L chain of GC194, 2 types of sequences were cloned. The
nucleotide sequences of the H chain variable regions of GC199,
GC202, GC33, GC179 and GC194 are shown in SEQ ID NOs: 55, 56, 57,
58 and 59, respectively, and the amino acid sequences thereof are
shown in SEQ ID NOs: 60, 61, 62, 63 and 64, respectively. The
nucleotide sequences of the L chain variable regions of GC199,
GC202, GC33, GC179, GC194(1) and GC194(2) are shown in SEQ ID NOs:
65, 66, 67, 68, 69 and 70 respectively, and the amino acid
sequences thereof are shown in SEQ ID NOs: 71, 72, 73, 74, 75 and
76, respectively.
[0202] Further, these amino acid sequences were examined for
homology by comparing with the database of the amino acid sequences
of known antibodies, whereby their CDR regions were determined as
follows.
TABLE-US-00002 Antibody CDR Amino Acid Sequence SEQ ID NO M13B3(H)
CDR1 NYAMS 103 CDR2 AINNNGDDTYYLDTVKD 104 CDR3 QGGAY 105 M3B8(H)
CDR1 TYGMGVG 106 CDR2 NIWWYDAKYYNSDLKS 107 CDR3 MGLAWFAY 108
M11F1(H) CDR1 IYGMGVG 109 CDR2 NIWWNDDKYYNSALKS 110 CDR3 IGYFYFDY
111 M5B9(H) CDR1 GYWMH 112 CDR2 AIYPGNSDTNYNQKFKG 113 CDR3
SGDLTGGLAY 114 M6B1(H) CDR1 SYAMS 115 CDR2 AINSNGGTTYYPDTMKD 116
CDR3 HNGGYENYGWFAY 117 M10D2(H) CDR1 SYWMH 118 CDR2
EIDPSDSYTYYNQKFRG 119 CDR3 SNLGDGHYRFPAFPY 120 L9G11(H) CDR1 SYWMH
118 CDR2 TIDPSDSETHYNLQFKD 121 CDR3 GAFYSSYSYWAWFAY 122 GC33(H)
CDR1 DYEMH 123 CDR2 ALDPKTGDTAYSQKFKG 124 CDR3 FYSYTY 125 GC179(H)
CDR1 INAMN 126 CDR2 RIRSESNNYATYYGDSVKD 127 CDR3 EVTTSFAY 128
GC194(H) CDR1 ASAMN 129 CDR2 RIRSKSNNYAIYYADSVKD 130 CDR3
DPGYYGNPWFAY 131 GC199(H) CDR1 DYSMH 132 CDR2 WINTETGEPTYADDFKG 133
CDR3 LY 134 GC202 (H) CDR1 TYGMGVG 106 CDR2 NIWWHDDKYYNSALKS 135
CDR3 IAPRYNKYEGFFAF 136 M13B3(L) CDR1 KSSQSLLDSDGKTYLN 137 CDR2
LVSKLDS 138 CDR3 WQGTHFPLT 139 M3B8(L) CDR1 KASQDINNYLS 140 CDR2
RANRLVD 141 CDR3 LQCDEFPPWT 142 M11F1(L) CDR1 RSSQSLVHSNGNTYLH 143
CDR2 KVSNRFS 144 CDR3 SQSTHVPWT 145 M5B9(L) CDR1 RSSKSLLHSNGITYLY
146 CDR2 QMSNLAS 147 CDR3 AQNLELPYT 148 M6B1(L) CDR1 KASQDINKNII
149 CDR2 YTSTLQP 150 CDR3 LQYDNLPRT 151 M10D2(L) CDR1 RASHSISNFLH
152 CDR2 YASQSIS 153 CDR3 QQSNIWSLT 154 L9G11(L) CDR1
RASESVEYYGTSLMQ 155 CDR2 GASNVES 156 CDR3 QQSRKVPYT 157 GC33(L)
CDR1 RSSQSLVHSNGNTYLH 143 CDR2 KVSNRFS 144 CDR3 SQNTHVPPT 158
GC179(L) CDR1 KSSKSLLHSNGNTYLN 159 CDR2 WMSNLAS 160 CDR3 MQHIEYPFT
161 GC194(L)1 CDR1 RSSKSLLHSYDITYLY 162 CDR2 QMSNLAS 147 CDR3
AQNLELPPT 163 GC194(L)2 CDR1 SASSSVSYMY 164 CDR2 DTSNLAS 165 CDR3
QQWSSYPLT 166 GC199(L) CDR1 KSSQSLLHSDGKTFLN 167 CDR2 LVSRLDS 168
CDR3 CQGTHFPRT 169 GC202(L) CDR1 RSSQSIVHSNGNTYLE 170 CDR2 KVSNRFS
144 CDR3 FQGSHVPWT 171
Example 19
Measurement of ADCC Activity Using Mouse Bone Marrow Derived
Effector Cell
19.1 Preparation of Mouse Bone Marrow Derived Effector Cell
Solution
[0203] Bone marrow cells were collected from the femur of an SCID
mouse (CLEA Japan, Inc., male, 10 weeks old), and suspended in 10%
FBS/RPMI 1640 medium at 5.times.10.sup.6 cells/mL. Mouse GM-CSF
(PeproTech) and human IL-2 (PeproTech) were added at 10 ng/mL and
50 ng/mL, respectively, and the cells were cultured in a 5% carbon
dioxide gas incubator at 37.degree. C. for 5 days. After the
cultivation, the cells were scraped off with a scraper and washed
with the medium once. Then, the cells were suspended in 10%
FBS/RPMI 1640 medium at 5.times.10.sup.6 cells/mL, and used as a
mouse bone marrow derived effector cell solution.
19.2 Preparation of Target Cell
[0204] A human hepatoma cell line, HuH-7, was maintained and
subcultured with DMEM medium (SIGMA) containing 10% FBS
(ThermoTrace). The cells were dissociated from the dish using Cell
Dissociation Buffer (Invitrogen), dispensed to each well of a
96-well U-bottomed plate (Falcon) at 1.times.10.sup.4 cells/well,
and cultured for 1 day. After the cultivation, 5.55 MBq of
chromium-51 was added, and the cells were cultured in a 5% carbon
dioxide gas incubator at 37.degree. C. for 1 hour. These cells were
washed with the medium once, and 50 .mu.L of 10% FBS/RPMI 1640
medium was added and used as a target cell.
19.3 Chromium Release Test (ADCC Activity)
[0205] To the target cell, 50 .mu.L of an antibody solution
prepared at different concentrations was added, and reacted on ice
for 15 minutes. Subsequently, 100 .mu.L of the mouse bone marrow
derived effector cell solution (5.times.10.sup.5 cells/well) was
added, and cells were cultured in a 5% carbon dioxide gas incubator
at 37.degree. C. for 4 hours. After the cultivation, the plate was
centrifuged, and the radioactivity in 100 .mu.L of the culture
supernatant was measured using a gamma counter. The specific
chromium release rate was obtained by the following formula.
Specific chromium release rate (%)=(A-C).times.100/(B-C) "A"
represents the mean value of the radioactivities (cpm) in each
well, "B" represents the mean value of the radioactivities (cpm) in
the wells in which 100 .mu.L of 2% NP-40 aqueous solution (Nonidet
P-40, Code No. 252-23, Nacalai Tesque) and 50 .mu.L of 10% FBS/RPMI
medium were added to the target cell, and "C" represents the mean
value of the radioactivities (cpm) in the wells in which 150 .mu.L
of 10% FBS/RPMI medium was added to the target cell. The test was
carried out in triplicate and the mean value and the standard
deviation were calculated for ADCC activity (%).
[0206] The results are shown in FIG. 9. It was revealed that GC33
antibody shows an ADCC activity when the concentration of antibody
is 0.1 .mu.g/mL or higher, and shows stronger activity than GC199
antibody.
Example 20
Antitumor Activity of GC33 Antibody to Mouse Model Transplanted
with Human Hepatoma
[0207] 20.1 Preparation of Mouse Model Transplanted with Human
Hepatoma
[0208] A human hepatoma cell line, HuH-7, was prepared at
5.times.10.sup.7 cells/mL in a solution containing DMEM medium and
MATRIGEL (BD Bioscience) at a ratio of 1:1. On the previous day,
100 .mu.L of an anti-asialo GM1 antibody solution (Wako Pure
Chemicals, one vial was dissolved with 1 mL of distilled water for
injection then added 4 mL of physiologic saline) was
intraperitoneally administered to a SCID mouse (male, 5 weeks old,
CLEA Japan, Inc.). The mouse was transplanted with 100 .mu.L of the
above-mentioned cell suspension (5.times.10.sup.6 cells/mouse)
subcutaneously in the abdominal area.
20.2 Preparation and Administration of Antibody
[0209] Starting from the day 20 after the cell transplantation, an
antibody solution prepared on the day of administration at 0.5
mg/mL (group of administration of 5 mg/kg) and at 0.1 mg/mL (group
of administration of 1 mg/kg) with PBS(-) was administered to the
mouse model transplanted with a human hepatoma cells at 10 mL/kg
via the tail vein once a week for 3 weeks. As a negative control,
PBS(-) (vehicle) was administered at 10 mL/kg via the tail vein
once a week for 3 weeks in a similar manner. Both groups consisted
of 6 mice each.
20.3 Evaluation of Antitumor Effect
[0210] The antitumor effect of GC33 antibody on the mouse model
transplanted with human hepatoma cells was evaluated with the
change in tumor volume with time and tumor weight at 1 week after
the final administration. The tumor volume was calculated by the
following formula.
Tumor volume=(major axis).times.(minor axis).times.(minor
axis)/2
[0211] As shown in FIG. 10, a significant inhibition of tumor
growth was observed in the GC33 antibody group compared with the
vehicle group.
[0212] Consequently, GC33 was shown to have an antitumor effect on
the mouse model transplanted with a human hepatoma cells.
Example 21
Preparation of GC33 Mouse-Human Chimeric Antibody
[0213] The H chain and the L chain of GC33 were amplified by PCR
using a synthetic oligonucleotide, which is complementary to the
5'-terminal nucleotide sequences and has a Kozak sequence and a
HindIII site, and a synthetic oligonucleotide, which is
complementary to the 3'-terminal nucleotide sequences and has a
BamHI site. After digestion with HindIII and BamHI, the obtained
PCR product was cloned into an expression vector, HEFg.gamma.1, in
which a human IgG1 constant region was inserted, and an expression
vector, HEFg.kappa., in which a human kappa chain constant region
was inserted (Sato et al., Mol Immunol. 1994; 371-381). The vectors
were introduced into a CHO cell (DG44 cell line) in accordance with
the above-mentioned method, and a stably expressing cell line was
established. The antibody was purified from the culture supernatant
using Hi Trap ProteinG HP (Amersham). The concentration of IgG in
the culture supernatant was measured by a human IgG sandwich ELISA
using goat anti-human IgG (BIOSOURCE) and goat anti-human IgG
alkaline phosphatase conjugate (BIOSOURCE), and the concentration
was determined by the comparison with a commercially available
human IgG (Cappel).
Example 22
Measurement of CDC Activity and ADCC Activity Using GC33
Mouse-Human Chimeric Antibody
[0214] In accordance with the methods described in Examples 16 and
17, the CDC activities and ADCC activities of GC33, M3C11 and M1E7
mouse-human chimeric antibodies were measured. As for the target
cell, the CHO cell expressing full-length GPC3 was used for
measuring the CDC activity and HepG2 was used for measuring the
ADCC activity. The results are shown in FIG. 11 and FIG. 12,
respectively. It was revealed that, in either test system, GC33
shows a strong CDC activity and ADCC activity compared with the
other two antibodies.
Example 23
Epitope Analysis for GC33
[0215] To determine the epitope for GC33 in detail, fusion proteins
of a further shorter C-terminal peptide of GPC3 and GST were
prepared, and analyzed by Western blotting. The prepared
GPC3-derived peptide sequences contained in the GST-fusion protein
are shown in FIG. 13. Since GC33 can bind to GC-4 (aa 537-563), but
cannot bind to GC-5 (aa 550-563), it was considered that the
epitope is located in a region containing at least part of the aa
537-550 region. First, the peptides GC-6 (G N S Q Q A T P K D N E I
S (SEQ ID NO: 93)), GC-7 (G N S Q Q A T P (SEQ ID NO: 94)), GC-8 (Q
Q A T P K D N (SEQ ID NO: 95)) and GC-9 (TPKDNEIS (SEQ ID NO: 96))
were prepared. A forward oligo DNA and a reverse oligo DNA were
prepared which were designed in such a manner that the cleavage
site of EcoRI recognition sequence is attached to the 5' end and
the cleavage site of SalI recognition sequence is attached to the
3' end, respectively. The synthesis of the oligo DNAs was done by
Espec Oligo Service. The DNA was purified with C-18 cartridge,
phosphorylated at the 5' end and used for the analysis. Twenty-five
microliters of the forward oligo DNA (10 .mu.M) and 25 .mu.L of the
reverse oligo DNA (10 .mu.M) were mixed and reacted at 94.degree.
C. for 5 minutes, at 37.degree. C. for 10 minutes, and at room
temperature for 15 minutes, then left at 4.degree. C. for 10
minutes to anneal the forward oligo DNA and the reverse oligo DNA.
The concentration of the oligos was determined by the absorbance
measurement at the molar ratio of the insert to the vector of 3:1.
The oligos were cloned into EcoRI- and SalI-digested pGEX4T-3, and
the nucleotide sequence was confirmed. A GST-fusion protein was
prepared in accordance with the above-mentioned method, and
purified using Gluthatione Sepharose 4B. The purified proteins were
separated by SDS-PAGE under reducing conditions, and analyzed by
Western blotting using GC33. As a result, the antibody GC33 could
not detect any GST-fusion protein strongly, suggesting that a
longer sequence at the C-terminal side is needed for the binding of
GC33 (FIG. 14). Based on the above prediction, GC-11 (A T P K D N E
I S T (SEQ ID NO: 97)), GC-12 (P K D N E I S T F H (SEQ ID NO:
98)), GC-13 (D N E I S T F H N L (SEQ ID NO: 99)) and GC-14 (E I S
T F H N L G N (SEQ ID NO: 100)) were prepared and evaluated in the
same manner. As a result, GC-11, GC-12 and GC-13 bound to GC33 more
strongly, suggesting that the epitope for GC33 is located in the
sequence from 544th to 553rd (P K D N E I S T F H) at the
C-terminus of GPC3.
Example 24
Humanization of GC33
[0216] Antibody sequence data were obtained from publicly disclosed
Kabat Database (ftp://ftp.ebi.ac.uk/pub/databases/kabat/) and from
ImMunoGeneTics Database (IMGT). The H chain variable region and the
L chain variable region were separately subjected to homology
search. As a result, the H chain variable region was found to have
a high homology with DN13 (Smithson et al., Mol Immunol. 1999; 36:
113-124), and the L chain variable region was found to have a high
homology with homo sapiens IGK mRNA for immunoglobulin kappa light
chain VLJ region, partial cds, clone: K64 of the accession number
of AB064105. The signal sequence of the accession number of 540357
that has a high homology with AB064105 was used as a signal
sequence of the L chain. The complementarity determining region
(hereinafter referred to as CDR) of GC33 were transplanted into the
frame work regions (hereinafter referred to as FR) of these human
antibodies to prepare a humanized antibody.
[0217] Specifically, synthetic oligo DNAs of approximately 50 bases
were designed in such a manner that approximately 20 bases of them
were hybridized and these synthetic oligo DNAs were assembled
together by the PCR method to prepare genes encoding each of the
variable regions. They were digested at the HindIII site inserted
in the end of the 5'-terminal synthetic oligo DNA and the BamHI
site inserted in the end of the 3'-terminal synthetic oligo DNA.
The fragments were cloned into an expression vector, HEFg.gamma.1,
in which a human IgG constant region was cloned, or an expression
vector, HEFg.kappa.1, in which a human kappa chain constant region
was cloned (Sato et. al., Mol Immunol. 1994; 371-381). The H chain
and the L chain of the humanized GC33 constructed as above were
named ver.a, respectively. The binding activity of the humanized
GC33, whose H chain and the L chain were both ver.a (ver.a/ver.a),
was lower than that of an antibody with mouse GC33 variable regions
(mouse/mouse). Antibodies were constructed in which the mouse GC33
sequence and the ver.a sequence were chimerically combined
(mouse/ver.a, ver.a/mouse) with regard to the H chain and the L
chain, and their binding activities were evaluated. As a result, a
decrease in binding activity was observed in ver.a/mouse,
indicating that the decrease in binding activity due to amino acid
replacement was attributed to the H chain (FIG. 15). Then, modified
H chains, ver.c, ver. f, ver.h, ver.i, ver. j, ver.k were prepared.
All these humanized GC33 showed a binding activity equivalent to
that of a chimeric antibody having the mouse GC33 variable region
(FIG. 15). The nucleotide sequences of the humanized GC33 H chain
variable regions, ver.a, ver.c, ver.f, ver.h, ver.i, ver.j, ver.k
were shown in SEQ ID NOs: 77, 78, 79, 80, 81, 82 and 83,
respectively, and the amino acid sequences thereof were shown in
SEQ ID NOs: 84, 85, 86, 87, 88, 89 and 90, respectively. The
nucleotide sequence and the amino acid sequence of a humanized GC33
L chain variable region, ver.a are shown in SEQ ID NOs: 91 and 92,
respectively. In humanized GC33 H chain variable regions ver.i,
ver.j and ver. k, the 6th glutamic acid residue is replaced with
glutamine residue. The heat stability of these antibodies was
significantly increased.
Example 25
Modification of Humanized GC33 L Chain
[0218] As for the deamidation of protein, the reaction rate
constant of deamidation is known to be dependent on the primary
sequence. It is also known that Asn-Gly is particularly susceptible
to deamidation (Rocinson et. al., Proc. Natl. Acad. Sci. USA 2001;
98; 944-949). As for Asn33 in the CDR1 of a humanized GC33 L chain
ver.a shown in SEQ ID NO: 91, the primary sequence is Asn-Gly,
which is predicted to be very susceptible to deamidation.
[0219] To evaluate the effect of deamidation of Asn33 on the
binding activity, a modified antibody was prepared in which Asn33
was replaced with Asp. A point mutation was introduced using a
Quick Change Site-Directed Mutagenesis Kit (Stratagene) was used.
More specifically, 50 .mu.L of a reaction mixture containing 125 ng
of a sense primer (CTT GTA CAC AGT GAC GGA AAC ACC TAT: SEQ ID NO:
172), 125 ng of an antisense primer (ATA GGT GTT TCC GTC ACT GTG
TAC AAG: SEQ ID NO: 173), 5 .mu.L of 10.times. reaction buffer, 1
.mu.L of dNTP mix, 10 ng of HEFg.kappa. into which a humanized GC33
L chain ver.a had been cloned and 1 .mu.L of Pfu Turbo DNA
Polymerase was subjected to PCR of 12 cycles consisting of
95.degree. C. for 30 seconds, 55.degree. C. for 1 minute and
68.degree. C. for 9 minutes. Subsequently, a restriction enzyme,
DpnI, was added and digestion was carried out at 37.degree. C. for
2 hours, and the digested product was introduced into XL1-Blue
competent cell attached to the kit, whereby a transformant was
obtained. The variable region was cleaved out from the clone in
which each mutation was properly introduced, and cloned into
HEFg.kappa. again. It was introduced into a COS7 cell using Fugene
6 (Roche) together with HEFg.gamma.1, in which a humanized GC33 H
chain ver.k had been cloned. The antibody transiently expressed in
the cell was recovered from the culture supernatant. The
concentration of antibody was determined by a sandwich ELISA using
the anti-human IgG antibody. The binding activity of the modified
antibody was evaluated by an ELISA using an immunoplate coated with
the soluble form of GPC3 core protein. As shown in FIG. 18, the
binding activity was lost in the modified antibody (N33D) in which
Asn33 had been replaced with Asp, suggesting that the effect of the
deamidation of Asn33 on the binding activity was significant.
[0220] As a method of suppressing deamidation of Asn33, replacement
of Gly34 with another amino acid has been reported (International
Patent Application WO 03057881A1). In accordance with the
above-mentioned method, G34 was replaced with any of 17 amino acids
other than Cys and Met using a Quick Change Site-Directed
Mutagenesis Kit to prepare a series of modified antibodies, namely,
G34A, G34D, G34E, G34F, G34H, G34N, G34P, G34Q, G34I, G34K, G34L,
G34V, G34W, G34Y, G34R, G34S and G34T. These antibodies were
transiently expressed in COS7 cells, and the binding activity was
evaluated using the culture supernatant. It was found that the
binding activity is maintained even if G34 is replaced with another
amino acid, except for Pro (G34P) and Val (G34V).
[0221] The amino acid sequences of the light chain CDR1 of the
above-mentioned modified antibodies are shown in SEQ ID NO: 174
(G34A), SEQ ID NO: 175 (G34D), SEQ ID NO: 176 (G34E), SEQ ID NO:
177 (G34F), SEQ ID NO: 178 (G34H), SEQ ID NO: 179 (G34N), SEQ ID
NO: 180 (G34T), SEQ ID NO: 181 (G34Q), SEQ ID NO: 182 (G34I), SEQ
ID NO: 183 (G34K), SEQ ID NO: 184 (G34L), SEQ ID NO: 185 (G34S),
SEQ ID NO: 186 (G34W), SEQ ID NO: 187 (G34Y), SEQ ID NO: 188
(G34R), SEQ ID NO: 189 (G34V) and SEQ ID NO: 190 (G34P),
respectively. The amino acid sequences of the light chain variable
regions of the above-mentioned modified antibodies are shown in SEQ
ID NO: 191 (G34A), SEQ ID NO: 192 (G34D), SEQ ID NO: 193 (G34E),
SEQ ID NO: 194 (G34F), SEQ ID NO: 195 (G34H), SEQ ID NO: 196
(G34N), SEQ ID NO: 197 (G34T), SEQ ID NO: 198 (G34Q), SEQ ID NO:
199 (G34I), SEQ ID NO: 200 (G34K), SEQ ID NO: 201 (G34L), SEQ ID
NO: 202 (G34S), SEQ ID NO: 203 (G34W), SEQ ID NO: 204 (G34Y), SEQ
ID NO: 205 (G34R), SEQ ID NO: 206 (G34V) and SEQ ID NO: 207 (G34P),
respectively.
[0222] The antibody of the present invention can be used as a cell
growth inhibitor, an anticancer agent or an agent for diagnosis of
cancers.
Example 26
Preparation of Human Hepatoma Cell Line (SK-03) Expressing
Full-Length Human GPC3
[0223] To obtain a cell line for evaluating a biological activity
of the anti-GPC3 antibodies, a human hepatoma cell line expressing
full-length GPC3 was established.
[0224] One microgram of a full-length human GPC3 gene expression
vector treated with Pvu I was mixed with 2 .mu.L, of FuGENE (Roche)
to allow for complex formation. The complex was added to SK-HEP-1
cells (purchased from ATCC) for gene introduction. After incubation
in CO2 incubator for 24 hours, GPC3 expressing cells were selected
using Dulbecco's MEM (D-MEM, SIGMA) containing Geneticin at a final
concentration of 1 mg/mL and 10% FBS. The resulting
Geneticin-resistant colonies were collected and cell cloning was
carried out by the limiting dilution method. The expression of
human GPC3 of each cell clone was assayed by flow cytometory using
the chimeric antibody GC33 and FITC-labeled goat anti-human IgG
antibody (ICN). In this way, a stably expressing cell line SK-03
was obtained.
Example 27
Comparison of CDC Activity and ADCC Activity of Mouse-Human
Chimeric Antibodies
[0225] In order to directly compare the CDC activity and ADCC
activity of the mouse-human chimeric antibodies GC33, M3C11, and
M1E7 described in Example 22, the CDC activity and ADCC activity of
three antibodies were measured in the same test system according to
the method described in Examples 16 and 17. As for the target cell,
the CHO cell expressing full-length GPC3 was used for measuring the
CDC activity and SK-03 was used for measuring the ADCC activity.
The results are shown in FIG. 19 and FIG. 20, respectively. It was
revealed that, in either test system, GC33 shows a stronger CDC
activity and ADCC activity compared with the other two
antibodies.
INDUSTRIAL APPLICABILITY
[0226] The antibody of the present invention can be used as a cell
growth inhibitor, an anticancer agent and an agent for diagnosis of
cancers.
Sequence CWU 1
1
207131DNAArtificial SequencePCR primer 1gatatcatgg ccgggaccgt
gcgcaccgcg t 31231DNAArtificial SequencePCR primer 2gctagctcag
tgcaccagga agaagaagca c 3131743DNAHomo sapiens 3atggccggga
ccgtgcgcac cgcgtgcttg gtggtggcga tgctgctcag cttggacttc 60ccgggacagg
cgcagccccc gccgccgccg ccggacgcca cctgtcacca agtccgctcc
120ttcttccaga gactgcagcc cggactcaag tgggtgccag aaactcccgt
gccaggatca 180gatttgcaag tatgtctccc taagggccca acatgctgct
caagaaagat ggaagaaaaa 240taccaactaa cagcacgatt gaacatggaa
cagctgcttc agtctgcaag tatggagctc 300aagttcttaa ttattcagaa
tgctgcggtt ttccaagagg cctttgaaat tgttgttcgc 360catgccaaga
actacaccaa tgccatgttc aagaacaact acccaagcct gactccacaa
420gcttttgagt ttgtgggtga atttttcaca gatgtgtctc tctacatctt
gggttctgac 480atcaatgtag atgacatggt caatgaattg tttgacagcc
tgtttccagt catctatacc 540cagctaatga acccaggcct gcctgattca
gccttggaca tcaatgagtg cctccgagga 600gcaagacgtg acctgaaagt
atttgggaat ttccccaagc ttattatgac ccaggtttcc 660aagtcactgc
aagtcactag gatcttcctt caggctctga atcttggaat tgaagtgatc
720aacacaactg atcacctgaa gttcagtaag gactgtggcc gaatgctcac
cagaatgtgg 780tactgctctt actgccaggg actgatgatg gttaaaccct
gtggcggtta ctgcaatgtg 840gtcatgcaag gctgtatggc aggtgtggtg
gagattgaca agtactggag agaatacatt 900ctgtcccttg aagaacttgt
gaatggcatg tacagaatct atgacatgga gaacgtactg 960cttggtctct
tttcaacaat ccatgattct atccagtatg tccagaagaa tgcaggaaag
1020ctgaccacca ctattggcaa gttatgtgcc cattctcaac aacgccaata
tagatctgct 1080tattatcctg aagatctctt tattgacaag aaagtattaa
aagttgctca tgtagaacat 1140gaagaaacct tatccagccg aagaagggaa
ctaattcaga agttgaagtc tttcatcagc 1200ttctatagtg ctttgcctgg
ctacatctgc agccatagcc ctgtggcgga aaacgacacc 1260ctttgctgga
atggacaaga actcgtggag agatacagcc aaaaggcagc aaggaatgga
1320atgaaaaacc agttcaatct ccatgagctg aaaatgaagg gccctgagcc
agtggtcagt 1380caaattattg acaaactgaa gcacattaac cagctcctga
gaaccatgtc tatgcccaaa 1440ggtagagttc tggataaaaa cctggatgag
gaagggtttg aaagtggaga ctgcggtgat 1500gatgaagatg agtgcattgg
aggctctggt gatggaatga taaaagtgaa gaatcagctc 1560cgcttccttg
cagaactggc ctatgatctg gatgtggatg atgcgcctgg aaacagtcag
1620caggcaactc cgaaggacaa cgagataagc acctttcaca acctcgggaa
cgttcattcc 1680ccgctgaagc ttctcaccag catggccatc tcggtggtgt
gcttcttctt cctggtgcac 1740tga 17434580PRTHomo sapiens 4Met Ala Gly
Thr Val Arg Thr Ala Cys Leu Val Val Ala Met Leu Leu 1 5 10 15 Ser
Leu Asp Phe Pro Gly Gln Ala Gln Pro Pro Pro Pro Pro Pro Asp 20 25
30 Ala Thr Cys His Gln Val Arg Ser Phe Phe Gln Arg Leu Gln Pro Gly
35 40 45 Leu Lys Trp Val Pro Glu Thr Pro Val Pro Gly Ser Asp Leu
Gln Val 50 55 60 Cys Leu Pro Lys Gly Pro Thr Cys Cys Ser Arg Lys
Met Glu Glu Lys 65 70 75 80 Tyr Gln Leu Thr Ala Arg Leu Asn Met Glu
Gln Leu Leu Gln Ser Ala 85 90 95 Ser Met Glu Leu Lys Phe Leu Ile
Ile Gln Asn Ala Ala Val Phe Gln 100 105 110 Glu Ala Phe Glu Ile Val
Val Arg His Ala Lys Asn Tyr Thr Asn Ala 115 120 125 Met Phe Lys Asn
Asn Tyr Pro Ser Leu Thr Pro Gln Ala Phe Glu Phe 130 135 140 Val Gly
Glu Phe Phe Thr Asp Val Ser Leu Tyr Ile Leu Gly Ser Asp 145 150 155
160 Ile Asn Val Asp Asp Met Val Asn Glu Leu Phe Asp Ser Leu Phe Pro
165 170 175 Val Ile Tyr Thr Gln Leu Met Asn Pro Gly Leu Pro Asp Ser
Ala Leu 180 185 190 Asp Ile Asn Glu Cys Leu Arg Gly Ala Arg Arg Asp
Leu Lys Val Phe 195 200 205 Gly Asn Phe Pro Lys Leu Ile Met Thr Gln
Val Ser Lys Ser Leu Gln 210 215 220 Val Thr Arg Ile Phe Leu Gln Ala
Leu Asn Leu Gly Ile Glu Val Ile 225 230 235 240 Asn Thr Thr Asp His
Leu Lys Phe Ser Lys Asp Cys Gly Arg Met Leu 245 250 255 Thr Arg Met
Trp Tyr Cys Ser Tyr Cys Gln Gly Leu Met Met Val Lys 260 265 270 Pro
Cys Gly Gly Tyr Cys Asn Val Val Met Gln Gly Cys Met Ala Gly 275 280
285 Val Val Glu Ile Asp Lys Tyr Trp Arg Glu Tyr Ile Leu Ser Leu Glu
290 295 300 Glu Leu Val Asn Gly Met Tyr Arg Ile Tyr Asp Met Glu Asn
Val Leu 305 310 315 320 Leu Gly Leu Phe Ser Thr Ile His Asp Ser Ile
Gln Tyr Val Gln Lys 325 330 335 Asn Ala Gly Lys Leu Thr Thr Thr Ile
Gly Lys Leu Cys Ala His Ser 340 345 350 Gln Gln Arg Gln Tyr Arg Ser
Ala Tyr Tyr Pro Glu Asp Leu Phe Ile 355 360 365 Asp Lys Lys Val Leu
Lys Val Ala His Val Glu His Glu Glu Thr Leu 370 375 380 Ser Ser Arg
Arg Arg Glu Leu Ile Gln Lys Leu Lys Ser Phe Ile Ser 385 390 395 400
Phe Tyr Ser Ala Leu Pro Gly Tyr Ile Cys Ser His Ser Pro Val Ala 405
410 415 Glu Asn Asp Thr Leu Cys Trp Asn Gly Gln Glu Leu Val Glu Arg
Tyr 420 425 430 Ser Gln Lys Ala Ala Arg Asn Gly Met Lys Asn Gln Phe
Asn Leu His 435 440 445 Glu Leu Lys Met Lys Gly Pro Glu Pro Val Val
Ser Gln Ile Ile Asp 450 455 460 Lys Leu Lys His Ile Asn Gln Leu Leu
Arg Thr Met Ser Met Pro Lys 465 470 475 480 Gly Arg Val Leu Asp Lys
Asn Leu Asp Glu Glu Gly Phe Glu Ser Gly 485 490 495 Asp Cys Gly Asp
Asp Glu Asp Glu Cys Ile Gly Gly Ser Gly Asp Gly 500 505 510 Met Ile
Lys Val Lys Asn Gln Leu Arg Phe Leu Ala Glu Leu Ala Tyr 515 520 525
Asp Leu Asp Val Asp Asp Ala Pro Gly Asn Ser Gln Gln Ala Thr Pro 530
535 540 Lys Asp Asn Glu Ile Ser Thr Phe His Asn Leu Gly Asn Val His
Ser 545 550 555 560 Pro Leu Lys Leu Leu Thr Ser Met Ala Ile Ser Val
Val Cys Phe Phe 565 570 575 Phe Leu Val His 580 531DNAArtificial
SequencePCR primer 5atagaattcc accatggccg ggaccgtgcg c
31631DNAArtificial SequencePCR primer 6ataggatccc ttcagcgggg
aatgaacgtt c 31721DNAArtificial SequencePCR primer 7gggccagtgg
atagacagat g 21824DNAArtificial SequencePCR primer 8caggggccag
tggatagacc gatg 24924DNAArtificial SequencePCR primer 9caggggccag
tggatagact gatg 241023DNAArtificial SequencePCR primer 10gctcactgga
tggtgggaag atg 23111392DNAMus musculus 11atgaacttcg ggctcacctt
gattttcctt gtccttactt taaaaggtgt ccagtgtgag 60gtgcaactgg tggagtctgg
gggaggctta gtgaagcctg gaggatccct gaaactctcc 120tgtgcagcct
ctggattcac tttcagtcgc tatgccatgt cttgggttcg ccagattcca
180gagaagatac tggagtgggt cgcagccatt gatagtagtg gtggtgacac
ctactattta 240gacactgtga aggaccgatt caccatctcc agagacaatg
ccaataatac cctgcacctg 300caaatgcgca gtctgaggtc tgaggacaca
gccttgtatt actgtgtaag acaggggggg 360gcttactggg gccaagggac
tctggtcact gtctctgcag ctagcaccaa gggcccatcg 420gtcttccccc
tggcaccctc ctccaagagc acctctgggg gcacagcggc cctgggctgc
480ctggtcaagg actacttccc cgaaccggtg acggtgtcgt ggaactcagg
cgccctgacc 540agcggcgtgc acaccttccc ggctgtccta cagtcctcag
gactctactc cctcagcagc 600gtggtgaccg tgccctccag cagcttgggc
acccagacct acatctgcaa cgtgaatcac 660aagcccagca acaccaaggt
ggacaagaaa gttgagccca aatcttgtga caaaactcac 720acatgcccac
cgtgcccagc acctgaactc ctggggggac cgtcagtctt cctcttcccc
780ccaaaaccca aggacaccct catgatctcc cggacccctg aggtcacatg
cgtggtggtg 840gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt
acgtggacgg cgtggaggtg 900cataatgcca agacaaagcc gcgggaggag
cagtacaaca gcacgtaccg tgtggtcagc 960gtcctcaccg tcctgcacca
ggactggctg aatggcaagg agtacaagtg caaggtctcc 1020aacaaagccc
tcccagcccc catcgagaaa accatctcca aagccaaagg gcagccccga
1080gaaccacagg tgtacaccct gcccccatcc cgggatgagc tgaccaagaa
ccaggtcagc 1140ctgacctgcc tggtcaaagg cttctatccc agcgacatcg
ccgtggagtg ggagagcaat 1200gggcagccgg agaacaacta caagaccacg
cctcccgtgc tggactccga cggctccttc 1260ttcctctaca gcaagctcac
cgtggacaag agcaggtggc agcaggggaa cgtcttctca 1320tgctccgtga
tgcatgaggc tctgcacaac cactacacgc agaagagcct ctccctgtct
1380ccgggtaaat ga 139212342DNAMus musculus 12gaggtgcacc tggtggagtc
tgggggaggc ttagtgaagc ctggagggtc cctgaaactc 60tcctgtgcag cctctggatt
cactttcagt aactatgcca tgtcttgggt tcgccagact 120ccagagaaga
ggctggagtg ggtcgcagcc attaataata atggtgatga cacctactat
180ttagacactg tgaaggaccg attcaccatc tccagagaca atgccaagaa
caccctgtac 240ctgcaaatga gcagtctgag gtctgaggac acagccctgt
attactgtgt aagacaaggg 300ggggcttact ggggccaagg gactctggtc
actgtctctg ca 342131413DNAMus musculus 13atgggatgga actggatctt
tattttaatc ctgtcagtaa ctacaggtgt ccactctgag 60gtccagctgc agcagtctgg
acctgagctg gtgaagcctg gggcttcagt gaagatatcc 120tgcaaggctt
ctggttactc attcactggc tactacatgc actgggtgaa gcaaagtcct
180gaaaagagcc ttgagtggat tggagagatt aatcctagca ctggtggtac
tacctacaac 240cagaagttca aggccaaggc cacattgact gtagacaaat
cctccagcac agcctacatg 300cagctcaaga gcctgacatc tgaggactct
gcagtctatt actgtgcaag gaggggcgga 360ttaactggga cgagcttctt
tgcttactgg ggccaaggga ctctggtcac tgtctctgca 420gctagcacca
agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg
480ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt
gacggtgtcg 540tggaactcag gcgccctgac cagcggcgtg cacaccttcc
cggctgtcct acagtcctca 600ggactctact ccctcagcag cgtggtgacc
gtgccctcca gcagcttggg cacccagacc 660tacatctgca acgtgaatca
caagcccagc aacaccaagg tggacaagaa agttgagccc 720aaatcttgtg
acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga
780ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc
ccggacccct 840gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc
ctgaggtcaa gttcaactgg 900tacgtggacg gcgtggaggt gcataatgcc
aagacaaagc cgcgggagga gcagtacaac 960agcacgtacc gtgtggtcag
cgtcctcacc gtcctgcacc aggactggct gaatggcaag 1020gagtacaagt
gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc
1080aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc
ccgggatgag 1140ctgaccaaga accaggtcag cctgacctgc ctggtcaaag
gcttctatcc cagcgacatc 1200gccgtggagt gggagagcaa tgggcagccg
gagaacaact acaagaccac gcctcccgtg 1260ctggactccg acggctcctt
cttcctctac agcaagctca ccgtggacaa gagcaggtgg 1320cagcagggga
acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg
1380cagaagagcc tctccctgtc tccgggtaaa tga 141314354DNAMus musculus
14caggtcactc tgaaagagtc tggccctggg atattgcagc cctcccagac cctcagtctg
60acttgttctt tctctgggtt ttcactgagc acttatggta tgggtgtagg ttggattcgt
120cagccttcag ggatgggtct ggagtggctg gccaacattt ggtggtatga
tgctaagtac 180tataactctg acctgaagag ccggctcaca atctccaagg
atacctccaa caaccaggtg 240ttcctcaaga tctccagtgt ggacacttca
gatactgcca catactactg tgctcaaatg 300ggactggcct ggtttgctta
ctggggccaa gggactctgg tcactgtctc tgca 35415354DNAMus musculus
15caggtcactc tgaaagagtc tggccctggg atattgcagc cctcccagac cctcagtctg
60acttgttctt tctctgggtt ttcactgagc atttatggta tgggtgtagg ttggattcgt
120cagccttcag ggaagggtct ggagtggctg gccaacattt ggtggaatga
tgataagtac 180tataactcag ccctgaagag ccggctcaca atctccaagg
atacctccaa caaccaggta 240ttcctcaaga tctccagtgt ggacactgca
gatactgcca catactactg tgctcaaata 300ggttacttct actttgacta
ctggggccaa ggcaccactc tcacagtctc ctca 354161416DNAMus musculus
16atgaacttcg ggctcacctt gattttcctc gtccttactt taaaaggtgt ccagtgtgag
60gtgcagctgg tggagtctgg gggagactta gtgaagcctg gagggaccct gaaactctcc
120tgtgcagcct ctggatccac tttcagtaac tatgccatgt cttgggttcg
ccagactcca 180gagaagaggc tggagtgggt cgcagccatt gatagtaatg
gaggtaccac ctactatcca 240gacactatga aggaccgatt caccatttcc
agagacaatg ccaagaacac cctgtacctg 300caaatgaaca gtctgaggtc
tgaagacaca gccttttatc actgtacaag acataatgga 360gggtatgaaa
actacggctg gtttgcttac tggggccaag ggactctggt cactgtctct
420gcagctagca ccaagggccc atcggtcttc cccctggcac cctcctccaa
gagcacctct 480gggggcacag cggccctggg ctgcctggtc aaggactact
tccccgaacc ggtgacggtg 540tcgtggaact caggcgccct gaccagcggc
gtgcacacct tcccggctgt cctacagtcc 600tcaggactct actccctcag
cagcgtggtg accgtgccct ccagcagctt gggcacccag 660acctacatct
gcaacgtgaa tcacaagccc agcaacacca aggtggacaa gaaagttgag
720cccaaatctt gtgacaaaac tcacacatgc ccaccgtgcc cagcacctga
actcctgggg 780ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca
ccctcatgat ctcccggacc 840cctgaggtca catgcgtggt ggtggacgtg
agccacgaag accctgaggt caagttcaac 900tggtacgtgg acggcgtgga
ggtgcataat gccaagacaa agccgcggga ggagcagtac 960aacagcacgt
accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc
1020aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga
gaaaaccatc 1080tccaaagcca aagggcagcc ccgagaacca caggtgtaca
ccctgccccc atcccgggat 1140gagctgacca agaaccaggt cagcctgacc
tgcctggtca aaggcttcta tcccagcgac 1200atcgccgtgg agtgggagag
caatgggcag ccggagaaca actacaagac cacgcctccc 1260gtgctggact
ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg
1320tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca
caaccactac 1380acgcagaaga gcctctccct gtctccgggt aaatga
141617366DNAMus musculus 17gaggtgcagc tggtggagtc tgggggagac
ttagtgaagc ctggagggtc cctgaaactc 60tcctgtgcag cctctggatt cactttcagt
agctatgcca tgtcttgggt tcgccagact 120ccagagaaga ggctggagtg
ggtcgcagcc attaatagta atggaggtac cacctactat 180ccagacacta
tgaaggaccg attcaccatc tccagagaca atgccaagaa caccctgtac
240ctgcaaatga gcagtctgag gtctgaagac tcagccttgt attactgtac
aagacataat 300ggagggtatg aaaactacgg ctggtttgct tactggggcc
aagggactct ggtcactgtc 360tctgca 366181413DNAMus musculus
18atggaatcta actggatact tccttttatt ctgtcggtag cttcaggggt ctactcagag
60gttcagctcc agcagtctgg gactgtgctg gcaaggcctg gggcttcagt gaagatgtcc
120tgcaaggctt ctggctacac ctttactggc tactggatgc gctgggtaaa
acagaggcct 180ggacagggtc tggaatggat tggcgctatt tatcctggaa
atagtgatac aacatacaac 240cagaagttca agggcaaggc caaactgact
gcagtcacat ctgtcagcac tgcctacatg 300gaactcagca gcctgacaaa
tgaggactct gcggtctatt actgttcaag atcgggggac 360ctaactgggg
ggtttgctta ctggggccaa gggactctgg tcactgtctc tacagccaaa
420gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag
cacctctggg 480ggcacagcgg ccctgggctg cctggtcaag gactacttcc
ccgaaccggt gacggtgtcg 540tggaactcag gcgccctgac cagcggcgtg
cacaccttcc cggctgtcct acagtcctca 600ggactctact ccctcagcag
cgtggtgacc gtgccctcca gcagcttggg cacccagacc 660tacatctgca
acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc
720aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact
cctgggggga 780ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc
tcatgatctc ccggacccct 840gaggtcacat gcgtggtggt ggacgtgagc
cacgaagacc ctgaggtcaa gttcaactgg 900tacgtggacg gcgtggaggt
gcataatgcc aagacaaagc cgcgggagga gcagtacaac 960agcacgtacc
gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag
1020gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa
aaccatctcc 1080aaagccaaag ggcagccccg agaaccacag gtgtacaccc
tgcccccatc ccgggatgag 1140ctgaccaaga accaggtcag cctgacctgc
ctggtcaaag gcttctatcc cagcgacatc 1200gccgtggagt gggagagcaa
tgggcagccg gagaacaact acaagaccac gcctcccgtg 1260ctggactccg
acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg
1320cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa
ccactacacg 1380cagaagagcc tctccctgtc tccgggtaaa tga 141319357DNAMus
musculus 19gaggttcagc tccagcagtc tgggactgtg ctggcaaggc ctggggcttc
agtgaagatg 60tcctgcaagg cttctggcta cacctttacc ggctactgga tgcactgggt
aaaacagagg 120cctggacagg gtctggaatg gattggcgct atttatcctg
gaaatagtga tactaactac 180aaccagaagt tcaagggcaa ggccaaactg
actgcagtca catctgccag cactgcctac 240atggagctca gcagcctgac
aaatgaggac gctgcggtct atcactgtac aagatcgggg 300gacctaactg
gggggcttgc ttactggggc caagggactc tggtcactgt ctctgca 35720372DNAMus
musculus 20caggtccagc tgcagcagcc tggggctgaa ctggtgaagc ctggggcttc
agtgaaactg 60tcctgcaagg cttctggata caccttcact agctactgga tgcattgggt
gaagcagagg 120cctggacaag gccttgagtg gatcggagag attgatcctt
ctgatagtta tacttactac 180aatcaaaagt tcaggggcaa ggccacattg
actgtagaca aatcctccaa cacagcctac 240atgcaactca gcagcctgac
atctgaggac tctgcggtct attactgttc aagatcaaat 300ctgggtgatg
gtcactaccg gtttcctgcg tttccttact ggggccaagg gactctggtc
360actgtctctg ca 37221372DNAMus musculus 21caggtccaac tgcagcagcc
tggggctgaa ctggtgaaac ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta
caccttcacc agctactgga tgcactgggt gaaacagagg 120cctggacaag
gccttgaatg gattggtaca attgaccctt ctgatagtga aactcactac
180aatctacagt tcaaggacac ggccacattg actgtagaca aatcctccag
cacagcctac 240atgcagctca gcagcctgac atctgaggac tctgcggtct
attattgtat aagaggcgcc 300ttctatagtt cctatagtta ctgggcctgg
tttgcttact ggggccaagg gactctggtc 360actgtctctg ca 37222463PRTMus
musculus
22Met Asn Phe Gly Leu Thr Leu Ile Phe Leu Val Leu Thr Leu Lys Gly 1
5 10 15 Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Lys 20 25 30 Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe 35 40 45 Ser Arg Tyr Ala Met Ser Trp Val Arg Gln Ile
Pro Glu Lys Ile Leu 50 55 60 Glu Trp Val Ala Ala Ile Asp Ser Ser
Gly Gly Asp Thr Tyr Tyr Leu 65 70 75 80 Asp Thr Val Lys Asp Arg Phe
Thr Ile Ser Arg Asp Asn Ala Asn Asn 85 90 95 Thr Leu His Leu Gln
Met Arg Ser Leu Arg Ser Glu Asp Thr Ala Leu 100 105 110 Tyr Tyr Cys
Val Arg Gln Gly Gly Ala Tyr Trp Gly Gln Gly Thr Leu 115 120 125 Val
Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 130 135
140 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
145 150 155 160 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
Trp Asn Ser 165 170 175 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala Val Leu Gln Ser 180 185 190 Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val Pro Ser Ser Ser 195 200 205 Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His Lys Pro Ser Asn 210 215 220 Thr Lys Val Asp Lys
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 225 230 235 240 Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 245 250 255
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 260
265 270 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
Glu 275 280 285 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys 290 295 300 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val Ser 305 310 315 320 Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys 325 330 335 Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 340 345 350 Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 355 360 365 Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 370 375 380
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 385
390 395 400 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser 405 410 415 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg 420 425 430 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu 435 440 445 His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly Lys 450 455 460 23114PRTMus musculus 23Glu
Val His Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10
15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr
20 25 30 Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu
Trp Val 35 40 45 Ala Ala Ile Asn Asn Asn Gly Asp Asp Thr Tyr Tyr
Leu Asp Thr Val 50 55 60 Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn
Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ser
Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Val Arg Gln Gly Gly Ala
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ala
24470PRTMus musculus 24Met Gly Trp Asn Trp Ile Phe Ile Leu Ile Leu
Ser Val Thr Thr Gly 1 5 10 15 Val His Ser Glu Val Gln Leu Gln Gln
Ser Gly Pro Glu Leu Val Lys 20 25 30 Pro Gly Ala Ser Val Lys Ile
Ser Cys Lys Ala Ser Gly Tyr Ser Phe 35 40 45 Thr Gly Tyr Tyr Met
His Trp Val Lys Gln Ser Pro Glu Lys Ser Leu 50 55 60 Glu Trp Ile
Gly Glu Ile Asn Pro Ser Thr Gly Gly Thr Thr Tyr Asn 65 70 75 80 Gln
Lys Phe Lys Ala Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser 85 90
95 Thr Ala Tyr Met Gln Leu Lys Ser Leu Thr Ser Glu Asp Ser Ala Val
100 105 110 Tyr Tyr Cys Ala Arg Arg Gly Gly Leu Thr Gly Thr Ser Phe
Phe Ala 115 120 125 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala
Ala Ser Thr Lys 130 135 140 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly 145 150 155 160 Gly Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro 165 170 175 Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 180 185 190 Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 195 200 205 Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 210 215
220 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
225 230 235 240 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu 245 250 255 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp 260 265 270 Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp 275 280 285 Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly 290 295 300 Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 305 310 315 320 Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 325 330 335
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 340
345 350 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu 355 360 365 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
Thr Lys Asn 370 375 380 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile 385 390 395 400 Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr 405 410 415 Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 420 425 430 Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 435 440 445 Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 450 455 460
Ser Leu Ser Pro Gly Lys 465 470 25118PRTMus musculus 25Gln Val Thr
Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln 1 5 10 15 Thr
Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Tyr 20 25
30 Gly Met Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Met Gly Leu Glu
35 40 45 Trp Leu Ala Asn Ile Trp Trp Tyr Asp Ala Lys Tyr Tyr Asn
Ser Asp 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser
Asn Asn Gln Val 65 70 75 80 Phe Leu Lys Ile Ser Ser Val Asp Thr Ser
Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Gln Met Gly Leu Ala Trp
Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala
115 26118PRTMus musculus 26Gln Val Thr Leu Lys Glu Ser Gly Pro Gly
Ile Leu Gln Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Phe
Ser Gly Phe Ser Leu Ser Ile Tyr 20 25 30 Gly Met Gly Val Gly Trp
Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu 35 40 45 Trp Leu Ala Asn
Ile Trp Trp Asn Asp Asp Lys Tyr Tyr Asn Ser Ala 50 55 60 Leu Lys
Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Asn Asn Gln Val 65 70 75 80
Phe Leu Lys Ile Ser Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr 85
90 95 Cys Ala Gln Ile Gly Tyr Phe Tyr Phe Asp Tyr Trp Gly Gln Gly
Thr 100 105 110 Thr Leu Thr Val Ser Ser 115 27471PRTMus musculus
27Met Asn Phe Gly Leu Thr Leu Ile Phe Leu Val Leu Thr Leu Lys Gly 1
5 10 15 Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val
Lys 20 25 30 Pro Gly Gly Thr Leu Lys Leu Ser Cys Ala Ala Ser Gly
Ser Thr Phe 35 40 45 Ser Asn Tyr Ala Met Ser Trp Val Arg Gln Thr
Pro Glu Lys Arg Leu 50 55 60 Glu Trp Val Ala Ala Ile Asp Ser Asn
Gly Gly Thr Thr Tyr Tyr Pro 65 70 75 80 Asp Thr Met Lys Asp Arg Phe
Thr Ile Ser Arg Asp Asn Ala Lys Asn 85 90 95 Thr Leu Tyr Leu Gln
Met Asn Ser Leu Arg Ser Glu Asp Thr Ala Phe 100 105 110 Tyr His Cys
Thr Arg His Asn Gly Gly Tyr Glu Asn Tyr Gly Trp Phe 115 120 125 Ala
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Ser Thr 130 135
140 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
145 150 155 160 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu 165 170 175 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His 180 185 190 Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser 195 200 205 Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr Tyr Ile Cys 210 215 220 Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 225 230 235 240 Pro Lys
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260
265 270 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val 275 280 285 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp 290 295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr 305 310 315 320 Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp 325 330 335 Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350 Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360 365 Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 370 375 380
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 385
390 395 400 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys 405 410 415 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser 420 425 430 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser 435 440 445 Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser 450 455 460 Leu Ser Leu Ser Pro Gly
Lys 465 470 28122PRTMus musculus 28Glu Val Gln Leu Val Glu Ser Gly
Gly Asp Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp
Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Ala
Ile Asn Ser Asn Gly Gly Thr Thr Tyr Tyr Pro Asp Thr Met 50 55 60
Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65
70 75 80 Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Ser Ala Leu Tyr
Tyr Cys 85 90 95 Thr Arg His Asn Gly Gly Tyr Glu Asn Tyr Gly Trp
Phe Ala Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ala
115 120 29470PRTMus musculus 29Met Glu Ser Asn Trp Ile Leu Pro Phe
Ile Leu Ser Val Ala Ser Gly 1 5 10 15 Val Tyr Ser Glu Val Gln Leu
Gln Gln Ser Gly Thr Val Leu Ala Arg 20 25 30 Pro Gly Ala Ser Val
Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Gly Tyr
Trp Met Arg Trp Val Lys Gln Arg Pro Gly Gln Gly Leu 50 55 60 Glu
Trp Ile Gly Ala Ile Tyr Pro Gly Asn Ser Asp Thr Thr Tyr Asn 65 70
75 80 Gln Lys Phe Lys Gly Lys Ala Lys Leu Thr Ala Val Thr Ser Val
Ser 85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Asn Glu Asp
Ser Ala Val 100 105 110 Tyr Tyr Cys Ser Arg Ser Gly Asp Leu Thr Gly
Gly Phe Ala Tyr Trp 115 120 125 Gly Gln Gly Thr Leu Val Thr Val Ser
Thr Ala Lys Ala Ser Thr Lys 130 135 140 Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys Ser Thr Ser Gly 145 150 155 160 Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 165 170 175 Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 180 185 190
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 195
200 205 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn 210 215 220 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro 225 230 235 240 Lys Ser Cys Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu 245 250 255 Leu Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp 260 265 270 Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp 275 280 285 Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 290 295 300 Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 305 310 315
320 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
325 330 335 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro 340 345 350 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu 355 360 365 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn 370 375 380 Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile 385 390 395 400 Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 405
410 415 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys 420 425 430 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys 435 440 445 Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu 450 455 460 Ser Leu Ser Pro Gly Lys 465 470
30119PRTMus musculus 30Glu Val Gln Leu Gln Gln Ser Gly Thr Val Leu
Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Trp Met His Trp Val Lys Gln
Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile Tyr Pro
Gly Asn Ser Asp Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys
Ala Lys Leu Thr Ala Val Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met
Glu Leu Ser Ser Leu Thr Asn Glu Asp Ala Ala Val Tyr His Cys 85 90
95 Thr Arg Ser Gly Asp Leu Thr Gly Gly Leu Ala Tyr Trp Gly Gln Gly
100 105 110 Thr Leu Val Thr Val Ser Ala 115 31124PRTMus musculus
31Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1
5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser
Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu
Glu Trp Ile 35 40 45 Gly Glu Ile Asp Pro Ser Asp Ser Tyr Thr Tyr
Tyr Asn Gln Lys Phe 50 55 60 Arg Gly Lys Ala Thr Leu Thr Val Asp
Lys Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr
Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Ser Asn Leu
Gly Asp Gly His Tyr Arg Phe Pro Ala Phe Pro 100 105 110 Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ala 115 120 32124PRTMus musculus
32Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1
5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser
Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu
Glu Trp Ile 35 40 45 Gly Thr Ile Asp Pro Ser Asp Ser Glu Thr His
Tyr Asn Leu Gln Phe 50 55 60 Lys Asp Thr Ala Thr Leu Thr Val Asp
Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr
Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ile Arg Gly Ala Phe
Tyr Ser Ser Tyr Ser Tyr Trp Ala Trp Phe Ala 100 105 110 Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ala 115 120 33717DNAMus musculus
33atgagtcctg cccagttcct gtttctgtta gtgctctgga ttcgggaaac caacggtgat
60gttgtgatga cccagactcc actcactttg tcggttacca ttggacaacc agcctccatc
120tcttgcaagt caagtcagag cctcttagat agtgatggaa agacatattt
gaattggttg 180ttacagaggc caggccagtc tccaaagcgc ctaatctatc
tggtgtctaa attggactct 240ggagcccctg acaggttcac tggcagtgga
tcagggacag atttcacact gaaaatcagt 300agagtggagg ctgaggattt
gggaatttat tattgctggc aaggtacaca ttttccgctc 360acgttcggtg
ctgggaccaa gctggagctg aaacgtacgg tggctgcacc atctgtcttc
420atcttcccgc catctgatga gcagttgaaa tctggaactg cctctgttgt
gtgcctgctg 480aataacttct atcccagaga ggccaaagta cagtggaagg
tggataacgc cctccaatcg 540ggtaactccc aggagagtgt cacagagcag
gacagcaagg acagcaccta cagcctcagc 600agcaccctga cgctgagcaa
agcagactac gagaaacaca aagtctacgc ctgcgaagtc 660acccatcagg
gcctgagctc gcccgtcaca aagagcttca acaggggaga gtgttga 71734336DNAMus
musculus 34gatgttgtga tgacccagtc tccactcact ttgtcgatta ccattggaca
accagcctcc 60atctcttgca agtcaagtca gagcctctta gatagtgatg gaaagacata
tttgaattgg 120ttgttacaga ggccaggcca gtctccaaag cgcctaatct
atctggtgtc taaactggac 180tctggagtcc ctgacaggtt cactggcagt
ggatcaggga cagatttctc actgaaaatc 240agcagagtgg aggctgagga
tttgggaatt tattattgct ggcaaggtac acattttccg 300ctcacgttcg
gtgctgggac caagctggag ctgaaa 33635717DNAMus musculus 35atgagtcctg
tccagttcct gtttctgtta atgctctgga ttcaggaaac caacggtgat 60gttgtgatga
cccagactcc actgtctttg tcggttacca ttggacaacc agcctctatc
120tcttgcaagt caagtcagag cctcttatat agtaatggaa agacatattt
gaattggtta 180caacagaggc ctggccaggc tccaaagcac ctaatgtatc
aggtgtccaa actggaccct 240ggcatccctg acaggttcag tggcagtgga
tcagaaacag attttacact taaaatcagc 300agagtggagg ctgaagattt
gggagtttat tactgcttgc aaagtacata ttatccgctc 360acgttcggtg
ctgggaccaa gctggagctg aaacgtacgg tggctgcacc atctgtcttc
420atcttcccgc catctgatga gcagttgaaa tctggaactg cctctgttgt
gtgcctgctg 480aataacttct atcccagaga ggccaaagta cagtggaagg
tggataacgc cctccaatcg 540ggtaactccc aggagagtgt cacagagcag
gacagcaagg acagcaccta cagcctcagc 600agcaccctga cgctgagcaa
agcagactac gagaaacaca aagtctacgc ctgcgaagtc 660acccatcagg
gcctgagctc gcccgtcaca aagagcttca acaggggaga gtgttga 71736324DNAMus
musculus 36gacatcaaga tgacccagtc tccatcttcc atgtatgcat ctctaggaga
gagagtcact 60atcacttgca aggcgagtca ggacattaat aactatttaa gctggttcca
gcagaaacca 120gggaaatctc ctaagaccct gatctatcgt gcaaacagat
tggtagatgg ggtcccatca 180aggttcagtg gcagtggatc tgggcaagat
tattctctca ccatcagcag cctggagtat 240gaagatatgg gaattaatta
ttgtctacag tgtgatgagt ttcctccgtg gacgttcggt 300ggaggcacca
agctggaaat caaa 32437336DNAMus musculus 37gatgttgtga tgacccaaac
tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60atctcttgca gatctagtca
gagccttgta cacagtaatg gaaacaccta tttacattgg 120tacctgcaga
agccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt
180tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac
actcaagatc 240agcagagtgg aggctgagga tctgggagtt tatttctgct
ctcaaagtac acatgttccg 300tggacgttcg gtggaggcac caagctggaa atcaaa
33638705DNAMus musculus 38atgagaccct ccattcagtt cctggggctc
ttgttgttct ggcttcatgg tgttcagtgt 60gacatccaga tgacacagtc tccatcctca
ctgtctgcat ctctgggagg caaagtcacc 120atcacttgca aggcaagtca
ggacattaac aagaatatag tttggtacca acacaagcct 180ggaaaaggtc
ctaggctgct catatggtac acatctacat tacagccagg catcccatca
240aggttcagtg gaagtgggtc tgggagagat tattccttca gcatcagcaa
cctggagcct 300gaagatattg caacttatta ctgtctacag tatgataatc
ttccacggac gttcggtgga 360ggcaccaaac tggaaatcaa acgtacggtg
gctgcaccat ctgtcttcat cttcccgcca 420tctgatgagc agttgaaatc
tggaactgcc tctgttgtgt gcctgctgaa taacttctat 480cccagagagg
ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag
540gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag
caccctgacg 600ctgagcaaag cagactacga gaaacacaaa gtctacgcct
gcgaagtcac ccatcagggc 660ctgagctcgc ccgtcacaaa gagcttcaac
aggggagagt gttga 70539321DNAMus musculus 39gacatccaga tgacacagtc
tccatcctca ctgtctgcat ctctgggagg caaagtcacc 60atcacttgca aggcaagtca
ggacattaac aagaatataa tttggtacca acacaagcct 120ggaaaaggtc
ctaggctgct catatggtac acatctacat tacagccagg catcccatca
180aggttcagtg gaagtgggtc tgggagagat tattccttca gcatcagcaa
cctggagcct 240gaagatattg caacttatta ctgtctacag tatgataatc
ttccacggac gttcggtgga 300ggcaccaagc tggaaatcaa a 32140720DNAMus
musculus 40atgaggttct ctgctcagct tctggggctg cttgtgctct ggatccctgg
atccactgca 60gatattgtga tgacgcaggc tgcattctcc aatccagtca ctcttggaac
atcaacttcc 120atctcctgca ggtctagtaa gagtctccta catagtaatg
gcatcactta tttgtattgg 180tatctgcaga agccaggcca gtctcctcag
ctcctgattt atcagatgtc caaccttgcc 240tcaggagtcc cagacaggtt
cagtagcagt gggtcaggaa ctgatttcac actgagaatc 300agcagagtgg
aggctgagga tgtgggtgtt tattactgtg ctcaaaatct agaacttccg
360tatacgttcg gatcggggac caagctggaa ataaaacgta cggtggctgc
accatctgtc 420ttcatcttcc cgccatctga tgagcagttg aaatctggaa
ctgcctctgt tgtgtgcctg 480ctgaataact tctatcccag agaggccaaa
gtacagtgga aggtggataa cgccctccaa 540tcgggtaact cccaggagag
tgtcacagag caggacagca aggacagcac ctacagcctc 600agcagcaccc
tgacgctgag caaagcagac tacgagaaac acaaagtcta cgcctgcgaa
660gtcacccatc agggcctgag ctcgcccgtc acaaagagct tcaacagggg
agagtgttga 72041336DNAMus musculus 41gatattgtga tgacgcaggc
tgcattctcc aatccagtca ctcttggaac atcagcttcc 60atctcctgca ggtctagtaa
gagtctccta catagtaatg gcatcactta tttgtattgg 120tttctgcaga
agccaggcca gtctcctcag ctcctgattt atcagatgtc caaccttgcc
180tcaggagtcc cagacaggtt cagtagcagt gggtcaggaa ctgatttcac
actgagaatc 240agcagagtgg aggctgagga tgtgggtgtt tattactgtg
ctcaaaatct agaacttccg 300tatacgttcg gatcggggac caagctggaa ataaaa
33642321DNAMus musculus 42gatattgtgc taactcagtc tccagccacc
ctgtctgtga ctccaggaga cagagtcagt 60ctttcctgca gggccagcca tagtattagc
aacttcctac actggtatcc acaaaaatca 120catgagtctc caaggcttct
catcaagtat gcttcccagt ccatctctgg gatcccctcc 180aggttcagtg
gcaatggatc agggacagat ttcactctca gtatcaacag tgtggagact
240gaagattttg gaatgtattt ctgtcaacag agtaacatct ggtcgctcac
gttcggtgct 300gggaccaagc tggagctgaa a 32143333DNAMus musculus
43gacattgtgc tcacccaatc tccaacttct ttggctgtgt ctctagggca gagtgtcacc
60atctcctgca gagccagtga aagtgttgaa tattatggca ctagtttaat gcagtggtac
120caacagaaac caggacagcc acccaaactc ctcatctatg gtgcatccaa
cgtagaatct 180ggggtccctg ccaggtttag tggcagtggg tctgggacag
acttcagcct caacatccat 240cctgtggagg aggatgatat tgcaatgtat
ttctgtcagc aaagtaggaa ggttccgtat 300acgttcggat cggggaccaa
gctggaaata aaa 33344238PRTMus musculus 44Met Ser Pro Ala Gln Phe
Leu Phe Leu Leu Val Leu Trp Ile Arg Glu 1 5 10 15 Thr Asn Gly Asp
Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val 20 25 30 Thr Ile
Gly Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu 35 40 45
Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro 50
55 60 Gly Gln Ser Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp
Ser 65 70 75 80 Gly Ala Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr
Asp Phe Thr 85 90 95 Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu
Gly Ile Tyr Tyr Cys 100 105 110 Trp Gln Gly Thr His Phe Pro Leu Thr
Phe Gly Ala Gly Thr Lys Leu 115 120 125 Glu Leu Lys Arg Thr Val Ala
Ala Pro Ser Val Phe Ile Phe Pro Pro 130 135 140 Ser Asp Glu Gln Leu
Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu 145 150 155 160 Asn Asn
Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn 165 170 175
Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser 180
185 190 Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys
Ala 195 200 205 Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr
His Gln Gly 210 215 220 Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg
Gly Glu Cys 225 230 235 45112PRTMus musculus 45Asp Val Val Met Thr
Gln Ser Pro Leu Thr Leu Ser Ile Thr Ile Gly 1 5 10 15 Gln Pro Ala
Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asp
Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40
45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro
50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu
Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr
Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Leu Thr Phe Gly Ala Gly
Thr Lys Leu Glu Leu Lys 100 105 110 46238PRTMus musculus 46Met Ser
Pro Val Gln Phe Leu Phe Leu Leu Met Leu Trp Ile Gln Glu 1 5 10 15
Thr Asn Gly Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val 20
25 30 Thr Ile Gly Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser
Leu 35 40 45 Leu Tyr Ser Asn Gly Lys Thr Tyr Leu Asn Trp Leu Gln
Gln Arg Pro 50 55 60 Gly Gln Ala Pro Lys His Leu Met Tyr Gln Val
Ser Lys Leu Asp Pro 65 70 75 80 Gly Ile Pro Asp Arg Phe Ser Gly Ser
Gly Ser Glu Thr Asp Phe Thr 85 90 95 Leu Lys Ile Ser Arg Val Glu
Ala Glu Asp Leu Gly Val Tyr Tyr Cys 100 105 110 Leu Gln Ser Thr Tyr
Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu 115 120 125 Glu Leu Lys
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro 130 135 140 Ser
Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu 145 150
155 160 Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp
Asn 165 170 175 Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu
Gln Asp Ser 180 185 190 Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu
Thr Leu Ser Lys Ala 195 200 205 Asp Tyr Glu Lys His Lys Val Tyr Ala
Cys Glu Val Thr His Gln Gly 210 215 220 Leu Ser Ser Pro Val Thr Lys
Ser Phe Asn Arg Gly Glu Cys 225 230 235 47108PRTMus musculus 47Asp
Ile Lys Met Thr Gln Ser Pro Ser Ser Met Tyr Ala Ser Leu Gly 1 5 10
15 Glu Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Asn Tyr
20 25 30 Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Thr
Leu Ile 35 40 45 Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr
Ile Ser Ser Leu Glu Tyr 65 70 75 80 Glu Asp Met Gly Ile Asn Tyr Cys
Leu Gln Cys Asp Glu Phe Pro Pro 85 90 95 Trp Thr Phe Gly Gly Gly
Thr Lys Leu Glu Ile Lys 100 105 48112PRTMus musculus 48Asp Val Val
Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp
Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25
30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly
Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val
Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Trp Thr Phe Gly
Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 49234PRTMus musculus
49Met Arg Pro Ser Ile Gln Phe Leu Gly Leu Leu Leu Phe Trp Leu His 1
5 10 15 Gly Val Gln Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser 20 25 30 Ala Ser Leu Gly Gly Lys Val Thr Ile Thr Cys Lys Ala
Ser Gln Asp 35 40 45 Ile Asn Lys Asn Ile Val Trp Tyr Gln His Lys
Pro Gly Lys Gly Pro 50 55 60 Arg Leu Leu Ile Trp Tyr Thr Ser Thr
Leu Gln Pro Gly Ile Pro Ser 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser
Gly Arg Asp Tyr Ser Phe Ser Ile Ser 85 90 95 Asn Leu Glu Pro Glu
Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp 100 105 110 Asn Leu Pro
Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 115 120 125 Thr
Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 130 135
140 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
145 150 155 160 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser 165 170 175 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr 180 185 190 Tyr Ser Leu Ser Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys 195 200 205 His Lys Val Tyr Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro 210 215 220 Val Thr Lys Ser
Phe Asn Arg Gly Glu Cys 225 230 50107PRTMus musculus 50Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Gly
Lys Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Lys Asn 20 25
30 Ile Ile Trp Tyr Gln His Lys Pro Gly Lys Gly Pro Arg Leu Leu Ile
35 40 45 Trp Tyr Thr Ser Thr Leu Gln Pro Gly Ile Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser
Asn Leu Glu Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln
Tyr Asp Asn Leu Pro Arg 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu
Glu Ile Lys 100 105 51239PRTMus musculus 51Met Arg Phe Ser Ala Gln
Leu Leu Gly Leu Leu Val Leu Trp Ile Pro 1 5 10 15 Gly Ser Thr Ala
Asp Ile Val Met Thr Gln Ala Ala Phe Ser Asn Pro 20 25 30 Val Thr
Leu Gly Thr Ser Thr Ser Ile Ser Cys Arg Ser Ser Lys Ser 35 40 45
Leu Leu His Ser Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys 50
55 60 Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu
Ala 65 70 75 80 Ser Gly Val Pro Asp Arg Phe Ser Ser Ser Gly Ser Gly
Thr Asp Phe 85 90 95 Thr Leu Arg Ile Ser Arg Val Glu Ala Glu Asp
Val Gly Val Tyr Tyr 100 105 110 Cys Ala Gln Asn Leu Glu Leu Pro Tyr
Thr Phe Gly Ser Gly Thr Lys 115 120 125 Leu Glu Ile Lys Arg Thr Val
Ala Ala Pro Ser Val Phe Ile Phe Pro 130 135 140 Pro Ser Asp Glu Gln
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 145 150 155 160 Leu Asn
Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 165 170 175
Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 180
185 190 Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser
Lys 195 200 205 Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val
Thr His Gln 210 215 220 Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn
Arg Gly Glu Cys 225 230 235 52112PRTMus musculus 52Asp Ile Val Met
Thr Gln Ala Ala Phe Ser Asn Pro Val Thr Leu Gly 1 5 10 15 Thr Ser
Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20 25 30
Asn Gly Ile Thr Tyr Leu Tyr Trp Phe Leu Gln Lys Pro Gly Gln Ser 35
40 45 Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val
Pro 50 55 60 Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr
Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Tyr Cys Ala Gln Asn 85 90 95 Leu Glu Leu Pro Tyr Thr Phe Gly Ser
Gly Thr Lys Leu Glu Ile Lys 100 105 110 53107PRTMus musculus 53Asp
Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly 1 5 10
15 Asp Arg Val Ser Leu Ser Cys Arg Ala Ser His Ser Ile Ser Asn Phe
20 25 30 Leu His Trp Tyr Pro Gln Lys Ser His Glu Ser Pro Arg Leu
Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser
Arg Phe Ser Gly 50 55 60 Asn Gly Ser Gly Thr Asp Phe Thr Leu Ser
Ile Asn Ser Val Glu Thr 65 70 75 80 Glu Asp Phe Gly Met Tyr Phe Cys
Gln Gln Ser Asn Ile Trp Ser Leu 85 90 95 Thr Phe Gly Ala Gly Thr
Lys Leu Glu Leu Lys 100 105 54111PRTMus musculus 54Asp Ile Val Leu
Thr Gln Ser Pro Thr Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Ser
Val Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Glu Tyr Tyr 20 25 30
Gly Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35
40 45 Lys Leu Leu Ile Tyr Gly Ala Ser Asn Val Glu Ser Gly Val Pro
Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu
Asn Ile His 65 70 75 80 Pro Val Glu Glu Asp Asp Ile Ala Met Tyr Phe
Cys Gln Gln Ser Arg 85 90 95 Lys Val Pro Tyr Thr Phe Gly Ser Gly
Thr Lys Leu Glu Ile Lys 100 105 110 55333DNAMus musculus
55cagatccagt tggagcagtc tggacctgag ctgaagaagc ctggagagac agtcaagatc
60tcctgcaagg cttctggtta tattttcaga gactattcaa tgcactgggt gaagcaggct
120ccaggaaagg gtttaaagtg gatgggctgg ataaacactg agacgggtga
gccaacatat 180gcagatgact tcaagggacg gtttgccttc tctttggaaa
cctctgccag cactgcctat 240ttgcagatca acaacctcaa aaatgaggac
acggctacat atttctgtac tagcctttac 300tggggccaag ggactctggt
cactgtctct gca 33356372DNAMus musculus 56caggtcactc tgaaagagtc
tggccctggg atattgcagc cctcccagac cctcagtctg 60acttgttctt tctctgggtt
ttcactgagc acttatggta tgggtgtagg ttggattcgt 120cagccttcag
ggaagggtct ggagtggctg gccaacattt ggtggcatga tgataagtac
180tataactcag ccctgaagag ccggctcaca atctccaagg atatctccaa
caaccaggta 240ttcctcaaga tctccagtgt ggacactgca gatactgcca
catactactg tgctcaaata 300gcccctcgat ataataagta cgaaggcttt
tttgctttct ggggccaagg gactctggtc 360actgtctctg ca 37257345DNAMus
musculus 57caggttcaac tgcagcagtc tggggctgag ctggtgaggc ctggggcttc
agtgaagctg 60tcctgcaagg cttcgggcta cacatttact gactatgaaa tgcactgggt
gaagcagaca 120cctgtgcatg gcctaaaatg gattggagct cttgatccta
aaactggtga tactgcctac 180agtcagaagt tcaagggcaa ggccacactg
actgcagaca aatcctccag cacagcctac 240atggagctcc gcagcctgac
atctgaggac tctgccgtct attactgtac aagattctac 300tcctatactt
actggggcca agggactctg gtcactgtct ctgca 34558357DNAMus musculus
58gaggtgcagc ttgttgagac tggtggagga ctggtgcagc ctgaagggtc attgaaactc
60tcatgtgcag cttctggatt cagcttcaat atcaatgcca tgaactgggt ccgccaggct
120ccaggaaagg gtttggaatg ggttgctcgc ataagaagtg aaagtaataa
ttatgcaaca 180tattatggcg attcagtgaa agacaggttc accatctcca
gagatgattc acaaaacatg 240ctctatctac aaatgaacaa cttgaaaact
gaggacacag ccatatatta ctgtgtgaga 300gaggtaacta catcgtttgc
ttattggggc caagggactc tggtcactgt ctctgca 35759369DNAMus musculus
59gaggtgcagc ttgttgagac tggtggagga ttggtgcagc ctaaagggtc attgaaactc
60tcatgtgcag cctctggatt caccttcaat gccagtgcca tgaactgggt ccgccaggct
120ccaggaaagg gtttggaatg ggttgctcgc ataagaagta aaagtaataa
ttatgcaata 180tattatgccg attcagtgaa agacaggttc accatctcca
gagatgattc acaaagcatg 240ctctatctgc aaatgaacaa cttgaaaact
gaggacacag ccatgtatta ctgtgtgaga 300gatccgggct actatggtaa
cccctggttt gcttactggg gccaagggac tctggtcact 360gtctctgca
36960111PRTMus musculus 60Gln Ile Gln Leu Glu Gln Ser Gly Pro Glu
Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala
Ser Gly Tyr Ile Phe Arg Asp Tyr 20 25 30 Ser Met His Trp Val Lys
Gln Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn
Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60 Lys Gly
Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80
Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85
90 95 Thr Ser Leu Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala
100 105 110 61124PRTMus musculus 61Gln Val Thr Leu Lys Glu Ser Gly
Pro Gly Ile Leu Gln Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys
Ser Phe Ser Gly Phe Ser Leu Ser Thr Tyr 20 25 30 Gly Met Gly Val
Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu 35 40 45 Trp Leu
Ala Asn Ile Trp Trp His Asp Asp Lys Tyr Tyr Asn Ser Ala 50 55 60
Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Ile Ser Asn Asn Gln Val 65
70 75 80 Phe Leu Lys Ile Ser Ser Val Asp Thr Ala Asp Thr Ala Thr
Tyr Tyr 85 90 95 Cys Ala Gln Ile Ala Pro Arg Tyr Asn Lys Tyr Glu
Gly Phe Phe Ala 100 105 110 Phe Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ala 115 120 62115PRTMus musculus 62Gln Val Gln Leu Gln Gln Ser
Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Met His
Trp Val Lys Gln Thr Pro Val His Gly Leu Lys Trp Ile 35 40 45 Gly
Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe 50 55
60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr
Tyr Cys 85 90 95 Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly
Thr Leu Val Thr 100 105 110 Val Ser Ala 115 63119PRTMus musculus
63Glu Val Gln Leu Val Glu Thr Gly Gly Gly Leu Val Gln Pro Glu Gly 1
5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Asn Ile
Asn 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Glu Ser Asn Asn Tyr Ala
Thr Tyr Tyr Gly Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser
Arg Asp Asp Ser Gln Asn Met 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn
Leu Lys Thr Glu Asp Thr Ala Ile Tyr 85 90 95 Tyr Cys Val Arg Glu
Val Thr Thr Ser Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ala 115 64123PRTMus musculus 64Glu Val Gln Leu Val Glu
Thr Gly Gly Gly Leu Val Gln Pro Lys Gly 1 5 10 15 Ser Leu Lys Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Ala Ser 20 25 30 Ala Met
Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ala Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Ile Tyr Tyr Ala Asp 50
55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser
Met 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr
Ala Met Tyr 85 90 95 Tyr Cys Val Arg Asp Pro Gly Tyr Tyr Gly Asn
Pro Trp Phe Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ala 115 120 65336DNAMus musculus 65gatgttgtga tgacccagac
tccactcact ttgtcggtta cccttggaca accagcctcc 60atctcttgca agtcaagtca
gagcctctta catagtgatg gaaagacatt tttgaattgg 120ttattacaga
ggccaggcca gtctccaaag cgcctaatct atctggtgtc tagactggac
180tctggagtcc ctgacaggtt cactggcagt ggatcaggga cagatttcac
actgaaaatc 240agcagagtgg aggctgagga tttgggagtt tattattgct
gccaaggtac acattttcct 300cggacgttcg gtggaggcac caggctggaa atcaaa
33666336DNAMus musculus 66gatgttttga tgacccaaac tccactctcc
ctgcctgtca gtcttggaga tcaagcctcc 60atctcttgca gatctagtca gagcattgta
catagtaatg gaaacaccta tttagaatgg 120tacctgcaga aaccaggcca
gtctccaaag ctcctgatct acaaagtttc caaccgattt 180tctggggtcc
cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc
240agcagagtgg aggctgagga tctgggagtt tattactgct ttcaaggttc
acatgttccg 300tggacgttcg gtggaggcac caagctggaa atcaaa
33667336DNAMus musculus 67gatgttgtga tgacccaaac tccactctcc
ctgcctgtca gtcttggaga tcaagcctcc 60atctcttgca gatctagtca gagccttgta
cacagtaatg gaaacaccta tttacattgg 120tacctgcaga agccaggcca
gtctccaaag ctcctgatct acaaagtttc caaccgattt 180tctggggtcc
cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc
240agcagagtgg aggctgagga tctgggagtt tatttctgct ctcaaaatac
acatgttcct 300cctacgttcg gatcggggac caagctggaa ataaaa
33668336DNAMus musculus 68gatattgtga tgactcagtc tgcaccctct
gtacctgtca ctcctggaga gtcagtatcc 60atctcctgca agtctagtaa gagtctcctg
catagtaatg gcaacactta cttgaattgg 120ttcctgcaga ggccaggcca
gtctcctcaa ctcctgattt attggatgtc caaccttgcc 180tcaggagtcc
cagacaggtt cagtggcagt gggtcaggaa ctgctttcac actgagaatc
240agtagagtgg aggctgagga tgtgggtgtt tattactgta tgcaacatat
agaataccct 300ttcacgttcg gcacggggac aaaattggaa ataaaa
33669336DNAMus musculus 69gatattgtga tgacgcaggc tgcattctcc
aatccagtca ctcttggaac atcagcttcc 60atctcctgca ggtctagtaa gagtctccta
catagttatg acatcactta tttgtattgg 120tatctgcaga agccaggcca
gtctcctcag ctcctgattt atcagatgtc caaccttgcc 180tcaggagtcc
cagacaggtt cagtagcagt gggtcaggaa ctgatttcac actgagaatc
240agcagagtgg aggctgagga tgtgggtgtt tattactgtg ctcaaaatct
agaacttcct 300ccgacgttcg gtggaggcac caagctggaa atcaaa
33670318DNAMus musculus 70caaattgttc tcacccagtc tccagcaatc
atgtctgcat ttccagggga gaaggtcacc 60atgacctgca gtgccagctc aagtgttagt
tacatgtact ggtaccagca gaagtcagga 120tcctccccca gactcctgat
ttatgacaca tccaacctgg cttctggagt ccctgttcgc 180ttcagtggca
gtgggtctgg gacctcttac tctctcacaa tcagccgaat ggaggctgaa
240gatgctgcca cttattactg ccagcagtgg agtagttacc cgctcacgtt
cggtggtggg 300accgagctgg agctgaaa 31871112PRTMus musculus 71Asp Val
Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Leu Gly 1 5 10 15
Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu His Ser 20
25 30 Asp Gly Lys Thr Phe Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln
Ser 35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Arg Leu Asp Ser
Gly Val Pro 50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly
Val Tyr Tyr Cys Cys Gln Gly 85 90 95 Thr His Phe Pro Arg Thr Phe
Gly Gly Gly Thr Arg Leu Glu Ile Lys 100 105 110 72112PRTMus
musculus 72Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser
Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser
Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu
Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val
Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu
Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His
Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110
73112PRTMus musculus 73Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu
Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser
Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His
Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile
Tyr Lys Val
Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu
Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Asn 85 90 95 Thr His
Val Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110
74112PRTMus musculus 74Asp Ile Val Met Thr Gln Ser Ala Pro Ser Val
Pro Val Thr Pro Gly 1 5 10 15 Glu Ser Val Ser Ile Ser Cys Lys Ser
Ser Lys Ser Leu Leu His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Asn
Trp Phe Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile
Tyr Trp Met Ser Asn Leu Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe
Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Ile 65 70 75 80 Ser
Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His 85 90
95 Ile Glu Tyr Pro Phe Thr Phe Gly Thr Gly Thr Lys Leu Glu Ile Lys
100 105 110 75112PRTMus musculus 75Asp Ile Val Met Thr Gln Ala Ala
Phe Ser Asn Pro Val Thr Leu Gly 1 5 10 15 Thr Ser Ala Ser Ile Ser
Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20 25 30 Tyr Asp Ile Thr
Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln
Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro 50 55 60
Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile 65
70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala
Gln Asn 85 90 95 Leu Glu Leu Pro Pro Thr Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys 100 105 110 76106PRTMus musculus 76Gln Ile Val Leu
Thr Gln Ser Pro Ala Ile Met Ser Ala Phe Pro Gly 1 5 10 15 Glu Lys
Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30
Tyr Trp Tyr Gln Gln Lys Ser Gly Ser Ser Pro Arg Leu Leu Ile Tyr 35
40 45 Asp Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly
Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met
Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser
Ser Tyr Pro Leu Thr 85 90 95 Phe Gly Gly Gly Thr Glu Leu Glu Leu
Lys 100 105 77345DNAArtificial SequenceMouse-human chimeric
antibody H chain 77caggtgcagc tggtggagtc tggagctgag gtgaagaagc
ctggggcctc agtgaaggtc 60tcctgcaagg cttctggata caccttcacc gactatgaaa
tgcactgggt gcgacaggcc 120cctggacaag ggcttgagtg gatgggagct
cttgatccta aaactggtga tactgcctac 180agtcagaagt tcaagggcag
agtcacgatt accgcggacg aatccacgag cacagcctac 240atggagctga
gcagcctgag atctgaggac acggccgtgt attactgtgc gagattctac
300tcctatactt actggggcca gggaaccctg gtcaccgtct cctca
34578345DNAArtificial SequenceMouse-human chimeric antibody H chain
78caggtgcagc tggtggagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc
60tcctgcaagg cttctggata caccttcacc gactatgaaa tgcactgggt gcgacaggcc
120cctggacaag ggcttgagtg gatgggagct cttgatccta aaactggtga
tactgcctac 180agtcagaagt tcaagggcag agtcacgctg accgcggacg
aatccacgag cacagcctac 240atggagctga gcagcctgag atctgaggac
acggccgtgt attactgtac aagattctac 300tcctatactt actggggcca
gggaaccctg gtcaccgtct cctca 34579345DNAArtificial
SequenceMouse-human chimeric antibody H chain 79caggtgcagc
tggtggagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60tcctgcaagg
cttctggata caccttcacc gactatgaaa tgcactgggt gcgacaggcc
120cctggacaag ggcttgagtg gatgggagct cttgatccta aaactggtga
tactgcctac 180agtcagaagt tcaagggcag agtcacgctg accgcggaca
aatccacgag cacagcctac 240atggagctga gcagcctgag atctgaggac
acggccgtgt attactgtac aagattctac 300tcctatactt actggggcca
gggaaccctg gtcaccgtct cctca 34580345DNAArtificial
SequenceMouse-human chimeric antibody H chain 80caggtgcagc
tggtggagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60tcctgcaagg
cttctggata caccttcacc gactatgaaa tgcactgggt gcgacaggcc
120cctggacaag ggcttgagtg gatgggagct cttgatccta aaactggtga
tactgcctac 180agtcagaagt tcaagggcag agtcacgctg accgcggaca
aatccacgag cacagcctac 240atggagctga gcagcctgac atctgaggac
acggccgtgt attactgtac aagattctac 300tcctatactt actggggcca
gggaaccctg gtcaccgtct cctca 34581345DNAArtificial
SequenceMouse-human chimeric antibody H chain 81caggtgcagc
tggtgcagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60tcctgcaagg
cttctggata caccttcacc gactatgaaa tgcactgggt gcgacaggcc
120cctggacaag ggcttgagtg gatgggagct cttgatccta aaactggtga
tactgcctac 180agtcagaagt tcaagggcag agtcacgctg accgcggacg
aatccacgag cacagcctac 240atggagctga gcagcctgag atctgaggac
acggccgtgt attactgtac aagattctac 300tcctatactt actggggcca
gggaaccctg gtcaccgtct cctca 34582345DNAArtificial
SequenceMouse-human chimeric antibody H chain 82caggtgcagc
tggtgcagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60tcctgcaagg
cttctggata caccttcacc gactatgaaa tgcactgggt gcgacaggcc
120cctggacaag ggcttgagtg gatgggagct cttgatccta aaactggtga
tactgcctac 180agtcagaagt tcaagggcag agtcacgctg accgcggaca
aatccacgag cacagcctac 240atggagctga gcagcctgag atctgaggac
acggccgtgt attactgtac aagattctac 300tcctatactt actggggcca
gggaaccctg gtcaccgtct cctca 34583345DNAArtificial
SequenceMouse-human chimeric antibody H chain 83caggtgcagc
tggtgcagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60tcctgcaagg
cttctggata caccttcacc gactatgaaa tgcactgggt gcgacaggcc
120cctggacaag ggcttgagtg gatgggagct cttgatccta aaactggtga
tactgcctac 180agtcagaagt tcaagggcag agtcacgctg accgcggaca
aatccacgag cacagcctac 240atggagctga gcagcctgac atctgaggac
acggccgtgt attactgtac aagattctac 300tcctatactt actggggcca
gggaaccctg gtcaccgtct cctca 34584115PRTArtificial
SequenceMouse-human chimeric antibody H chain 84Gln Val Gln Leu Val
Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45 Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe
50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr
Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly
Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115
85115PRTArtificial SequenceMouse-human chimeric antibody H chain
85Gln Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
Tyr 20 25 30 Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Met 35 40 45 Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala
Tyr Ser Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp
Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Phe Tyr Ser
Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser
115 86115PRTArtificial SequenceMouse-human chimeric antibody H
chain 86Gln Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly
Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Thr Asp Tyr 20 25 30 Glu Met His Trp Val Arg Gln Ala Pro Gly Gln
Gly Leu Glu Trp Met 35 40 45 Gly Ala Leu Asp Pro Lys Thr Gly Asp
Thr Ala Tyr Ser Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr
Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Phe
Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val
Ser Ser 115 87115PRTArtificial SequenceMouse-human chimeric
antibody H chain 87Gln Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys
Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Met His Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ala Leu Asp Pro Lys
Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe 50 55 60 Lys Gly Arg Val
Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu
Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr 100
105 110 Val Ser Ser 115 88115PRTArtificial SequenceMouse-human
chimeric antibody H chain 88Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Met His Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ala Leu Asp
Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe 50 55 60 Lys Gly
Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val
Thr 100 105 110 Val Ser Ser 115 89115PRTArtificial
SequenceMouse-human chimeric antibody H chain 89Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45 Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe
50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr
Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly
Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115
90115PRTArtificial SequenceMouse-human chimeric antibody H chain
90Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
Tyr 20 25 30 Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Met 35 40 45 Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala
Tyr Ser Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp
Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Phe Tyr Ser
Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser
115 91336DNAArtificial SequenceMouse-human chimeric antibody L
chain 91gatgttgtga tgactcagtc tccactctcc ctgcccgtca cccctggaga
gccggcctcc 60atctcctgca gatctagtca gagccttgta cacagtaatg gaaacaccta
tttacattgg 120tacctgcaga agccagggca gtctccacag ctcctgatct
ataaagtttc caaccgattt 180tctggggtcc ctgacaggtt cagtggcagt
ggatcaggca cagattttac actgaaaatc 240agcagagtgg aggctgagga
tgttggggtt tattactgct ctcaaaatac acatgttcct 300cctacgtttg
gccaggggac caagctggag atcaaa 33692112PRTArtificial
SequenceMouse-human chimeric antibody L chain 92Asp Val Val Met Thr
Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala
Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn
Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40
45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr
Cys Ser Gln Asn 85 90 95 Thr His Val Pro Pro Thr Phe Gly Gln Gly
Thr Lys Leu Glu Ile Lys 100 105 110 9314PRTHomo sapiens 93Gly Asn
Ser Gln Gln Ala Thr Pro Lys Asp Asn Glu Ile Ser 1 5 10 948PRTHomo
sapiens 94Gly Asn Ser Gln Gln Ala Thr Pro 1 5 958PRTHomo sapiens
95Gln Gln Ala Thr Pro Lys Asp Asn 1 5 968PRTHomo sapiens 96Thr Pro
Lys Asp Asn Glu Ile Ser 1 5 9710PRTHomo sapiens 97Ala Thr Pro Lys
Asp Asn Glu Ile Ser Thr 1 5 10 9810PRTHomo sapiens 98Pro Lys Asp
Asn Glu Ile Ser Thr Phe His 1 5 10 9910PRTHomo sapiens 99Asp Asn
Glu Ile Ser Thr Phe His Asn Leu 1 5 10 10010PRTHomo sapiens 100Glu
Ile Ser Thr Phe His Asn Leu Gly Asn 1 5 10 10127PRTHomo sapiens
101Gly Asn Ser Gln Gln Ala Thr Pro Lys Asp Asn Glu Ile Ser Thr Phe
1 5 10 15 His Asn Leu Gly Asn Val His Ser Pro Leu Lys 20 25
10214PRTHomo sapiens 102Ser Thr Phe His Asn Leu Gly Asn Val His Ser
Pro Leu Lys 1 5 10 1035PRTMus musculus 103Asn Tyr Ala Met Ser 1 5
10417PRTMus musculus 104Ala Ile Asn Asn Asn Gly Asp Asp Thr Tyr Tyr
Leu Asp Thr Val Lys 1 5 10 15 Asp 1055PRTMus musculus 105Gln Gly
Gly Ala Tyr 1 5 1067PRTMus musculus 106Thr Tyr Gly Met Gly Val Gly
1 5 10716PRTMus musculus 107Asn Ile Trp Trp Tyr Asp Ala Lys Tyr Tyr
Asn Ser Asp Leu Lys Ser 1 5 10 15 1088PRTMus musculus 108Met Gly
Leu Ala Trp Phe Ala Tyr 1 5 1097PRTMus musculus 109Ile Tyr Gly Met
Gly Val Gly 1 5 11016PRTMus musculus 110Asn Ile Trp Trp Asn Asp Asp
Lys Tyr Tyr Asn Ser Ala Leu Lys Ser 1 5 10 15 1118PRTMus musculus
111Ile Gly Tyr Phe Tyr Phe Asp Tyr 1 5 1125PRTMus musculus 112Gly
Tyr Trp Met His 1 5 11317PRTMus musculus 113Ala Ile Tyr Pro Gly Asn
Ser Asp Thr Asn Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly 11410PRTMus
musculus 114Ser Gly Asp Leu Thr Gly Gly Leu Ala Tyr 1 5 10
1155PRTMus musculus 115Ser Tyr Ala Met Ser 1 5 11617PRTMus musculus
116Ala Ile Asn Ser Asn Gly Gly Thr Thr Tyr Tyr Pro Asp Thr Met Lys
1 5 10 15 Asp 11713PRTMus musculus 117His Asn Gly Gly Tyr Glu Asn
Tyr Gly Trp Phe Ala Tyr 1 5 10 1185PRTMus musculus 118Ser Tyr Trp
Met His 1 5 11917PRTMus musculus 119Glu Ile Asp Pro Ser Asp Ser Tyr
Thr Tyr Tyr Asn Gln Lys Phe Arg 1 5 10 15 Gly 12015PRTMus musculus
120Ser Asn Leu Gly Asp Gly His Tyr Arg Phe Pro Ala Phe Pro Tyr 1 5
10
15 12117PRTMus musculus 121Thr Ile Asp Pro Ser Asp Ser Glu Thr His
Tyr Asn Leu Gln Phe Lys 1 5 10 15 Asp 12215PRTMus musculus 122Gly
Ala Phe Tyr Ser Ser Tyr Ser Tyr Trp Ala Trp Phe Ala Tyr 1 5 10 15
1235PRTMus musculus 123Asp Tyr Glu Met His 1 5 12417PRTMus musculus
124Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe Lys
1 5 10 15 Gly 1256PRTMus musculus 125Phe Tyr Ser Tyr Thr Tyr 1 5
1265PRTMus musculus 126Ile Asn Ala Met Asn 1 5 12719PRTMus musculus
127Arg Ile Arg Ser Glu Ser Asn Asn Tyr Ala Thr Tyr Tyr Gly Asp Ser
1 5 10 15 Val Lys Asp 1288PRTMus musculus 128Glu Val Thr Thr Ser
Phe Ala Tyr 1 5 1295PRTMus musculus 129Ala Ser Ala Met Asn 1 5
13019PRTMus musculus 130Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Ile
Tyr Tyr Ala Asp Ser 1 5 10 15 Val Lys Asp 13112PRTMus musculus
131Asp Pro Gly Tyr Tyr Gly Asn Pro Trp Phe Ala Tyr 1 5 10
1325PRTMus musculus 132Asp Tyr Ser Met His 1 5 13317PRTMus musculus
133Trp Ile Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe Lys
1 5 10 15 Gly 1342PRTMus musculus 134Leu Tyr 1 13516PRTMus musculus
135Asn Ile Trp Trp His Asp Asp Lys Tyr Tyr Asn Ser Ala Leu Lys Ser
1 5 10 15 13614PRTMus musculus 136Ile Ala Pro Arg Tyr Asn Lys Tyr
Glu Gly Phe Phe Ala Phe 1 5 10 13716PRTMus musculus 137Lys Ser Ser
Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn 1 5 10 15
1387PRTMus musculus 138Leu Val Ser Lys Leu Asp Ser 1 5 1399PRTMus
musculus 139Trp Gln Gly Thr His Phe Pro Leu Thr 1 5 14011PRTMus
musculus 140Lys Ala Ser Gln Asp Ile Asn Asn Tyr Leu Ser 1 5 10
1417PRTMus musculus 141Arg Ala Asn Arg Leu Val Asp 1 5 14210PRTMus
musculus 142Leu Gln Cys Asp Glu Phe Pro Pro Trp Thr 1 5 10
14316PRTMus musculus 143Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly
Asn Thr Tyr Leu His 1 5 10 15 1447PRTMus musculus 144Lys Val Ser
Asn Arg Phe Ser 1 5 1459PRTMus musculus 145Ser Gln Ser Thr His Val
Pro Trp Thr 1 5 14616PRTMus musculus 146Arg Ser Ser Lys Ser Leu Leu
His Ser Asn Gly Ile Thr Tyr Leu Tyr 1 5 10 15 1477PRTMus musculus
147Gln Met Ser Asn Leu Ala Ser 1 5 1489PRTMus musculus 148Ala Gln
Asn Leu Glu Leu Pro Tyr Thr 1 5 14911PRTMus musculus 149Lys Ala Ser
Gln Asp Ile Asn Lys Asn Ile Ile 1 5 10 1507PRTMus musculus 150Tyr
Thr Ser Thr Leu Gln Pro 1 5 1516PRTMus musculus 151Leu Gln Tyr Asp
Asn Leu 1 5 15211PRTMus musculus 152Arg Ala Ser His Ser Ile Ser Asn
Phe Leu His 1 5 10 1537PRTMus musculus 153Tyr Ala Ser Gln Ser Ile
Ser 1 5 1549PRTMus musculus 154Gln Gln Ser Asn Ile Trp Ser Leu Thr
1 5 15515PRTMus musculus 155Arg Ala Ser Glu Ser Val Glu Tyr Tyr Gly
Thr Ser Leu Met Gln 1 5 10 15 1567PRTMus musculus 156Gly Ala Ser
Asn Val Glu Ser 1 5 1579PRTMus musculus 157Gln Gln Ser Arg Lys Val
Pro Tyr Thr 1 5 1589PRTMus musculus 158Ser Gln Asn Thr His Val Pro
Pro Thr 1 5 15916PRTMus musculus 159Lys Ser Ser Lys Ser Leu Leu His
Ser Asn Gly Asn Thr Tyr Leu Asn 1 5 10 15 1607PRTMus musculus
160Trp Met Ser Asn Leu Ala Ser 1 5 1619PRTMus musculus 161Met Gln
His Ile Glu Tyr Pro Phe Thr 1 5 16216PRTMus musculus 162Arg Ser Ser
Lys Ser Leu Leu His Ser Tyr Asp Ile Thr Tyr Leu Tyr 1 5 10 15
1639PRTMus musculus 163Ala Gln Asn Leu Glu Leu Pro Pro Thr 1 5
16410PRTMus musculus 164Ser Ala Ser Ser Ser Val Ser Tyr Met Tyr 1 5
10 1657PRTMus musculus 165Asp Thr Ser Asn Leu Ala Ser 1 5
1669PRTMus musculus 166Gln Gln Trp Ser Ser Tyr Pro Leu Thr 1 5
16716PRTMus musculus 167Lys Ser Ser Gln Ser Leu Leu His Ser Asp Gly
Lys Thr Phe Leu Asn 1 5 10 15 1687PRTMus musculus 168Leu Val Ser
Arg Leu Asp Ser 1 5 1696PRTMus musculus 169Cys Gln Gly Thr His Phe
1 5 17016PRTMus musculus 170Arg Ser Ser Gln Ser Ile Val His Ser Asn
Gly Asn Thr Tyr Leu Glu 1 5 10 15 1719PRTMus musculus 171Phe Gln
Gly Ser His Val Pro Trp Thr 1 5 17227DNAArtificial SequencePCR
primer 172cttgtacaca gtgacggaaa cacctat 2717327DNAArtificial
SequencePCR primer 173ataggtgttt ccgtcactgt gtacaag
2717416PRTArtificial Sequencemutant antibody L chain 174Arg Ser Ser
Gln Ser Leu Val His Ser Asn Ala Asn Thr Tyr Leu His 1 5 10 15
17516PRTArtificial Sequencemutant antibody L chain 175Arg Ser Ser
Gln Ser Leu Val His Ser Asn Asp Asn Thr Tyr Leu His 1 5 10 15
17616PRTArtificial Sequencemutant antibody L chain 176Arg Ser Ser
Gln Ser Leu Val His Ser Asn Glu Asn Thr Tyr Leu His 1 5 10 15
17716PRTArtificial Sequencemutant antibody L chain 177Arg Ser Ser
Gln Ser Leu Val His Ser Asn Phe Asn Thr Tyr Leu His 1 5 10 15
17816PRTArtificial Sequencemutant antibody L chain 178Arg Ser Ser
Gln Ser Leu Val His Ser Asn His Asn Thr Tyr Leu His 1 5 10 15
17916PRTArtificial Sequencemutant antibody L chain 179Arg Ser Ser
Gln Ser Leu Val His Ser Asn Asn Asn Thr Tyr Leu His 1 5 10 15
18016PRTArtificial Sequencemutant antibody L chain 180Arg Ser Ser
Gln Ser Leu Val His Ser Asn Thr Asn Thr Tyr Leu His 1 5 10 15
18116PRTArtificial Sequencemutant antibody L chain 181Arg Ser Ser
Gln Ser Leu Val His Ser Asn Gln Asn Thr Tyr Leu His 1 5 10 15
18217PRTArtificial Sequencemutant antibody L chain 182Arg Ser Ser
Gln Ser Leu Val His Ser Asn Gly Ile Asn Thr Tyr Leu 1 5 10 15 His
18316PRTArtificial Sequencemutant antibody L chain 183Arg Ser Ser
Gln Ser Leu Val His Ser Asn Lys Asn Thr Tyr Leu His 1 5 10 15
18416PRTArtificial Sequencemutant antibody L chain 184Arg Ser Ser
Gln Ser Leu Val His Ser Asn Leu Asn Thr Tyr Leu His 1 5 10 15
18516PRTArtificial Sequencemutant antibody L chain 185Arg Ser Ser
Gln Ser Leu Val His Ser Asn Ser Asn Thr Tyr Leu His 1 5 10 15
18616PRTArtificial Sequencemutant antibody L chain 186Arg Ser Ser
Gln Ser Leu Val His Ser Asn Trp Asn Thr Tyr Leu His 1 5 10 15
18716PRTArtificial Sequencemutant antibody L chain 187Arg Ser Ser
Gln Ser Leu Val His Ser Asn Tyr Asn Thr Tyr Leu His 1 5 10 15
18816PRTArtificial Sequencemutant antibody L chain 188Arg Ser Ser
Gln Ser Leu Val His Ser Asn Arg Asn Thr Tyr Leu His 1 5 10 15
18916PRTArtificial Sequencemutant antibody L chain 189Arg Ser Ser
Gln Ser Leu Val His Ser Asn Val Asn Thr Tyr Leu His 1 5 10 15
19016PRTArtificial Sequencemutant antibody L chain 190Arg Ser Ser
Gln Ser Leu Val His Ser Asn Pro Asn Thr Tyr Leu His 1 5 10 15
191112PRTArtificial Sequencemutant antibody L chain 191Asp Val Val
Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu
Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25
30 Asn Ala Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly
Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val
Tyr Tyr Cys Ser Gln Asn 85 90 95 Thr His Val Pro Pro Thr Phe Gly
Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 192112PRTArtificial
Sequencemutant antibody L chain 192Asp Val Val Met Thr Gln Ser Pro
Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser
Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Asp Asn Thr
Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln
Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65
70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser
Gln Asn 85 90 95 Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys
Leu Glu Ile Lys 100 105 110 193112PRTArtificial Sequencemutant
antibody L chain 193Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro
Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser
Gln Ser Leu Val His Ser 20 25 30 Asn Glu Asn Thr Tyr Leu His Trp
Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr
Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg
Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn 85 90 95
Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100
105 110 194112PRTArtificial Sequencemutant antibody L chain 194Asp
Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10
15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
20 25 30 Asn Phe Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly
Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe
Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val
Gly Val Tyr Tyr Cys Ser Gln Asn 85 90 95 Thr His Val Pro Pro Thr
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110
195112PRTArtificial Sequencemutant antibody L chain 195Asp Val Val
Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu
Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25
30 Asn His Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly
Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val
Tyr Tyr Cys Ser Gln Asn 85 90 95 Thr His Val Pro Pro Thr Phe Gly
Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 196112PRTArtificial
Sequencemutant antibody L chain 196Asp Val Val Met Thr Gln Ser Pro
Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser
Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Asn Asn Thr
Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln
Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65
70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser
Gln Asn 85 90 95 Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys
Leu Glu Ile Lys 100 105 110 197112PRTArtificial Sequencemutant
antibody L chain 197Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro
Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser
Gln Ser Leu Val His Ser 20 25 30 Asn Thr Asn Thr Tyr Leu His Trp
Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr
Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg
Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn 85 90 95
Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100
105 110 198112PRTArtificial Sequencemutant antibody L chain 198Asp
Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10
15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
20 25 30 Asn Gln Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly
Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe
Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val
Gly Val Tyr Tyr Cys Ser Gln Asn 85 90 95 Thr His Val Pro Pro Thr
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110
199112PRTArtificial Sequencemutant antibody L chain 199Asp Val Val
Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu
Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25
30 Asn Ile Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly
Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val
Tyr Tyr Cys Ser Gln Asn 85 90 95 Thr His Val Pro Pro Thr Phe Gly
Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 200112PRTArtificial
Sequencemutant antibody L chain 200Asp Val Val Met Thr Gln Ser Pro
Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser
Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Lys Asn Thr
Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln
Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65
70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser
Gln Asn 85 90 95 Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys
Leu Glu Ile Lys 100
105 110 201112PRTArtificial Sequencemutant antibody L chain 201Asp
Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10
15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
20 25 30 Asn Leu Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly
Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe
Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val
Gly Val Tyr Tyr Cys Ser Gln Asn 85 90 95 Thr His Val Pro Pro Thr
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110
202112PRTArtificial Sequencemutant antibody L chain 202Asp Val Val
Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu
Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25
30 Asn Ser Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly
Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val
Tyr Tyr Cys Ser Gln Asn 85 90 95 Thr His Val Pro Pro Thr Phe Gly
Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 203112PRTArtificial
Sequencemutant antibody L chain 203Asp Val Val Met Thr Gln Ser Pro
Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser
Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Trp Asn Thr
Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln
Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65
70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser
Gln Asn 85 90 95 Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys
Leu Glu Ile Lys 100 105 110 204112PRTArtificial Sequencemutant
antibody L chain 204Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro
Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser
Gln Ser Leu Val His Ser 20 25 30 Asn Tyr Asn Thr Tyr Leu His Trp
Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr
Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg
Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn 85 90 95
Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100
105 110 205112PRTArtificial Sequencemutant antibody L chain 205Asp
Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10
15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
20 25 30 Asn Arg Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly
Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe
Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val
Gly Val Tyr Tyr Cys Ser Gln Asn 85 90 95 Thr His Val Pro Pro Thr
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110
206112PRTArtificial Sequencemutant antibody L chain 206Asp Val Val
Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu
Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25
30 Asn Val Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly
Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val
Tyr Tyr Cys Ser Gln Asn 85 90 95 Thr His Val Pro Pro Thr Phe Gly
Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 207112PRTArtificial
Sequencemutant antibody L chain 207Asp Val Val Met Thr Gln Ser Pro
Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser
Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Pro Asn Thr
Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln
Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65
70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser
Gln Asn 85 90 95 Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys
Leu Glu Ile Lys 100 105 110
* * * * *