U.S. patent application number 15/582112 was filed with the patent office on 2019-10-17 for therapeutic dll4 binding proteins.
This patent application is currently assigned to AbbVie Inc.. The applicant listed for this patent is AbbVie Inc.. Invention is credited to Ming-Jiu Chen, Jije Gu, Chung-Ming Hsieh, Yingchun Li, Susan Morgan-Lappe.
Application Number | 20190315855 15/582112 |
Document ID | / |
Family ID | 44531508 |
Filed Date | 2019-10-17 |
United States Patent
Application |
20190315855 |
Kind Code |
A1 |
Chen; Ming-Jiu ; et
al. |
October 17, 2019 |
Therapeutic DLL4 Binding Proteins
Abstract
DLL4 binding proteins are described herein, including
antibodies, CDR-grafted antibodies, humanized antibodies, and DLL4
binding fragments thereof, proteins that bind DLL4 with high
affinity, and DLL4 binding proteins that neutralize DLL4 and/or
VEGF activity. The DLL4 binding proteins are useful for treating or
preventing cancers and tumors and especially for treating or
preventing tumor angiogenesis.
Inventors: |
Chen; Ming-Jiu; (Shrewsbury,
MA) ; Hsieh; Chung-Ming; (Newton, MA) ; Gu;
Jije; (Shrewsbury, MA) ; Morgan-Lappe; Susan;
(Riverwoods, IL) ; Li; Yingchun; (Buffalo Grove,
IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AbbVie Inc. |
North Chicago |
IL |
US |
|
|
Assignee: |
AbbVie Inc.
North Chicago
IL
|
Family ID: |
44531508 |
Appl. No.: |
15/582112 |
Filed: |
April 28, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15295427 |
Oct 17, 2016 |
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15582112 |
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14798849 |
Jul 14, 2015 |
9469689 |
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15295427 |
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13037932 |
Mar 1, 2011 |
9115195 |
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14798849 |
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61309494 |
Mar 2, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/76 20130101;
C07K 2317/92 20130101; A61P 35/00 20180101; A61K 45/06 20130101;
C07K 2317/73 20130101; C07K 2317/24 20130101; C07K 16/28 20130101;
A61K 2039/505 20130101; A61K 39/3955 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 39/395 20060101 A61K039/395; A61K 45/06 20060101
A61K045/06 |
Claims
1. A binding protein capable of binding DLL4, comprising at least
one amino acid sequence selected from the group of amino acid
sequences consisting of SEQ ID NO:157, SEQ ID NO:158, SEQ ID
NO:159, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163,
and SEQ ID NO:164.
2. A binding protein comprising an antigen binding domain wherein
the binding protein is capable of binding human DLL4, said antigen
binding domain comprising at least one CDR, wherein: CDR-H1 is
selected from the group consisting of:
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5 (SEQ ID NO:151), wherein;
X.sub.1 is N, H, or Y; X.sub.2 is F; X.sub.3 is P; X.sub.4 is M;
and X.sub.5 is A or S; residues 31-35 of SEQ ID NO:157 (CDR-H1
38H12); residues 31-35 of SEQ ID NO:161 (CDR-H1 37D10); residues
31-35 of SEQ ID NO:163 (CDR-H1 32C7); residues 31-35 of SEQ ID
NO:165 (CDR-H1 14G1); residues 31-35 of SEQ ID NO:167 (CDR-H1
14A11); residues 31-35 of SEQ ID NO:169 (CDR-H1 15D6); residues
31-35 of SEQ ID NO:171 (CDR-H1 VH.1 1A11); residues 31-35 of SEQ ID
NO:172 (CDR-H1 VH.1a 1A11); residues 31-35 of SEQ ID NO:173 (CDR-H1
VH.1b 1A11); residues 31-35 of SEQ ID NO:174 (CDR-H1 VH.2a 1A11);
residues 31-35 of SEQ ID NO:179 (CDR-H1 VH.1 38H12); residues 31-35
of SEQ ID NO:180 (CDR-H1 VH.1A 38H12); residues 31-35 of SEQ ID
NO:181 (CDR-H1 VH.1b 38H12); residues 31-35 of SEQ ID NO:182
(CDR-H1 VH.2a 38H12); residues 31-35 of SEQ ID NO:187 (CDR-H1
h1A11VH.1); residues 31-35 of SEQ ID NO:188 (CDR-H1 h1A11.A6);
residues 31-35 of SEQ ID NO:189 (CDR-H1 h1A11.A8); residues 31-35
of SEQ ID NO:190 (CDR-H1 h1A11.C6); residues 31-35 of SEQ ID NO:191
(CDR-H1 h1A11.A11); residues 31-35 of SEQ ID NO:192 (CDR-H1
h1A11.B5); residues 31-35 of SEQ ID NO:193 (CDR-H1 h1A11.E12);
residues 31-35 of SEQ ID NO:194 (CDR-H1 h1A11.G3); residues 31-35
of SEQ ID NO:195 (CDR-H1 h1A11.F5); and residues 31-35 of SEQ ID
NO:196 (CDR-H1 h1A11.H2); CDR-H2 is selected from the group
consisting of:
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X-
.sub.10-X.sub.11-X.sub.12-X.sub.13-X.sub.14-X.sub.15-X.sub.16-X.sub.17
(SEQ ID NO:152), wherein; X.sub.1 is T or S; X.sub.2 is I; X.sub.3
is S; X.sub.4 is S or G; X.sub.5 is S; X.sub.6 is D; X.sub.7 is G,
A, D, S, or E; X.sub.8 is T or W; X.sub.9 is T, P, or A; X.sub.10
is Y, S, T, or N; X.sub.11 is Y or I; X.sub.12 is R or G; X.sub.13
is D; X.sub.14 is 5; X.sub.15 is V; X.sub.16 is K; and X.sub.17 is
G; residues 50-66 of SEQ ID NO:157 (CDR-H2 38H12); residues 50-68
of SEQ ID NO:161 (CDR-H2 37D10); residues 50-66 of SEQ ID NO:163
(CDR-H2 32C7); residues 50-66 of SEQ ID NO:165 (CDR-H2 14G1);
residues 50-66 of SEQ ID NO:167 (CDR-H2 14A11); residues 50-66 of
SEQ ID NO:169 (CDR-H2 15D6); residues 50-66 of SEQ ID NO:171
(CDR-H2 VH.1 1A11); residues 50-66 of SEQ ID NO:172 (CDR-H2 VH.1a
1A11); residues 50-66 of SEQ ID NO:173 (CDR-H2 VH.1b 1A11);
residues 50-66 of SEQ ID NO:174 (CDR-H2 VH.2a 1A11); residues 50-66
of SEQ ID NO:179 (CDR-H2 VH.1 38H12); residues 50-66 of SEQ ID
NO:180 (CDR-H2 VH.1A 38H12); residues 50-66 of SEQ ID NO:181
(CDR-H2 VH.1b 38H12); residues 31-35 of SEQ ID NO:182 (CDR-H1 VH.2a
38H12); residues 50-66 of SEQ ID NO:187 (CDR-H2 h1A11VH.1);
residues 50-66 of SEQ ID NO:188 (CDR-H2 h1A11.A6); residues 50-66
of SEQ ID NO:189 (CDR-H2 h1A11.A8); residues 50-66 of SEQ ID NO:190
(CDR-H2 h1A11.C6); residues 50-66 of SEQ ID NO:191 (CDR-H2
h1A11.A11); residues 50-66 of SEQ ID NO:192 (CDR-H2 h1A11.B5);
residues 50-66 of SEQ ID NO:193 (CDR-H2 h1A11.E12); residues 50-66
of SEQ ID NO:194 (CDR-H2 h1A11.G3); residues 50-66 of SEQ ID NO:195
(CDR-H2 h1A11.F5); and residues 50-66 of SEQ ID NO:196 (CDR-H2
h1A11.H2); CDR-H3 is selected from the group consisting of:
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9
(SEQ ID NO:153), wherein; X.sub.1 is G; X.sub.2 is Y; X.sub.3 is Y;
X.sub.4 is N; X.sub.5 is S; X.sub.6 is P; X.sub.7 is F; X.sub.8 is
A; and X.sub.9 is Y, F, or S; residues 99-107 of SEQ ID NO:157
(CDR-H3 38H12); residues 101-111 of SEQ ID NO:161 (CDR-H3 37D10);
residues 99-105 of SEQ ID NO:163 (CDR-H3 32C7); residues 99-105 of
SEQ ID NO:165 (CDR-H3 14G1); residues 99-110 of SEQ ID NO:167
(CDR-H3 14A11); residues 99-110 of SEQ ID NO:169 (CDR-H3 15D6);
residues 99-107 of SEQ ID NO:171 (CDR-H3 VH.1 1A11); residues
99-107 of SEQ ID NO:172 (CDR-H3 VH.1a 1A11); residues 99-107 of SEQ
ID NO:173 (CDR-H3 VH.1b 1A11); residues 99-107 of SEQ ID NO:174
(CDR-H3 VH.2a 1A11); residues 99-107 of SEQ ID NO:179 (CDR-H3 VH.1
38H12); residues 99-107 of SEQ ID NO:180 (CDR-H3 VH.1A 38H12);
residues 99-107 of SEQ ID NO:181 (CDR-H2 VH.1b 38H12); residues
99-107 of SEQ ID NO:182 (CDR-H1 VH.2a 38H12); residues 99-107 of
SEQ ID NO:187 (CDR-H3 h1A11VH.1); residues 99-107 of SEQ ID NO:188
(CDR-H3 h1A11.A6); residues 99-107 of SEQ ID NO:189 (CDR-H3
h1A11.A8); residues 99-107 of SEQ ID NO:190 (CDR-H3 h1A11.C6);
residues 99-107 of SEQ ID NO:191 (CDR-H3 h1A11.A11); residues
99-107 of SEQ ID NO:192 (CDR-H3 h1A11.B5); residues 99-107 of SEQ
ID NO:193 (CDR-H3 h1A11.E12); residues 99-107 of SEQ ID NO:194
(CDR-H3 h1A11.G3); residues 99-107 of SEQ ID NO:195 (CDR-H3
h1A11.F5); and residues 99-107 of SEQ ID NO:196 (CDR-H3 h1A11.H2);
CDR-L1 is selected from the group consisting of:
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X-
.sub.10-X.sub.11 (SEQ ID NO:154), wherein; X.sub.1 is R; X.sub.2 is
A; X.sub.3 is S; X.sub.4 is E or Q; X.sub.5 is D or E; X.sub.6 is
I; X.sub.7 is Y or W; X.sub.8 is S, I, Y, N, or R; X.sub.9 is N;
X.sub.10 is L; and X.sub.11 is A; residues 24-34 of SEQ ID NO:158
(CDR-L1 38H12); residues 24-34 of SEQ ID NO:162 (CDR-L1 37D10);
residues 24-34 of SEQ ID NO:164 (CDR-L1 32C7); residues 24-34 of
SEQ ID NO:166 (CDR-L1 14G1); residues 23-37 of SEQ ID NO:168
(CDR-L1 14A11); residues 23-37 of SEQ ID NO:170 (CDR-L1 15D6);
residues 24-34 of SEQ ID NO:175 (CDR-L1 VL.1 1A11); residues 24-34
of SEQ ID NO:176 (CDR-L1 VL.1a 1A11); residues 24-34 of SEQ ID
NO:177 (CDR-L1 VL.1b 1A11); residues 24-34 of SEQ ID NO:178 (CDR-L1
VL.2a 1A11); residues 24-34 of SEQ ID NO:183 (CDR-L1 VL.1 38H12);
residues 24-34 of SEQ ID NO:184 (CDR-L1 VL.1a 38H12); residues
24-34 of SEQ ID NO:185 (CDR-L1 VL.1b 38H12); residues 24-34 of SEQ
ID NO:186 (CDR-L1 VL.2a 38H12); residues 24-34 of SEQ ID NO:197
(CDR-L1 h1A11VL.1); residues 24-34 of SEQ ID NO:198 (CDR-L1
h1A11.A2); residues 24-34 of SEQ ID NO:199 (CDR-L1 h1A11.A12);
residues 24-34 of SEQ ID NO:200 (CDR-L1 h1A11.A7); residues 24-34
of SEQ ID NO:201 (CDR-L1 h1A11.B4); residues 24-34 of SEQ ID NO:202
(CDR-L1 h1A11.B5); and residues 24-34 of SEQ ID NO:203 (CDR-L1
h1A11.E12); CDR-L2 is selected from group consisting of:
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7 (SEQ ID
NO:155), wherein; X.sub.1 is D; X.sub.2 is T; X.sub.3 is N or S;
X.sub.4 is N, D, S, I, Y, or V; X.sub.5 is L; X.sub.6 is A; and
X.sub.7 is D; residues 50-56 of SEQ ID NO:158 (CDR-L2 38H12);
residues 50-56 of SEQ ID NO:162 (CDR-L2 37D10); residues 50-56 of
SEQ ID NO:164 (CDR-L2 32C7); residues 50-56 of SEQ ID NO:166
(CDR-L2 14G1); residues 53-59 of SEQ ID NO:168 (CDR-L2 14A11);
residues 53-59 of SEQ ID NO:170 (CDR-L2 15D6); residues 50-56 of
SEQ ID NO:175 (CDR-L2 VL.1 1A11); residues 50-56 of SEQ ID NO:176
(CDR-L2 VL.1a 1A11); residues 50-56 of SEQ ID NO:177 (CDR-L2 VL.1b
1A11); residues 50-56 of SEQ ID NO:178 (CDR-L2 VL.2a 1A11);
residues 50-56 of SEQ ID NO:183 (CDR-L2 VL.1 38H12); residues 50-56
of SEQ ID NO:184 (CDR-L2 VL.1a 38H12); residues 50-56 of SEQ ID
NO:185 (CDR-L2 VL.1b 38H12); residues 50-56 of SEQ ID NO:186
(CDR-L2 VL.2a 38H12); residues 50-56 of SEQ ID NO:197 (CDR-L2
h1A11VL.1); residues 50-56 of SEQ ID NO:198 (CDR-L2 h1A11.A2);
residues 50-56 of SEQ ID NO:199 (CDR-L2 h1A11.A12); residues 50-56
of SEQ ID NO:200 (CDR-L2 h1A11.A7); residues 50-56 of SEQ ID NO:201
(CDR-L2 h1A11.B4); residues 50-56 of SEQ ID NO:202 (CDR-L2
h1A11.B5); and residues 50-56 of SEQ ID NO:203 (CDR-L2 h1A11.E12);
and CDR-L3 is selected from the group consisting of:
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9
(SEQ ID NO:156), wherein; X.sub.1 is Q; X.sub.2 is Q; X.sub.3 is Y;
X.sub.4 is N, D, or T; X.sub.5 is N, Y, or W; X.sub.6 is Y or V;
X.sub.7 is P; X.sub.8 is P; and X.sub.9 is T. residues 89-97 of SEQ
ID NO:158 (CDR-L3 38H12); residues 89-97 of SEQ ID NO:162 (CDR-L3
37D10); residues 89-97 of SEQ ID NO:164 (CDR-L3 32C7); residues
89-98 of SEQ ID NO:166 (CDR-L3 14G1); residues 92-100 of SEQ ID
NO:168 (CDR-L3 14A11); residues 92-100 of SEQ ID NO:170 (CDR-L3
15D6); residues 89-97 of SEQ ID NO:175 (CDR-L3 VL.1 1A11); residues
89-97 of SEQ ID NO:176 (CDR-L3 VL.1a 1A11); residues 89-97 of SEQ
ID NO:177 (CDR-L3 VL.1b 1A11); residues 89-97 of SEQ ID NO:178
(CDR-L3 VL.2a 1A11); residues 89-97 of SEQ ID NO:183 (CDR-L3 VL.1
38H12); residues 89-97 of SEQ ID NO:184 (CDR-L3 VL.1a 38H12);
residues 89-97 of SEQ ID NO:185 (CDR-L3 VL.1b 38H12); residues
89-97 of SEQ ID NO:186 (CDR-L3 VL.2a 38H12); residues 89-97 of SEQ
ID NO:197 (CDR-L3 h1A11VL.1); residues 89-97 of SEQ ID NO:198
(CDR-L3 h1A11.A2); residues 89-97 of SEQ ID NO:199 (CDR-L3
h1A11.A12); residues 89-97 of SEQ ID NO:200 (CDR-L3 h1A11.A7);
residues 89-97 of SEQ ID NO:201 (CDR-L3 h1A11.B4); residues 89-97
of SEQ ID NO:202 (CDR-L3 h1A11.B5); and residues 89-97 of SEQ ID
NO:203 (CDR-L3 h1A11.E12).
3. The binding protein according to claim 2, wherein said at least
one CDR comprises an amino acid sequence selected from the group
consisting of: residues 31-35 of SEQ ID NO:157 (CDR-H1 38H12);
residues 50-66 of SEQ ID NO:157 (CDR-H2 38H12); residues 99-107 of
SEQ ID NO:157 (CDR-H3 38H12); residues 24-34 of SEQ ID NO:158
(CDR-L1 38H12); residues 50-56 of SEQ ID NO:158 (CDR-L2 38H12);
residues 89-97 of SEQ ID NO:158 (CDR-L3 38H12); residues 31-35 of
SEQ ID NO:159 (CDR-H1 1A11); residues 50-66 of SEQ ID NO:159
(CDR-H2 1A11); residues 99-107 of SEQ ID NO:159 (CDR-H3 1A11);
residues 24-34 of SEQ ID NO:160 (CDR-L1 1A11); residues 50-56 of
SEQ ID NO:160 (CDR-L2 1A11); residues 89-97 of SEQ ID NO:160
(CDR-L3 1A11); residues 31-35 of SEQ ID NO:161 (CDR-H1 37D10);
residues 50-68 of SEQ ID NO:161 (CDR-H2 37D10); residues 101-111 of
SEQ ID NO:161 (CDR-H3 37D10); residues 24-34 of SEQ ID NO:162
(CDR-L1 37D10); residues 50-56 of SEQ ID NO:162 (CDR-L2 37D10);
residues 89-97 of SEQ ID NO:162 (CDR-L3 37D10); residues 31-35 of
SEQ ID NO:163 (CDR-H1 32C7); residues 50-66 of SEQ ID NO:163
(CDR-H2 32C7); residues 99-105 of SEQ ID NO:163 (CDR-H3 32C7);
residues 24-34 of SEQ ID NO:164 (CDR-L1 32C7); residues 50-56 of
SEQ ID NO:164 (CDR-L2 32C7); residues 89-98 of SEQ ID NO:164
(CDR-L3 32C7); residues 31-35 of SEQ ID NO:165 (CDR-H1 14G1);
residues 50-66 of SEQ ID NO:165 (CDR-H2 14G1); residues 99-105 of
SEQ ID NO:165 (CDR-H3 14G1); residues 24-34 of SEQ ID NO:166
(CDR-L1 14G1); residues 50-56 of SEQ ID NO:166 (CDR-L2 14G1);
residues 89-98 of SEQ ID NO:166 (CDR-L3 14G1); residues 31-35 of
SEQ ID NO:167 (CDR-H1 14A11); residues 50-66 of SEQ ID NO:167
(CDR-H2 14A11); residues 99-110 of SEQ ID NO:167 (CDR-H3 14A11);
residues 23-37 of SEQ ID NO:168 (CDR-L1 14A11); residues 53-59 of
SEQ ID NO:168 (CDR-L2 14A11); residues 92-100 of SEQ ID NO:168
(CDR-L3 14A11); residues 31-35 of SEQ ID NO:169 (CDR-H1 15D6);
residues 50-66 of SEQ ID NO:169 (CDR-H2 15D6); residues 99-110 of
SEQ ID NO:169 (CDR-H3 15D6); residues 23-37 of SEQ ID NO:170
(CDR-L1 15D6); residues 53-59 of SEQ ID NO:170 (CDR-L2 15D6);
residues 92-100 of SEQ ID NO:170 (CDR-L3 15D6); residues 31-35 of
SEQ ID NO:171 (CDR-H1 VH.1 1A11); residues 50-66 of SEQ ID NO:171
(CDR-H2 VH.1 1A11); residues 99-107 of SEQ ID NO:171 (CDR-H3 VH.1
1A11); residues 31-35 of SEQ ID NO:172 (CDR-H1 VH.1a 1A11);
residues 50-66 of SEQ ID NO:172 (CDR-H2 VH.1a 1A11); residues
99-107 of SEQ ID NO:172 (CDR-H3 VH.1a 1A11); residues 31-35 of SEQ
ID NO:173 (CDR-H1 VH.1b 1A11); residues 50-66 of SEQ ID NO:173
(CDR-H2 VH.1b 1A11); residues 99-107 of SEQ ID NO:173 (CDR-H3 VH.1b
1A11); residues 31-35 of SEQ ID NO:174 (CDR-H1 VH.2a 1A11);
residues 50-66 of SEQ ID NO:174 (CDR-H2 VH.2a 1A11); residues
99-107 of SEQ ID NO:174 (CDR-H3 VH.2a 1A11); residues 24-34 of SEQ
ID NO:175 (CDR-L1 VL.1 1A11); residues 50-56 of SEQ ID NO:175
(CDR-L2 VL.1 1A11); residues 89-97 of SEQ ID NO:175 (CDR-L3 VL.1
1A11); residues 24-34 of SEQ ID NO:176 (CDR-L1 VL.1a 1A11);
residues 50-56 of SEQ ID NO:176 (CDR-L2 VL.1a 1A11); residues 89-97
of SEQ ID NO:176 (CDR-L3 VL.1a 1A11); residues 24-34 of SEQ ID
NO:177 (CDR-L1 VL.1b 1A11); residues 50-56 of SEQ ID NO:177 (CDR-L2
VL.1b 1A11); residues 89-97 of SEQ ID NO:177 (CDR-L3 VL.1b 1A11);
residues 24-34 of SEQ ID NO:178 (CDR-L1 VL.2a 1A11); residues 50-56
of SEQ ID NO:178 (CDR-L2 VL.2a 1A11); residues 89-97 of SEQ ID
NO:178 (CDR-L3 VL.2a 1A11); residues 31-35 of SEQ ID NO:179 (CDR-H1
VH.1 38H12); residues 50-66 of SEQ ID NO:179 (CDR-H2 VH.1 38H12);
residues 99-107 of SEQ ID NO:179 (CDR-H3 VH.1 38H12); residues
31-35 of SEQ ID NO:180 (CDR-H1 VH.1A 38H12); residues 50-66 of SEQ
ID NO:180 (CDR-H2 VH.1A 38H12); residues 99-107 of SEQ ID NO:180
(CDR-H3 VH.1A 38H12); residues 31-35 of SEQ ID NO:181 (CDR-H1 VH.1b
38H12); residues 50-66 of SEQ ID NO:181 (CDR-H2 VH.1b 38H12);
residues 99-107 of SEQ ID NO:181 (CDR-H3 VH.1b 38H12); residues
31-35 of SEQ ID NO:182 (CDR-H1 VH.2a 38H12); residues 50-66 of SEQ
ID NO:182 (CDR-H2 VH.2a 38H12); residues 99-107 of SEQ ID NO:182
(CDR-H3 VH.2a 38H12); residues 24-34 of SEQ ID NO:183 (CDR-L1 VL.1
38H12); residues 50-56 of SEQ ID NO:183 (CDR-L2 VL.1 38H12);
residues 89-97 of SEQ ID NO:183 (CDR-L3 VL.1 38H12); residues 24-34
of SEQ ID NO:184 (CDR-L1 VL.1a 38H12); residues 50-56 of SEQ ID
NO:184 (CDR-L2 VL.1a 38H12); residues 89-97 of SEQ ID NO:184
(CDR-L3 VL.1a 38H12); residues 24-34 of SEQ ID NO:185 (CDR-L1 VL.1b
38H12); residues 50-56 of SEQ ID NO:185 (CDR-L2 VL.1b 38H12);
residues 89-97 of SEQ ID NO:185 (CDR-L3 VL.1b 38H12); residues
24-34 of SEQ ID NO:186 (CDR-L1 VL.2a 38H12); residues 50-56 of SEQ
ID NO:186 (CDR-L2 VL.2a 38H12); residues 89-97 of SEQ ID NO:186
(CDR-L3 VL.2a 38H12); residues 31-35 of SEQ ID NO:187 (CDR-H1
h1A11VH.1), residues 50-66 of SEQ ID NO:187 (CDR-H2 h1A11VH.1);
residues 99-107 of SEQ ID NO:187 (CDR-H3 h1A11VH.1); residues 31-35
of SEQ ID NO:188 (CDR-H1 h1A11.A6), residues 50-66 of SEQ ID NO:188
(CDR-H2 h1A11.A6); residues 99-107 of SEQ ID NO:188 (CDR-H3
h1A11.A6); residues 31-35 of SEQ ID NO:189 (CDR-H1 h1A11.A8),
residues 50-66 of SEQ ID NO:189 (CDR-H2 h1A11.A8); residues 99-107
of SEQ ID NO:189 (CDR-H3 h1A11.A8); residues 31-35 of SEQ ID NO:190
(CDR-H1 h1A11.C6), residues 50-66 of SEQ ID NO:190 (CDR-H2
h1A11.C6); residues 99-107 of SEQ ID NO:190 (CDR-H3 h1A11.C6);
residues 31-35 of SEQ ID NO:191 (CDR-H1 h1A11.A11), residues 50-66
of SEQ ID NO:191 (CDR-H2 h1A11.A11); residues 99-107 of SEQ ID
NO:191 (CDR-H3 h1A11.A11); residues 31-35 of SEQ ID NO:192 (CDR-H1
h1A11.B5), residues 50-66 of SEQ ID NO:192 (CDR-H2 h1A11.B5);
residues 99-107 of SEQ ID NO:192 (CDR-H3 h1A11.B5); residues 31-35
of SEQ ID NO:193 (CDR-H1 h1A11.E12), residues 50-66 of SEQ ID
NO:193 (CDR-H2 h1A11.E12); residues 99-107 of SEQ ID NO:193 (CDR-H3
h1A11.E12); residues 31-35 of SEQ ID NO:194 (CDR-H1 h1A11.G3),
residues 50-66 of SEQ ID NO:194 (CDR-H2 h1A11.G3); residues 99-107
of SEQ ID NO:194 (CDR-H3 h1A11.G3); residues 31-35 of SEQ ID NO:195
(CDR-H1 h1A11.F5), residues 50-66 of SEQ ID NO:195 (CDR-H2
h1A11.F5); residues 99-107 of SEQ ID NO:195 (CDR-H3 h1A11.F5);
residues 31-35 of SEQ ID NO:196 (CDR-H1 h1A11.H2), residues 50-66
of SEQ ID NO:196 (CDR-H2 h1A11.H2); residues 99-107 of SEQ ID
NO:196 (CDR-H3 h1A11.H2); residues 24-34 of SEQ ID NO:197 (CDR-L1
h1A11VL.1), residues 50-56 of SEQ ID NO:197 (CDR-L2 h1A11VL.1);
residues 89-97 of SEQ ID NO:197 (CDR-L3 h1A11VL.1); residues 24-34
of SEQ ID NO:198 (CDR-L1 h1A11.A2), residues 50-56 of SEQ ID NO:198
(CDR-L2 h1A11.A2); residues 89-97 of SEQ ID NO:198 (CDR-L3
h1A11.A2); residues 24-34 of SEQ ID NO:199 (CDR-L1 h1A11.A12),
residues 50-56 of SEQ ID NO:199 (CDR-L2 h1A11.A12); residues 89-97
of SEQ ID NO:199 (CDR-L3 h1A11.A12); residues 24-34 of SEQ ID
NO:200 (CDR-L1 h1A11.A7), residues 50-56 of SEQ ID NO:200 (CDR-L2
h1A11.A7); residues 89-97 of SEQ ID NO:200 (CDR-L3 h1A11.A7);
residues 24-34 of SEQ ID NO:201 (CDR-L1 h1A11.B4), residues 50-56
of SEQ ID NO:201 (CDR-L2 h1A11.B4); residues 89-97 of SEQ ID NO:201
(CDR-L3 h1A11.B4); residues 24-34 of SEQ ID NO:202 (CDR-L1
h1A11.B5), residues 50-56 of SEQ ID NO:202 (CDR-L2 h1A11.B5);
residues 89-97 of SEQ ID NO:202 (CDR-L3 h1A11.B5); residues 24-34
of SEQ ID NO:203 (CDR-L1 h1A11.E12), residues 50-56 of SEQ ID
NO:203 (CDR-L2 h1A11.E12); and residues 89-97 of SEQ ID NO:203
(CDR-L3 h1A11.E12).
4. The binding protein according to claim 2, wherein said binding
protein comprises at least three CDRs.
5. The binding protein according to claim 4, wherein said at least
three CDRs are selected from a variable domain CDR set selected
from the group consisting of: VH 38H12 CDR Set CDR-H1: residues
31-35 of SEQ ID NO:157 CDR-H2: residues 50-66 of SEQ ID NO:157
CDR-H3 residues 99-107 of SEQ ID NO:157 VL 38H12 CDR Set CDR-L1:
residues 24-34 of SEQ ID NO:158 CDR-L2: residues 50-56 of SEQ ID
NO:158 CDR-L3: residues 89-97 of SEQ ID NO:158 VH 1A11 CDR Set
CDR-H1: residues 31-35 of SEQ ID NO:159 CDR-H2: residues 50-66 of
SEQ ID NO:159 CDR-H3: residues 99-107 of SEQ ID NO:159 VL 1A11 CDR
Set CDR-L1: residues 24-34 of SEQ ID NO:160 CDR-L2: residues 50-56
of SEQ ID NO:160 CDR-L3: residues 89-97 of SEQ ID NO:160 VH 37D10
CDR Set CDR-H1: residues 31-35 of SEQ ID NO:161 CDR-H2: residues
50-68 of SEQ ID NO:161 CDR-H3: residues 101-111 of SEQ ID NO:161 VL
37D10 CDR Set CDR-L1: residues 24-34 of SEQ ID NO:162 CDR-L2:
residues 50-56 of SEQ ID NO:162 CDR-L3: residues 89-97 of SEQ ID
NO:162 VH 32C7 CDR Set CDR-H1: residues 31-35 of SEQ ID NO:163
CDR-H2: residues 50-66 of SEQ ID NO:163 CDR-H3: residues 99-105 of
SEQ ID NO:163 VL 32C7 CDR Set CDR-L1: residues 24-34 of SEQ ID
NO:164 CDR-L2: residues 50-56 of SEQ ID NO:164 CDR-L3: residues
89-98 of SEQ ID NO:164 VH 14G1 CDR Set CDR-H1: residues 31-35 of
SEQ ID NO:165 CDR-H2: residues 50-66 of SEQ ID NO:165 CDR-H3:
residues 99-105 of SEQ ID NO:165 VL 14G1 CDR Set CDR-L1: residues
24-34 of SEQ ID NO:166 CDR-L2: residues 50-56 of SEQ ID NO:166
CDR-L3: residues 89-97 of SEQ ID NO:166 VH 14A11 CDR Set CDR-H1:
residues 31-35 of SEQ ID NO:167 CDR-H2: residues 50-66 of SEQ ID
NO:167 CDR-H3: residues 99-110 of SEQ ID NO:167 VL 14A11 CDR Set
CDR-L1: residues 23-37 of SEQ ID NO:168 CDR-L2: residues 53-59 of
SEQ ID NO:168 CDR-L3: residues 92-100 of SEQ ID NO:168 VH 15D6 CDR
Set CDR-H1: residues 31-35 of SEQ ID NO:169 CDR-H2: residues 50-66
of SEQ ID NO:169 CDR-H3: residues 99-110 of SEQ ID NO:169 VL 15D6
CDR Set CDR-L1: residues 23-37 of SEQ ID NO:170 CDR-L2: residues
53-59 of SEQ ID NO:170 CDR-L3: residues 92-100 of SEQ ID NO:170 VH
VH.1 1A11 CDR Set CDR-H1: residues 31-35 of SEQ ID NO:171 CDR-H2:
residues 50-66 of SEQ ID NO:171 CDR-H3: residues 99-107 of SEQ ID
NO:171 VH VH.1a 1A11 CDR Set CDR-H1: residues 31-35 of SEQ ID
NO:172 CDR-H2: residues 50-66 of SEQ ID NO:172 CDR-H3: residues
99-107 of SEQ ID NO:172 VH VH.1b 1A11 CDR Set CDR-H1: residues
31-35 of SEQ ID NO:173 CDR-H2: residues 50-66 of SEQ ID NO:173
CDR-H3: residues 99-107 of SEQ ID NO:173 VH VH.2a 1A11 CDR Set
CDR-H1: residues 31-35 of SEQ ID NO:174 CDR-H2: residues 50-66 of
SEQ ID NO:174 CDR-H3: residues 99-107 of SEQ ID NO:174 VL VL.1 1A11
CDR Set CDR-L1: residues 24-34 of SEQ ID NO:175 CDR-L2: residues
50-56 of SEQ ID NO:175 CDR-L3: residues 89-97 of SEQ ID NO:175 VL
VL.1a 1A11 CDR Set CDR-L1: residues 24-34 of SEQ ID NO:176 CDR-L2:
residues 50-56 of SEQ ID NO:176 CDR-L3: residues 89-97 of SEQ ID
NO:176 VL VL.1b 1A11 CDR Set CDR-L1: residues 24-34 of SEQ ID
NO:177 CDR-L2: residues 50-56 of SEQ ID NO:177 CDR-L3: residues
89-97 of SEQ ID NO:177 VL VL.2a 1A11 CDR Set CDR-L1: residues 24-34
of SEQ ID NO:178 CDR-L2: residues 50-56 of SEQ ID NO:178 CDR-L3:
residues 89-97 of SEQ ID NO:178 VH VH.1 38H12 CDR Set CDR-H1:
residues 31-35 of SEQ ID NO:179 CDR-H2: residues 50-66 of SEQ ID
NO:179 CDR-H3: residues 99-107 of SEQ ID NO:179 VH VH.1a 38H12 CDR
Set CDR-H1: residues 31-35 of SEQ ID NO:180 CDR-H2: residues 50-66
of SEQ ID NO:180 CDR-H3: residues 99-107 of SEQ ID NO:180 VH VH.1b
38H12 CDR Set CDR-H1: residues 31-35 of SEQ ID NO:181 CDR-H2:
residues 50-66 of SEQ ID NO:181 CDR-H3: residues 99-107 of SEQ ID
NO:181 VH VH.2a 38H12 CDR Set CDR-H1: residues 31-35 of SEQ ID
NO:182 CDR-H2: residues 50-66 of SEQ ID NO:182 CDR-H3: residues
99-107 of SEQ ID NO:182 VL VL.1 38H12 CDR Set CDR-L1: residues
24-34 of SEQ ID NO:183 CDR-L2: residues 50-56 of SEQ ID NO:183
CDR-L3: residues 89-97 of SEQ ID NO:183 VL VL.1a 38H12 CDR Set
CDR-L1: residues 24-34 of SEQ ID NO:184 CDR-L2: residues 50-56 of
SEQ ID NO:184 CDR-L3: residues 89-97 of SEQ ID NO:184 VL VL.1b
38H12 CDR Set CDR-L1: residues 24-34 of SEQ ID NO:185 CDR-L2:
residues 50-56 of SEQ ID NO:185 CDR-L3: residues 89-97 of SEQ ID
NO:185 VL VL.2a 38H12 CDR Set CDR-L1: residues 24-34 of SEQ ID
NO:186 CDR-L2: residues 50-56 of SEQ ID NO:186 CDR-L3: residues
89-97 of SEQ ID NO:186 VH hA11VH.1 CDR Set CDR-H1: residues 31-35
of SEQ ID NO:187 CDR-H2: residues 50-66 of SEQ ID NO:187 CDR-H3:
residues 99-107 of SEQ ID NO:187 VH hA11.A6 CDR Set CDR-H1:
residues 31-35 of SEQ ID NO:188 CDR-H2: residues 50-66 of SEQ ID
NO:188 CDR-H3: residues 99-107 of SEQ ID NO:188 VH hA11.A8 CDR Set
CDR-H1: residues 31-35 of SEQ ID NO:189 CDR-H2: residues 50-66 of
SEQ ID NO:189 CDR-H3: residues 99-107 of SEQ ID NO:189 VH hA11.C6
CDR Set CDR-H1: residues 31-35 of SEQ ID NO:190 CDR-H2: residues
50-66 of SEQ ID NO:190 CDR-H3: residues 99-107 of SEQ ID NO:190 VH
hA11.A11 CDR Set CDR-H1: residues 31-35 of SEQ ID NO:191 CDR-H2:
residues 50-66 of SEQ ID NO:191 CDR-H3: residues 99-107 of SEQ ID
NO:191 VH hA11.B5 CDR Set CDR-H1: residues 31-35 of SEQ ID NO:192
CDR-H2: residues 50-66 of SEQ ID NO:192 CDR-H3: residues 99-107 of
SEQ ID NO:192 VH hA11.E12 CDR Set CDR-H1: residues 31-35 of SEQ ID
NO:193 CDR-H2: residues 50-66 of SEQ ID NO:193 CDR-H3: residues
99-107 of SEQ ID NO:193 VH hA11.G3 CDR Set CDR-H1: residues 31-35
of SEQ ID NO:194 CDR-H2: residues 50-66 of SEQ ID NO:194 CDR-H3:
residues 99-107 of SEQ ID NO:194 VH hA11.F5 CDR Set CDR-H1:
residues 31-35 of SEQ ID NO:195 CDR-H2: residues 50-66 of SEQ ID
NO:195 CDR-H3: residues 99-107 of SEQ ID NO:195 VH hA11.H2 CDR Set
CDR-H1: residues 31-35 of SEQ ID NO:196 CDR-H2: residues 50-66 of
SEQ ID NO:196 CDR-H3: residues 99-107 of SEQ ID NO:196 VL h1A11VL.1
CDR Set CDR-L1: residues 24-34 of SEQ ID NO:197 CDR-L2: residues
50-56 of SEQ ID NO:197 CDR-L3: residues 89-97 of SEQ ID NO:197 VL
h1A11.A2 CDR Set CDR-L1: residues 24-34 of SEQ ID NO:198 CDR-L2:
residues 50-56 of SEQ ID NO:198 CDR-L3: residues 89-97 of SEQ ID
NO:198 VL h1A11.A12 CDR Set CDR-L1: residues 24-34 of SEQ ID NO:199
CDR-L2: residues 50-56 of SEQ ID NO:199 CDR-L3: residues 89-97 of
SEQ ID NO:199 VL h1A11.A7 CDR Set CDR-L1: residues 24-34 of SEQ ID
NO:200 CDR-L2: residues 50-56 of SEQ ID NO:200 CDR-L3: residues
89-97 of SEQ ID NO:200 VL h1A11.B4 CDR Set CDR-L1: residues 24-34
of SEQ ID NO:201 CDR-L2: residues 50-56 of SEQ ID NO:201 CDR-L3:
residues 89-97 of SEQ ID NO:201 VL h1A11.B5 CDR Set CDR-L1:
residues 24-34 of SEQ ID NO:202 CDR-L2: residues 50-56 of SEQ ID
NO:202 CDR-L3: residues 89-97 of SEQ ID NO:202 and VL h1A11.E12 CDR
Set CDR-L1: residues 24-34 of SEQ ID NO:203 CDR-L2: residues 50-56
of SEQ ID NO:203 CDR-L3: residues 89-97 of SEQ ID NO: 203
6. The binding protein according to claim 5, comprising CDRs from
at least two variable domain CDR sets.
7. The binding protein according to claim 6, wherein said at least
two variable domain CDR sets are a pair of CDR sets selected from
the group consisting of: VH 38H12 CDR Set and VL 38H12 CDR Set, VH
1A11 CDR Set and VL 1A11 CDR Set, VH 37D10 CDR Set and VL 37D10 CDR
Set, VH 32C7 CDR Set and VL 32C7 CDR Set, VH 14G1 Set and VL 14G1
CDR Set, VH 14A11 CDR Set and VL 14A11 CDR Set, VH 15D6 CDR Set and
VL 15D6 CDR Set, VH VH.1 1A11 CDR Set and VL VL.1 1A11 CDR Set, VH
VH.1 1A11 CDR Set and VL VL.1a 1A11 CDR Set, VH VH.1 1A11 CDR Set
and VL VL.1b 1A11 CDR Set, VH VH.1 1A11 CDR Set and VL VL.2a 1A11
CDR Set, VH VH.1a 1A11 CDR Set and VL VL.1 1A11 CDR Set, VH VH.1a
1A11 CDR Set and VL VL.1a 1A11 CDR Set, VH VH.1a 1A11 CDR Set and
VL VL.1b 1A11 CDR Set, VH VH.1a 1A11 CDR Set and VL VL.2a 1A11 CDR
Set, VH VH.1b 1A11 CDR Set and VL VL.1 1A11 CDR Set, VH VH.1b 1A11
CDR Set and VL VL.1a 1A11 CDR Set, VH VH.1b 1A11 CDR Set and VL
VL.1b 1A11 CDR Set, VH VH.1b 1A11 CDR Set and VL VL.2a 1A11 CDR
Set, VH VH.2a 1A11 CDR Set and VL VL.1 1A11 CDR Set, VH VH.2a 1A11
CDR Set and VL VL.1a 1A11 CDR Set, VH VH.2a 1A11 CDR Set and VL
VL.1b 1A11 CDR Set, VH VH.2a 1A11 CDR Set and VL VL.2a 1A11 CDR
Set, VH VH.1 38H12 CDR Set and VL VL.1 38H12 CDR Set, VH VH.1 38H12
CDR Set and VL VL.1a 38H12 CDR Set, VH VH.1 38H12 CDR Set and VL
VL.1b 38H12 CDR Set, VH VH.1 38H12 CDR Set and VL VL.2a 38H12 CDR
Set, VH VH.1a 38H12 CDR Set and VL VL.1 38H12 CDR Set, VH VH.1a
38H12 CDR Set and VL VL.1a 38H12 CDR Set, VH VH.1a 38H12 CDR Set
and VL VL.1b 38H12 CDR Set, VH VH.1a 38H12 CDR Set and VL VL.2a
38H12 CDR Set, VH VH.1b 38H12 CDR Set and VL VL.1 38H12 CDR Set, VH
VH.1b 38H12 CDR Set and VL VL.1a 38H12 CDR Set, VH VH.1b 38H12 CDR
Set and VL VL.1b 38H12 CDR Set, VH VH.1b 38H12 CDR Set and VL VL.2a
38H12 CDR Set, VH VH.2a 38H12 CDR Set and VL VL.1 38H12 CDR Set, VH
VH.2a 38H12 CDR Set and VL VL.1a 38H12 CDR Set, VH VH.2a 38H12 CDR
Set and VL VL.1b 38H12 CDR Set, VH VH.2a 38H12 CDR Set and VL VL.2a
38H12 CDR Set, VH h1A11.A6 CDR Set and VL h1A11VL.1 CDR Set, VH
h1A11.C6 CDR Set and VL h1A11VL.1 CDR Set, VH h1A11.A11 CDR Set and
VL h1A11VL.1 CDR Set, VH h1A11.A8 CDR Set and VL h1A11VL.1 CDR Set,
VH h1A11VH.1 CDR Set and VL h1A11.B4 CDR Set, VH h1A11VH.1 CDR Set
and VL h1A11.A7 CDR Set, VH h1A11VH.1 CDR Set and VL h1A11.A12 CDR
Set, VH h1A11VH.1 CDR Set and VL h1A11.A2 CDR Set, VH h1A11.B5 CDR
Set and VL h1A11.B5 CDR Set, VH h1A11.E12 CDR Set and VL h1A11.E12
CDR Set, VH h1A11.G3 CDR Set and VL h1A11.E12 CDR Set, VH h1A11.F5
CDR Set and VL h1A11.E12 CDR Set, and VH h1A11.H2 CDR Set and VL
h1A11.E12 CDR Set.
8. The binding protein according to claim 4, further comprising a
human acceptor framework sequence.
9. The binding protein according to claim 5, further comprising a
human acceptor framework sequence.
10. The binding protein according to claim 6, further comprising a
human acceptor framework sequence.
11. The binding protein according to claim 7, further comprising a
human acceptor framework sequence.
12. The binding protein according to claim 8, wherein said human
acceptor framework sequence is selected from the group of acceptor
sequences listed in Tables 3 and 4.
13. The binding protein according to claim 9, wherein said human
acceptor framework sequence is selected from the group of acceptor
sequences listed in Tables 3 and 4.
14. The binding protein according to claim 10, wherein said human
acceptor framework sequence is selected from the group of acceptor
sequences listed in Tables 3 and 4
15. The binding protein according to claim 11, wherein said human
acceptor framework sequence is selected from the group of acceptor
sequences listed in Tables 3 and 4.
16. The binding protein according to claim 8, wherein said human
acceptor framework sequence is selected from any framework sequence
in a variable region sequence selected from the group consisting
of: TABLE-US-00037 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: 171
180 188 196 VH.1 1A11 VH.1a 38H12 VH h1A11.A6 VH h1A11.H2 SEQ ID
NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: 172 181 189 197 VH.1a 1A11
VH.1b 38H12 VH h1A11.A8 VL h1A11VL.1 SEQ ID NO: SEQ ID NO: SEQ ID
NO: SEQ ID NO: 173 182 190 198 VH.1b 1A11 VH.2a 38H12 VH h1A11.C6
VL h1A11.A2 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: 174 183 191
199 VH.2a 1A11 VL.1 38H12 VH h1A11.A11 VL h1A11.A12 SEQ ID NO: SEQ
ID NO: SEQ ID NO: SEQ ID NO: 175 184 192 200 VL.1 1A11 VL.1a VH
h1A11.B5 VL h1A11.A7 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO:
176 185 193 201 VL.1a 1A11 VL.1b VH h1A11.E12 VL h1A11.B4 SEQ ID
NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: 177 186 194 202 VL.1b 1A11
VL.2a VH h1A11.G3 VL h1A11.B5 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ
ID NO: 178 187 195 203 VL.2a 1A11 VH h1A11VH.1 VH h1A11.F5 VL
h1A11.E12 SEQ ID NO: 179 VH.1 38H12
17. The binding protein according to claim 9, wherein said human
acceptor framework sequence is selected from any framework sequence
in a variable region sequence selected from the group consisting
of: TABLE-US-00038 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: 171
180 188 196 VH.1 1A11 VH.1a 38H12 VH h1A11.A6 VH h1A11.H2 SEQ ID
NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: 172 181 189 197 VH.1a 1A11
VH.1b 38H12 VH h1A11.A8 VL h1A11VL.1 SEQ ID NO: SEQ ID NO: SEQ ID
NO: SEQ ID NO: 173 182 190 198 VH.1b 1A11 VH.2a 38H12 VH h1A11.C6
VL h1A11.A2 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: 174 183 191
199 VH.2a 1A11 VL.1 38H12 VH h1A11.A11 VL h1A11.A12 SEQ ID NO: SEQ
ID NO: SEQ ID NO: SEQ ID NO: 175 184 192 200 VL.1 1A11 VL.1a VH
h1A11.B5 VL h1A11.A7 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO:
176 185 193 201 VL.1a 1A11 VL.1b VH h1A11.E12 VL h1A1 1.B4 SEQ ID
NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: 177 186 194 202 VL.1b 1A11
VL.2a VH h1A11.G3 VL h1A11.B5 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ
ID NO: 178 187 195 203 VL.2a 1A11 VH h1A11VH.1 VH h1A11.F5 VL
h1A11.E12 SEQ ID NO: 179 VH.1 38H12
18. The binding protein according to claim 10, wherein said human
acceptor framework sequence is selected from any framework sequence
in a variable region sequence selected from the group consisting
of: TABLE-US-00039 SEQ ID NO: SEQ ID NO: 180 SEQ ID NO: 188 SEQ ID
NO: 196 171 VH.1a 38H12 VH h1A11.A6 VH h1A11.H2 VH.1 1A11 SEQ ID
NO: SEQ ID NO: 181 SEQ ID NO: 189 SEQ ID NO: 197 172 VH.1b 38H12 VH
h1A11.A8 VL h1A11VL.1 VH.1a 1A11 SEQ ID NO: SEQ ID NO: 182 SEQ ID
NO: 190 SEQ ID NO: 198 173 VH.2a 38H12 VH h1A11.C6 VL h1A11.A2
VH.1b 1A11 SEQ ID NO: SEQ ID NO: 183 SEQ ID NO: 191 SEQ ID NO: 199
174 VL.1 38H12 VH h1A11.A11 VL h1A11.A12 VH.2a 1A11 SEQ ID NO: SEQ
ID NO: 184 SEQ ID NO: 192 SEQ ID NO: 200 175 VL.1a VH h1A11.B5 VL
h1A11.A7 VL.1 1A11 SEQ ID NO: SEQ ID NO: 185 SEQ ID NO: 193 SEQ ID
NO: 201 176 VL.1b VH h1A11.E12 VL h1A11.B4 VL.1a 1A11 SEQ ID NO:
SEQ ID NO: 186 SEQ ID NO: 194 SEQ ID NO: 202 177 VL.2a VH h1A11.G3
VL h1A11.B5 VL.1b 1A11 SEQ ID NO: SEQ ID NO: 187 SEQ ID NO: 195 SEQ
ID NO: 203 178 VH h1A11VH.1 VH h1A11.F5 VL h1A11.E12 VL.2a 1A11 SEQ
ID NO: 179 VH.1 38H12
19. The binding protein according to claim 11, wherein said human
acceptor framework sequence is selected from any framework sequence
in a variable region sequence elected from the group consisting of:
TABLE-US-00040 SEQ ID NO: SEQ ID NO: 180 SEQ ID NO: 188 SEQ ID NO:
196 171 VH.1a 38H12 VH h1A11.A6 VH h1A11.H2 VH.1 1A11 SEQ ID NO:
SEQ ID NO: 181 SEQ ID NO: 189 SEQ ID NO: 197 172 VH.1b 38H12 VH
h1A11.A8 VL h1A11VL.1 VH.1a 1A11 SEQ ID NO: SEQ ID NO: 182 SEQ ID
NO: 190 SEQ ID NO: 198 173 VH.2a 38H12 VH h1A11.C6 VL h1A11.A2
VH.1b 1A11 SEQ ID NO: SEQ ID NO: 183 SEQ ID NO: 191 SEQ ID NO: 199
174 VL.1 38H12 VH h1A11.A11 VL h1A11.A12 VH.2a 1A11 SEQ ID NO: SEQ
ID NO: 184 SEQ ID NO: 192 SEQ ID NO: 200 175 VL.1a VH h1A11.B5 VL
h1A11.A7 VL.1 1A11 SEQ ID NO: SEQ ID NO: 185 SEQ ID NO: 193 SEQ ID
NO: 201 176 VL.1b VH h1A11.E12 VL h1A11.B4 VL.1a 1A11 SEQ ID NO:
SEQ ID NO: 186 SEQ ID NO: 194 SEQ ID NO: 202 177 VL.2a VH h1A11.G3
VL h1A11.B5 VL.1b 1A11 SEQ ID NO: SEQ ID NO: 187 SEQ ID NO: 195 SEQ
ID NO: 203 178 VH h1A11VH.1 VH h1A11.F5 VL h1A11.E12 VL.2a 1A11 SEQ
ID NO: 179 VH.1 38H12
20. The binding protein according to claim 8, wherein said binding
protein comprises at least one acceptor framework sequence selected
from the group consisting of: heavy chain framework-1 (H-FR1):
E-V-Q-L-V-E-S-G-G-G-L-V-Q-P-G-G-S-L-R-L-S-C-A-A-S-G-F-T-F-X.sub.30
(SEQ ID NO:143), wherein X.sub.30 is S, R, or G; heavy chain
framework-2 (H-FR2): W-V-R-Q-A-P-G-K-G-L-E-W-V-A (SEQ ID NO:144);
heavy chain framework-3 (H-FR3):
R-F-T-I-S-R-D-N-A-K-X.sub.11-S-L-Y-L-Q-M-N-S-L-R-A-E-D-T-A-V-Y-Y-C-X.sub.-
31-R (SEQ ID NO:145), wherein; X.sub.11 is N or S; and X.sub.31 is
A or S; heavy chain framework-4 (H-FR4): W-G-Q-G-T-L-V-T-V-S-S(SEQ
ID NO:146); light chain framework-1 (L-FR1):
D-I-Q-M-T-Q-S-P-S-S-L-S-A-S-V-G-D-R-V-T-I-T-C(SEQ ID NO:147); light
chain framework-2 (L-FR2):
W-Y-Q-Q-K-P-G-K-X.sub.9-P-K-L-L-I-X.sub.15 (SEQ ID NO:148),
wherein; X.sub.9 is A or S; and X.sub.15 is F or Y; light chain
framework-3 (L-FR3):
G-V-P-S-R-F-S-G-S-G-S-G-T-D-X.sub.15-T-L-T-I-S-S-L-Q-P-E-D-F-A-T-Y-Y-C
(SEQ ID NO:149), wherein; X.sub.15 is F or S; and light chain
framework-4 (L-FR4): F-G-Q-G-T-K-L-E-I-K (SEQ ID NO:150).
21. The binding protein according to claim 9, wherein said binding
protein comprises at least one acceptor framework sequence selected
from the group consisting of: heavy chain framework-1 (H-FR1):
E-V-Q-L-V-E-S-G-G-G-L-V-Q-P-G-G-S-L-R-L-S-C-A-A-S-G-F-T-F-X.sub.30
(SEQ ID NO:143), wherein X.sub.30 is S, R, or G; heavy chain
framework-2 (H-FR2): W-V-R-Q-A-P-G-K-G-L-E-W-V-A (SEQ ID NO:144);
heavy chain framework-3 (H-FR3):
R-F-T-I-S-R-D-N-A-K-X.sub.11-S-L-Y-L-Q-M-N-S-L-R-A-E-D-T-A-V-Y-Y-C-X.sub.-
31-R (SEQ ID NO:145), wherein; X.sub.11 is N or S; and X.sub.31 is
A or S; heavy chain framework-4 (H-FR4): W-G-Q-G-T-L-V-T-V-S-S(SEQ
ID NO:146); light chain framework-1 (L-FR1):
D-I-Q-M-T-Q-S-P-S-S-L-S-A-S-V-G-D-R-V-T-I-T-C(SEQ ID NO:147); light
chain framework-2 (L-FR2): (SEQ ID NO:148), wherein; X.sub.9 is A
or S; and X.sub.15 is F or Y; light chain framework-3 (L-FR3):
G-V-P-S-R-F-S-G-S-G-S-G-T-D-X.sub.15-T-L-T-I-S-S-L-Q-P-E-D-F-A-T-Y-Y-C
(SEQ ID NO:149), wherein; X.sub.15 is F or S; and light chain
framework-4 (L-FR4): F-G-Q-G-T-K-L-E-I-K (SEQ ID NO:150).
22. The binding protein according to claim 10, wherein said binding
protein comprises at least one acceptor framework sequence selected
from the group consisting of: heavy chain framework-1 (H-FR1):
E-V-Q-L-V-E-S-G-G-G-L-V-Q-P-G-G-S-L-R-L-S-C-A-A-S-G-F-T-F-X.sub.30
(SEQ ID NO:143), wherein X.sub.30 is S, R, or G; heavy chain
framework-2 (H-FR2): W-V-R-Q-A-P-G-K-G-L-E-W-V-A (SEQ ID NO:144);
heavy chain framework-3 (H-FR3):
R-F-T-I-S-R-D-N-A-K-X.sub.11-S-L-Y-L-Q-M-N-S-L-R-A-E-D-T-A-V-Y-Y-C-X.sub.-
31-R (SEQ ID NO:145), wherein; X.sub.11 is N or S; and X.sub.31 is
A or S; heavy chain framework-4 (H-FR4): W-G-Q-G-T-L-V-T-V-S-S(SEQ
ID NO:146); light chain framework-1 (L-FR1):
D-I-Q-M-T-Q-S-P-S-S-L-S-A-S-V-G-D-R-V-T-I-T-C(SEQ ID NO:147); light
chain framework-2 (L-FR2): (SEQ ID NO:148), wherein; X.sub.9 is A
or S; and X.sub.15 is F or Y; light chain framework-3 (L-FR3):
G-V-P-S-R-F-S-G-S-G-S-G-T-D-X.sub.15-T-L-T-I-S-S-L-Q-P-E-D-F-A-T-Y-Y-C
(SEQ ID NO:149), wherein; X.sub.15 is F or S; and light chain
framework-4 (L-FR4): F-G-Q-G-T-K-L-E-I-K (SEQ ID NO:150).
23. The binding protein according to claim 11, wherein said binding
protein comprises at least one acceptor framework sequence selected
from the group consisting of: heavy chain framework-1 (H-FR1):
E-V-Q-L-V-E-S-G-G-G-L-V-Q-P-G-G-S-L-R-L-S-C-A-A-S-G-F-T-F-X.sub.30
(SEQ ID NO:143), wherein X.sub.30 is S, R, or G; heavy chain
framework-2 (H-FR2): W-V-R-Q-A-P-G-K-G-L-E-W-V-A (SEQ ID NO:144);
heavy chain framework-3 (H-FR3):
R-F-T-I-S-R-D-N-A-K-X.sub.11-S-L-Y-L-Q-M-N-S-L-R-A-E-D-T-A-V-Y-Y-C-X.sub.-
31-R (SEQ ID NO:145), wherein; X.sub.11 is N or S; and X.sub.31 is
A or S; heavy chain framework-4 (H-FR4): W-G-Q-G-T-L-V-T-V-S-S(SEQ
ID NO:146); light chain framework-1 (L-FR1):
D-I-Q-M-T-Q-S-P-S-S-L-S-A-S-V-G-D-R-V-T-I-T-C(SEQ ID NO:147); light
chain framework-2 (L-FR2):
W-Y-Q-Q-K-P-G-K-X.sub.9-P-K-L-L-I-X.sub.15 (SEQ ID NO:148),
wherein; X.sub.9 is A or S; and X.sub.15 is F or Y; light chain
framework-3 (L-FR3):
G-V-P-S-R-F-S-G-S-G-S-G-T-D-X.sub.15-T-L-T-I-S-S-L-Q-P-E-D-F-A-T-Y-Y-C(SE-
Q ID NO:149), wherein; X.sub.15 is F or S; and light chain
framework-4 (L-FR4): F-G-Q-G-T-K-L-E-I-K (SEQ ID NO:150).
24. The binding protein according to claim 2, wherein said binding
protein comprises a human acceptor framework sequence and at least
one variable domain having an amino acid sequence selected from the
group consisting of: TABLE-US-00041 SEQ ID NO: 171 SEQ ID NO: 188
VH VH.1 1A11 VH h1A11.A6 SEQ ID NO: 172 SEQ ID NO: 189 VH VH.1a
1A11 VH h1A11.A8 SEQ ID NO: 173 SEQ ID NO: 190 VH VH.1b 1A11 VH
h1A11.C6 SEQ ID NO: 174 SEQ ID NO: 191 VH VH.2a 1A11 VH h1A11.A11
SEQ ID NO: 175 SEQ ID NO: 192 VL VL.1 1A11 VH h1A11.B5 SEQ ID NO:
176 SEQ ID NO: 193 VL VL.1a 1A11 VH h1A11.E12 SEQ ID NO: 177 SEQ ID
NO: 194 VL VL.1b VH h1A11.G3 SEQ ID NO: 178 SEQ ID NO: 195 VL VL.2a
1A11 VH h1A11.F5 SEQ ID NO: 179 SEQ ID NO: 196 VH VH.1 38H12 VH
h1A11.H2 SEQ ID NO: 180 SEQ ID NO: 197 VH VH.1a 38H12 VL h1A11VL.1
SEQ ID NO: 181 SEQ ID NO: 198 VH VH.1b 38H12 VL h1A11.A2 SEQ ID NO:
182 SEQ ID NO: 199 VH VH.2a 38H12 VL h1A11.A12 SEQ ID NO: 183 SEQ
ID NO: 200 VL VL.1 38H12 VL h1A11.A7 SEQ ID NO: 184 SEQ ID NO: 201
VL VL.1a 38H12 VL h1A11.B4 SEQ ID NO: 185 SEQ ID NO: 202 VL VL.1b
38H12 VL h1A11.B5 SEQ ID NO: 186 SEQ ID NO: 203 VL VL.2a 38H12 VL
h1A11.E12 SEQ ID NO: 187 VH h1A11VH.1
25. The binding protein according to claim 24, wherein said binding
protein comprises two variable domains, wherein said two variable
domains have amino acid sequences selected from the group
consisting of: TABLE-US-00042 SEQ ID NO: 171 and SEQ ID NO: 175 SEQ
ID NO: 180 and SEQ ID VH.1 and VL.1 1A11 (Table 12) NO: 186 VH.1a
and VL.2a (Table 16) SEQ ID NO: 171 and SEQ ID NO: 176 SEQ ID NO:
181 and SEQ ID VH.1 and VL.1a 1A11 (Table 12) NO: 183 VH.1b and
VL.1 (Table 16) SEQ ID NO: 171 and SEQ ID NO: 177 SEQ ID NO: 181
and SEQ ID VH.1 and VL.1b 1A11 (Table 12) NO: 184 VH.1b and VL.1a
(Table 16) SEQ ID NO: 171 and SEQ ID NO: 178 SEQ ID NO: 181 and SEQ
ID VH.1 and VL.2a 1A11(Table 12) NO: 185 VH.1b and VL.1b (Table 16)
SEQ ID NO: 172 and SEQ ID NO: 175 SEQ ID NO: 181 and SEQ ID VH.1a
and VL.1 1A11 (Table 12) NO: 186 VH.1b and VL.2a (Table 16) SEQ ID
NO: 172 and SEQ ID NO: 176 SEQ ID NO: 182 and SEQ ID VH.1a and
VL.1a 1A11 (Table 12) NO: 183 VH.2a and VL.1 (Table 16) SEQ ID NO:
172 and SEQ ID NO: 177 SEQ ID NO: 182 and SEQ ID VH.1a and VL.1b
1A11 (Table 12) NO: 184 VH.2a and VL.1a (Table 16) SEQ ID NO: 172
and SEQ ID NO: 178 SEQ ID NO: 182 and SEQ ID VH.1a and VL.2a 1A11
(Table 12) NO: 185 VH.2a and VL.1b (Table 16) SEQ ID NO: 173 and
SEQ ID NO: 175 SEQ ID NO: 182 and SEQ ID VH.1b and VL.1 1A11 (Table
12) NO: 186 VH.2a and VL.2a (Table 16) SEQ ID NO: 173 and SEQ ID
NO: 176 SEQ ID NO: 188 and SEQ ID VH.1b and VL.1a 1A11 (Table 12)
NO: 197 h1A11.A6 VH and h1A11VL.1 Tables 20/21 SEQ ID NO: 173 and
SEQ ID NO: 177 SEQ ID NO: 190 and SEQ ID VH.1b and VL.1b 1A11
(Table 12) NO: 197 h1A11.C6 VH and h1A11VL.1 Tables 20/21 SEQ ID
NO: 173 and SEQ ID NO: 178 SEQ ID NO: 191 and SEQ ID VH.1b and
VL.2a 1A11 (Table 12) NO: 197 h1A11.All VH and h1A11VL.1 Tables
20/21 SEQ ID NO: 174 and SEQ ID NO: 175 SEQ ID NO: 189 and SEQ ID
VH.2a and VL.1 1A11 (Table 12) NO: 197 h1A11.A8 VH and h1A11 VL.1
Tables 20/21 SEQ ID NO: 174 and SEQ ID NO: 176 SEQ ID NO: 187 and
SEQ ID VH.2a and VL.1a 1A11 (Table 12) NO: 201 h1A11VH.1 and
h1A11.B4 VL Tables 20/21 SEQ ID NO: 174 and SEQ ID NO: 177 SEQ ID
NO: 187 and SEQ ID VH.2a and VL.1b 1A11 (Table 12) NO: 200
h1A11VH.1 and h1All.A7 VL Tables 20/21 SEQ ID NO: 174 and SEQ ID
NO: 178 SEQ ID NO: 187 and SEQ ID VH.2a and VL.2a 1A11 (Table 12)
NO: 199 h1A11VH.1 and h1All.A12 VL Tables 20/21 SEQ ID NO: 179 and
SEQ ID NO: 183 SEQ ID NO: 187 and SEQ ID VH.1 and VL.1 38H12 (Table
16) NO: 198 h1A11VH.1 VH and h1All.A2 VL Tables 20/21 SEQ ID NO:
179 and SEQ ID NO: 184 SEQ ID NO: 192 and SEQ ID VH.1 and VL.1a
38H12 (Table 16) NO: 202 h1A11.B5 VH and h1All.B5 VL Tables 20/21
SEQ ID NO: 179 and SEQ ID NO: 185 SEQ ID NO: 193 and SEQ ID VH.1
and VL.1b 38H12 (Table 16) NO: 203 h1A11.E12 VH and h1All.E12 VL
Tables 20/21 SEQ ID NO: 179 and SEQ ID NO: 186 SEQ ID NO: 194 and
SEQ ID VH.1 and VL.2a 38H12 (Table 16) NO: 203 h1A11.G3 VH and
h1All.E12 VL Tables 20/21 SEQ ID NO: 180 and SEQ ID NO: 183 SEQ ID
NO: 195 and SEQ ID VH.1a and VL.1 38H12 (Table 16) NO: 203 h1A11.F5
VH and h1All.E12 VL Tables 20/21 SEQ ID NO: 180 and SEQ ID NO: 184
SEQ ID NO: 196 and SEQ ID VH.1a and VL.1a 38H12 (Table 16) NO: 203
h1A1 1.H2 VH and h1All.E12 VL Tables 20/21 SEQ ID NO: 180 and SEQ
ID NO: 185 VH.1a and VL.1b 38H12 (Table 16)
26. The binding protein according to claim 8, wherein said human
acceptor framework sequence comprises at least one Framework Region
amino acid substitution at a key residue, said key residue selected
from the group consisting of: a residue adjacent to a CDR; a
glycosylation site residue; a rare residue; a residue capable of
interacting with human DLL4; a residue capable of interacting with
a CDR; a canonical residue; a contact residue between heavy chain
variable region and light chain variable region; a residue within a
Vernier zone; and a residue in a region that overlaps between a
Chothia-defined variable heavy chain CDR1 and a Kabat-defined first
heavy chain framework.
27. The binding protein according to claim 11, wherein said human
acceptor framework sequence comprises at least one Framework Region
amino acid substitution at a key residue, said key residue selected
from the group consisting of: a residue adjacent to a CDR; a
glycosylation site residue; a rare residue; a residue capable of
interacting with human DLL4; a residue capable of interacting with
a CDR; a canonical residue; a contact residue between heavy chain
variable region and light chain variable region; a residue within a
Vernier zone; and a residue in a region that overlaps between a
Chothia-defined variable heavy chain DR1 and a Kabat-defined first
heavy chain framework.
28. The binding protein according to claim 25, wherein said human
acceptor framework sequence comprises at least one Framework Region
amino acid substitution at a key residue, said key residue selected
from the group consisting of: a residue adjacent to a CDR; a
glycosylation site residue; a rare residue; a residue capable of
interacting with human DLL4; a residue capable of interacting with
a CDR; a canonical residue; a contact residue between heavy chain
variable region and light chain variable region; a residue within a
Vernier zone; and a residue in a region that overlaps between a
Chothia-defined variable heavy chain CDR1 and a Kabat-defined first
heavy chain framework.
29. The binding protein according to claim 26, wherein the binding
protein comprises a consensus human variable domain sequence.
30. The binding protein according to claim 27, wherein the binding
protein comprises a consensus human variable domain sequence.
31. The binding protein according to claim 28, wherein the binding
protein comprises a consensus human variable domain sequence.
32. The binding protein according to claim 8, wherein said human
acceptor framework comprises at least one framework region amino
acid substitution, wherein the amino acid sequence of the framework
is at least 65% identical to a sequence of a human germline
acceptor framework and comprises at least 70 amino acid residues
identical to the human germline acceptor framework.
33. The binding protein according to claim 11, wherein said human
acceptor framework comprises at least one framework region amino
acid substitution, wherein the amino acid sequence of the framework
is at least 65% identical to a sequence of a human germline
acceptor framework and comprises at least 70 amino acid residues
identical to the human germline acceptor framework.
34. The binding protein according to claim 25, wherein said human
acceptor framework comprises at least one framework region amino
acid substitution, wherein the amino acid sequence of the framework
is at least 65% identical to a sequence of a human germline
acceptor framework and comprises at least 70 amino acid residues
identical to the human germline acceptor framework.
35. The binding protein according to claim 2, wherein said binding
protein comprises at least one variable domain having an amino acid
sequence selected from the group consisting of: TABLE-US-00043 SEQ
ID NO: 157 SEQ ID NO: 181 VH 38H12 VH VH.1b 38H12 SEQ ID NO: 158
SEQ ID NO: 182 VL 38H12 VH VH.2a 38H12 SEQ ID NO: 159 SEQ ID NO:
182 VH 1A11 VL VL.1 38H12 SEQ ID NO: 160 SEQ ID NO: 184 VL 1A11 VL
VL.1a 38H12 SEQ ID NO: 161 SEQ ID NO: 185 VH 37D10 VL VL.1b 38H12
SEQ ID NO: 162 SEQ ID NO: 186 VL 37D10 VL VL.2a 38H12 SEQ ID NO:
163 SEQ ID NO: 187 VH 32C7 VH h1A11VH.1 SEQ ID NO: 164 SEQ ID NO:
188 VL 32C7 VH h1A11.A6 SEQ ID NO: 165 SEQ ID NO: 189 VH 14G1 VH
h1A11.A8 SEQ ID NO: 166 SEQ ID NO: 190 VL 14G1 VH h1A11.C6 SEQ ID
NO: 167 SEQ ID NO: 191 VH 14A11 VH h1A11.A11 SEQ ID NO: 168 SEQ ID
NO: 192 VL 14A11 VH h1A11.B5 SEQ ID NO: 169 SEQ ID NO: 193 VH 15D6
VH h1A11.E12 SEQ ID NO: 170 SEQ ID NO: 194 VL 15D6 VH h1A11.G3 SEQ
ID NO: 171 SEQ ID NO: 195 VH VH.1 1A11 VH h1A11.F5 SEQ ID NO: 172
SEQ ID NO: 196 VH VH.1a 1A11 VH h1A11.H2 SEQ ID NO: 173 SEQ ID NO:
197 VH VH.1b 1A11 VL h1A11VL.1 SEQ ID NO: 174 SEQ ID NO: 198 VH
VH.2a 1A11 VL h1A11.A2 SEQ ID NO: 175 SEQ ID NO: 199 VL VL.1 1A11
VL h1A11.A12 SEQ ID NO: 176 SEQ ID NO: 200 VL VL.1a 1A11 VL
h1A11.A7 SEQ ID NO: 177 SEQ ID NO: 201 VL VL.1b VL h1A11.B4 SEQ ID
NO: 178 SEQ ID NO: 202 VL VL.2a 1A11 VL h1A11.B5 SEQ ID NO: 179 SEQ
ID NO: 203 VH VH.1 38H12 VL h1A11.E12 SEQ ID NO: 180 VH VH.1a
38H12
36. The binding protein according to claim 35, wherein said binding
protein comprises two variable domains, wherein said two variable
domains have amino acid sequences selected from the group
consisting of: TABLE-US-00044 SEQ ID NO: 157 and SEQ ID NO: 158 SEQ
ID NO: 179 and SEQ ID 38H12 NO: 186 VH.1 and VL.2a 38H12 SEQ ID NO:
159 and SEQ ID NO: 160 SEQ ID NO: 180 and SEQ ID 1A11 NO: 183 VH.1a
and VL.1 38H12 SEQ ID NO: 161 and SEQ ID NO: 162 SEQ ID NO: 180 and
SEQ ID 37D10 NO: 184 VH.1a and VL.1a 38H12 SEQ ID NO: 163 and SEQ
ID NO: 164 SEQ ID NO: 180 and SEQ ID 32C7 NO: 185 VH.1a and VL.1b
38H12 SEQ ID NO: 165 and SEQ ID NO: 166 SEQ ID NO: 180 and SEQ ID
14G1 NO: 186 VH.1a and VL.2a 38H12 SEQ ID NO: 167 and SEQ ID NO:
168 SEQ ID NO: 181 and SEQ ID 14A11 NO: 183 VH.1b and VL.1 38H12
SEQ ID NO: 169 and SEQ ID NO: 170 SEQ ID NO: 181 and SEQ ID 15D6
NO: 184 VH.1b and VL.1a 38H12 SEQ ID NO: 171 and SEQ ID NO: 175 SEQ
ID NO: 181 and SEQ ID VH.1 and VL.1 1A11 (Table 11) NO: 185 VH.1b
and VL.1b 38H12 SEQ ID NO: 171 and SEQ ID NO: 176 SEQ ID NO: 181
and SEQ ID VH.1 and VL.1a 1A11 NO: 186 VH.1b and VL.2a 38H12 SEQ ID
NO: 171 and SEQ ID NO: 177 SEQ ID NO: 182 and SEQ ID VH.1 and VL.1b
1A11 NO: 183 VH.2a and VL.1 38H12 SEQ ID NO: 171 and SEQ ID NO: 178
SEQ ID NO: 182 and SEQ ID VH.1 and VL.2a NO: 184 VH.2a and VL.1a
38H12 SEQ ID NO: 172 and SEQ ID NO: 175 SEQ ID NO: 182 and SEQ ID
VH.1a and VL.1 NO: 185 VH.2a and VL.1b 38H12 SEQ ID NO: 172 and SEQ
ID NO: 176 SEQ ID NO: 182 and SEQ ID VH.1a and VL.1a NO: 186 VH.2a
and VL.2a 38H12 SEQ ID NO: 172 and SEQ ID NO: 177 SEQ ID NO: 188
and SEQ ID VH.1a and VL.1b NO: 197 h1A11.A6 VH and h1A11VL.1 SEQ ID
NO: 172 and SEQ ID NO: 178 SEQ ID NO: 190 and SEQ ID VH.1a and
VL.2a NO: 197 h1A11.C6 VH and h1A11VL.1 SEQ ID NO: 173 and SEQ ID
NO: 175 SEQ ID NO: 191 and SEQ ID VH.1b and VL.1 NO: 197 h1A11.All
VH and h1A11VL.1 SEQ ID NO: 173 and SEQ ID NO: 176 SEQ ID NO: 189
and SEQ ID VH.1b and VL.1a NO: 197 h1A11.A8 VH and h1A11 VL.1 SEQ
ID NO: 173 and SEQ ID NO: 177 SEQ ID NO: 187 and SEQ ID VH.1b and
VL.1b NO: 201 h1A11VH.1 and h1A11.B4 VL SEQ ID NO: 173 and SEQ ID
NO: 178 SEQ ID NO: 187 and SEQ ID VH.1b and VL.2a NO: 200 h1A11VH.1
and h1All.A7 VL SEQ ID NO: 174 and SEQ ID NO: 175 SEQ ID NO: 187
and SEQ ID VH.2a and VL.1 NO: 199 h1A11VH.1 and h1All.A12 VL SEQ ID
NO: 174 and SEQ ID NO: 176 SEQ ID NO: 187 and SEQ ID VH.2a and
VL.1a NO: 198 h1A11VH.1 VH and h1All.A2 VL SEQ ID NO: 174 and SEQ
ID NO: 177 SEQ ID NO: 192 and SEQ ID VH.2a and VL.1b NO: 202
h1A11.B5 VH and h1All.B5 VL SEQ ID NO: 174 and SEQ ID NO: 178 SEQ
ID NO: 193 and SEQ ID VH.2a and VL.2a NO: 203 h1A11.E12 VH and
h1All.E12 VL SEQ ID NO: 179 and SEQ ID NO: 183 SEQ ID NO: 194 and
SEQ ID VH.1 and VL.1 38H12 NO: 203 h1A11.G3 VH and h1All.E12 VL SEQ
ID NO: 179 and SEQ ID NO: 184 SEQ ID NO: 195 and SEQ ID VH.1 and
VL.1a 38H12 NO: 203 h1A11.F5 VH and h1All.E12 VL SEQ ID NO: 179 and
SEQ ID NO: 185 SEQ ID NO: 196 and SEQ ID VH.1 and VL.1b 38H12 NO:
203 h1A11.H2 VH and h1All.E12 VL
37. The DLL4 binding protein according to claim 2, wherein said
binding protein is capable of blocking DLL4 interaction with a
Notch protein.
38. The DLL4 binding protein according to claim 36, wherein the
binding protein is capable of blocking DLL4 interaction with a
Notch protein selected from the group consisting of Notch-1,
Notch-2, Notch-3, Notch-4, and combinations thereof.
39. The DLL4 binding protein according to claim 2, wherein said
binding protein is capable of modulating a biological function of
DLL4.
40. The DLL4 binding protein according to claim 2, wherein said
binding protein is capable of neutralizing a biological function of
DLL4.
41. The DLL4 binding protein according to claim 2, wherein said
binding protein is capable of inhibiting VEGFR2 activity, VEGFR1
activity, or both.
42. The DLL4 binding protein according to claim 2, wherein said
binding protein is capable of diminishing the ability of DLL4 to
bind to its receptor.
43. The DLL4 binding protein according to claim 2, wherein said
binding protein is capable of inhibiting normal angiogenesis.
44. The DLL4 binding protein according to claim 2, wherein said
binding protein has an on rate constant (Kon) to DLL4 selected from
the group consisting of: at least about 10.sup.2M.sup.-1s.sup.-1;
at least about 10.sup.3M.sup.-1s.sup.-1; at least about
10.sup.4M.sup.-1s.sup.-1; at least about 10.sup.5M.sup.-1s.sup.-1;
and at least about 10.sup.6M.sup.-1s.sup.-1, as measured by surface
plasmon resonance.
45. The DLL4 binding protein according to claim 8, wherein said
binding protein has an on rate constant (Kon) to DLL4 selected from
the group consisting of: at least about 10.sup.2M.sup.-1s.sup.-1;
at least about 10.sup.3M.sup.-1s.sup.-1; at least about
10.sup.4M.sup.-1s.sup.-1; at least about 10.sup.5M.sup.-1s.sup.-1;
and at least about 10.sup.6M.sup.-1s.sup.-1, as measured by surface
plasmon resonance.
46. The DLL4 binding protein according to claim 24, wherein said
binding protein has an on rate constant (Kon) to DLL4 selected from
the group consisting of: at least about 10.sup.2M.sup.-1s.sup.-1;
at least about 10.sup.3M.sup.-1s.sup.-1; at least about
10.sup.4M.sup.-1s.sup.-1; at least about 10.sup.5M.sup.-1s.sup.-1;
and at least about 10.sup.6M.sup.-1s.sup.-1, as measured by surface
plasmon resonance.
47. The DLL4 binding protein according to claim 2, wherein said
binding protein has an off rate constant (Koff) to DLL4 selected
from the group consisting of: at most about 10.sup.-3s.sup.-1; at
most about 10.sup.-4s.sup.-1; at most about 10.sup.-5s.sup.-1; and
at most about 10.sup.-6s.sup.-1, as measured by surface plasmon
resonance.
48. The DLL4 binding protein according to claim 8, wherein said
binding protein has an off rate constant (Koff) to DLL4 selected
from the group consisting of: at most about 10.sup.-3s.sup.-1; at
most about 10.sup.-4s.sup.-1; at most about 10.sup.-5s.sup.-1; and
at most about 10.sup.-6s.sup.-1, as measured by surface plasmon
resonance.
49. The DLL4 binding protein according to claim 24, wherein said
binding protein has an off rate constant (Koff) to DLL4 selected
from the group consisting of: at most about 10.sup.-3s.sup.-1; at
most about 10.sup.-4s.sup.-1; at most about 10.sup.-5s.sup.-1; and
at most about 10.sup.-6s.sup.-1, as measured by surface plasmon
resonance.
50. The DLL4 binding protein according to claim 2, wherein said
binding protein has a dissociation constant (K.sub.D) to DLL4
selected from the group consisting of: at most about 10.sup.-7M; at
most about 10.sup.-8 M; at most about 10.sup.-9 M; at most about
10.sup.-10 M; at most about 10.sup.-11 M; at most about 10.sup.-12
M; and at most 10.sup.-13M.
51. The DLL4 binding protein according to claim 8, wherein said
binding protein has a dissociation constant (K.sub.D) to DLL4
selected from the group consisting of: at most about 10.sup.-7M; at
most about 10.sup.-8 M; at most about 10.sup.-9 M; at most about
10.sup.-10 M; at most about 10.sup.-11 M; at most about 10.sup.-12
M; and at most 10.sup.-13M.
52. The DLL4 binding protein according to claim 24, wherein said
binding protein has a dissociation constant (K.sub.D) to DLL4
selected from the group consisting of: at most about 10.sup.-7M; at
most about 10.sup.-8 M; at most about 10.sup.-9 M; at most about
10.sup.-10 M; at most about 10.sup.-11 M; at most about 10.sup.-12
M; and at most 10.sup.-13M.
53. An antibody construct comprising a binding protein described in
claim 2, said antibody construct further comprising a linker
polypeptide or an immunoglobulin constant domain.
54. The antibody construct according to claim 53, selected from the
group consisting of: an immunoglobulin molecule, a monoclonal
antibody, a chimeric antibody, a CDR-grafted antibody, a Fab, a
Fab', a F(ab').sub.2, an Fv, a disulfide linked Fv, an scFv, a
single domain antibody, a diabody, a multispecific antibody, a dual
specific antibody, and a bispecific antibody.
55. The antibody construct according to claim 53, wherein said
antibody construct comprises a heavy chain immunoglobulin constant
domain selected from the group consisting of: a human IgM constant
domain, a human IgG1 constant domain, a human IgG2 constant domain,
a human IgG3 constant domain, a human IgG4 constant domain, a human
IgE constant domain, and a human IgA constant domain.
56. The antibody construct according to claim 53, comprising an
immunoglobulin constant domain having an amino acid sequence
selected from the group consisting of: SEQ ID NO:3, SEQ ID NO:4,
SEQ ID NO:5, SEQ ID NO:6, and combinations thereof.
57. An antibody conjugate comprising an antibody construct as
described in claim 53, said antibody conjugate further comprising
an agent selected from the group consisting of: an imaging agent, a
therapeutic agent, a cytotoxic agent, and an immunoadhesion
molecule.
58. The antibody conjugate according to claim 57, wherein said
agent is an imaging agent selected from the group consisting of a
radiolabel, an enzyme, a fluorescent label, a luminescent label, a
bioluminescent label, a magnetic label, and biotin.
59. The antibody conjugate according to claim 57, wherein said
imaging agent is a radiolabel selected from the group consisting
of: .sup.3H, .sup.14C, .sup.35S, .sup.90Y, .sup.99Tc, .sup.111In,
.sup.125I, .sup.131I, .sup.177Lu, .sup.166Ho, and .sup.153Sm.
60. The antibody conjugate according to claim 57, wherein said
agent is a therapeutic or cytotoxic agent selected from the group
consisting of: an anti-metabolite, an alkylating agent, an
antibiotic, a growth factor, a cytokine, an anti-angiogenic agent,
an anti-mitotic agent, an anthracycline, a toxin, and an apoptotic
agent.
61. The antibody construct according to claim 53, wherein said
binding protein possesses a human glycosylation pattern.
62. The antibody conjugate according to claim 57, wherein said
binding protein possesses a human glycosylation pattern.
63. The binding protein according to claim 4, wherein said binding
protein exists as a crystal.
64. The antibody construct according to claim 53, wherein said
antibody construct exists as a crystal.
65. The antibody conjugate according to claim 57, wherein said
antibody construct exists as a crystal.
66. The binding protein according to claim 63, wherein said crystal
is a carrier-free pharmaceutical controlled release crystal.
67. The antibody construct according to claim 64, wherein said
crystal is a carrier-free pharmaceutical controlled release
crystal.
68. The antibody conjugate according to claim 65, wherein said
crystal is a carrier-free pharmaceutical controlled release
crystal.
69. The binding protein according to claim 63, wherein said binding
protein crystal has a greater half life in vivo than the soluble
counterpart of the binding protein.
70. The antibody construct according to claim 64, wherein said
antibody construct crystal has a greater half life in vivo than the
soluble counterpart of the antibody construct.
71. The antibody conjugate according to claim 65, wherein said
antibody conjugate crystal has a greater half life in vivo than the
soluble counterpart of the antibody conjugate.
72. The binding protein according to claim 63, wherein said binding
protein crystal retains biological activity of the non-crystal form
of the binding protein.
73. The antibody construct according to claim 64, wherein said
antibody construct crystal retains biological activity of the
non-crystal form of the antibody construct.
74. The antibody conjugate according to claim 65, wherein said
antibody conjugate crystal retains biological activity of the
non-crystal form of the antibody conjugate.
75. An isolated nucleic acid encoding a binding protein amino acid
sequence described in claim 2.
76. An isolated nucleic acid encoding a polypeptide selected from
the group consisting of: a polypeptide comprising a heavy chain
variable domain, wherein the heavy chain variable domain comprises
one or more of a CDR-H1, a CDR-H2, and a CDR-H3 as described in
claim 2; a polypeptide comprising a light chain variable domain,
wherein the light chain variable domain comprises one or more of a
CDR-L1, a CDR-L2, and a CDR-L3 as described in claim 2; and a
combination of both polypeptides.
77. A vector comprising the isolated nucleic acid according to
claim 76.
78. The vector according to claim 77, wherein the vector is
selected from the group consisting of: pcDNA, pTT, pTT3, pEFBOS,
pBV, pJV, and pBJ.
79. A host cell comprising the vector described in claim 77.
80. The host cell according to claim 79, wherein said host cell is
a prokaryotic cell.
81. The host cell according to claim 80, wherein said host cell is
Escherichia coli.
82. The host cell according to claim 79, wherein said host cell is
a eukaryotic cell.
83. The host cell according to claim 82, wherein said eukaryotic
cell is selected from the group consisting of: a protist cell, an
animal cell, a plant cell, and a fungal cell.
84. The host cell according to claim 83, wherein said eukaryotic
cell is an animal cell selected from the group consisting of: a
mammalian cell, an avian cell, and an insect cell.
85. The host cell according to claim 84, wherein said mammalian
cell is a CHO cell.
86. The host cell according to claim 84, wherein said mammalian
cell is a COS cell.
87. The host cell according to claim 83, wherein said fungal cell
is Saccharomyces cerevisiae.
88. The host cell according to claim 84, wherein said insect cell
is an Sf9 cell.
89. A method of producing a binding protein that binds human DLL4,
comprising culturing the host cell as described in claim 79 in a
culture medium under conditions sufficient to produce a binding
protein that binds human DLL4.
90. A DLL4 binding protein produced according to the method of
claim 89.
91. A composition for the release of a binding protein said
composition comprising: (a) a formulation, wherein said formulation
comprises a crystallized binding protein according as described in
claim 63, and an ingredient; and (b) at least one polymeric
carrier.
92. The composition according to claim 93, wherein said polymeric
carrier is a polymer selected from one or more of the group
consisting of: poly (acrylic acid), poly (cyanoacrylates), poly
(amino acids), poly (anhydrides), poly (depsipeptide), poly
(esters), poly (lactic acid), poly (lactic-co-glycolic acid) or
PLGA, poly (b-hydroxybutyrate), poly (caprolactone), poly
(dioxanone), poly (ethylene glycol), poly ((hydroxypropyl)
methacrylamide, poly [(organo)phosphazene], poly (ortho esters),
poly (vinyl alcohol), poly (vinylpyrrolidone), maleic
anhydride-alkyl vinyl ether copolymers, pluronic polyols, albumin,
alginate, cellulose and cellulose derivatives, collagen, fibrin,
gelatin, hyaluronic acid, oligosaccharides, glycaminoglycans,
sulfated polysaccharides, blends and copolymers thereof.
93. The composition according to claim 91, wherein said ingredient
is selected from the group consisting of albumin, sucrose,
trehalose, lactitol, gelatin,
hydroxypropyl-.quadrature.-cyclodextrin, methoxypolyethylene glycol
and polyethylene glycol.
94. A method for treating a mammal comprising the step of
administering to the mammal an effective amount of the composition
as described in claim 91.
95. A pharmaceutical composition comprising a DLL4 binding protein
as described in claim 2, and a pharmaceutically acceptable
carrier.
96. The pharmaceutical composition of claim 95, further comprising
at least one additional agent for treating a disorder in which DLL4
activity is detrimental.
97. The pharmaceutical composition of claim 95, wherein said
additional agent is selected from the group consisting of: a
therapeutic agent; an imaging agent; an antineoplastic agent; a
chemotherapeutic agent; an angiogenesis inhibitor; an anti-VEGF
antibody; an anti-EGFR antibody; an anti-cMet antibody; an
anti-ErbB3 antibody; an anti-HER2 antibody; an anti-CD20 antibody;
aflibercept; a kinase inhibitor; a co-stimulation molecule blocker;
an anti-B7.2 antibody; a CTLA4-Ig; an adhesion molecule blocker; an
anti-E selectin antibody; an anti-L selectin antibody; an
anti-cytokine antibody or functional fragment thereof; an
anti-IL-18 antibody; an anti-TNF antibody; anti-IL-6 antibody;
methotrexate; a corticosteroid; a cyclosporin; a rapamycin; FK506;
a DNA alkylating agent; cisplatin; carboplatin; an anti-tubulin
agent; paclitaxel; docetaxel; doxorubicin; gemcitabine; gemzar; an
anthracycline; adriamycin; a topoisiomersase I inhibitor; a
topoisomerase II inhibitor; 5-fluorouracil (5-FU); leucovorin;
irinotecan; a receptor tyrosine kinase inhibitor, an apoptosis
inhibitor; a Bcl2/Bclx inhibitor; erlotinib, gefitinib, a COX-2
inhibitor, celecoxib, cyclosporin; rapamycin; a detectable label or
reporter molecule; a TNF antagonist; an antirheumatic; a muscle
relaxant; a narcotic; an analgesic; an anesthetic; a sedative; a
local anesthetic; a neuromuscular blocker; an antimicrobial agent;
an antipsoriatic agent; a corticosteroid; an anabolic steroid; an
erythropoietin; an immunization; an immunoglobulin; an
immunosuppressive agent; a growth hormone; a hormone replacement
drug; a radiopharmaceutical drug; an antidepressant; an
antipsychotic drug; a stimulant; an asthma medication; a beta
agonist; an inhaled steroid; an epinephrine; an epinephrine analog
thereof; a cytokine; and a cytokine antagonist.
98. A method for reducing human DLL4.quadrature. activity
comprising contacting human DLL4 with the binding protein as
described in claim 2 such that human DLL4 activity is reduced.
99. A method for reducing human DLL4 activity in a human subject
suffering from a disorder in which DLL4 activity is detrimental,
comprising administering to the human subject a binding protein as
described in claim 2 such that human DLL4 activity in the human
subject is reduced.
100. A method for treating a subject for a disease or a disorder in
which DLL4 activity is detrimental by administering to the subject
a DLL4 binding protein as described in claim 2 such that treatment
is achieved.
101. The method of claim 100, wherein said disorder is selected
from the group consisting of: breast cancer, colon cancer, rectal
cancer, lung cancer, oropharynx cancer, hypopharynx cancer,
esophageal cancer, stomach cancer, pancreas cancer, liver cancer,
gallbladder cancer, bile duct cancer, small intestine cancer,
urinary tract cancer, female genital tract cancer, male genital
tract cancer, endocrine gland cancer, skin cancer, hemangioma,
melanoma, sarcoma, brain tumor, nerve cancer, eye tumor, meninges
cancer, solid tumors from hematopoietic malignancy, tumor
metastases, ocular neovascularization, edema, rheumatoid arthritis,
atherosclerotic plaques, Crohn's disease, inflammatory bowel
disease, refractory ascites, psoriasis, sarcoidosis, arterial
arteriosclerosis, sepsis, peptic ulcers, burns, pancreatitis,
polycystic ovarian disease (POD), endometriosis, uterine fibroid,
benign prostate hypertrophy, T-cell acute lymphoblastic leukemia
(T-ALL), cerebral autosomal dominant arteriopathy with subcortical
infarcts and leukoencephalopathy (CADASIL), multiple sclerosis
(MS), tetralogy of Fallot (TOF), Alagille syndrome (AS), macular
degeneration and age-related macular degeneration diseases, and
other angiogenesis independent and dependent diseases characterized
by aberrant DLL4 activity.
102. The method according to claim 101, wherein the disorder is a
primary and metastatic cancer.
103. The method according to claim 101, wherein the urinary tract
cancer is selected from the group consisting of renal cancer,
bladder cancer, and urothelium cancer.
104. The method according to claim 101, wherein the female genital
tract cancer is selected from the group consisting of cervical
cancer, uterine cancer, ovarian cancer, choriocarcinoma, and
gestational trophoblastic disease.
105. The method according to claim 101, wherein the male genital
tract cancer is selected from the group consisting of prostate
cancer, seminal vesicles cancer, testicular cancer, and germ cell
tumor.
106. The method according to claim 101, wherein the endocrine gland
cancer is selected from the group consisting of thyroid cancer,
adrenal cancer, and pituitary gland cancer.
107. The method according to claim 101, wherein the sarcoma is
selected from the group consisting of a bone sarcoma, a soft tissue
sarcoma, and Kaposi's sarcoma.
108. The method according to claim 101, wherein the meninges cancer
is selected from the group consisting of an astrocytoma, a glioma,
a glioblastoma, a retinoblastoma, a neuroma, a neuroblastoma, a
Schwannoma, and a meningioma.
109. The method according to claim 101, wherein the solid tumor
from a hematopoietic malignancy is a leukemia, a Hodgkin's
leukemia, a non-Hodgkin's leukemia, a lymphoma, a Hodgkin's
lymphoma, and a non-Hodgkin's lymphomas.
110. The method according to claim 101, wherein the ocular
neovascularization is selected from the group consisting of
diabetic blindness, a retinopathy, an age-related macular
degeneration, and a rubeosis.
111. The method according to claim 101, wherein said administering
to the subject is by at least one mode selected from the group
consisting of: parenteral, subcutaneous, intramuscular,
intravenous, intraarterial, intraarticular, intrabronchial,
intraabdominal, intracapsular, intracartilaginous, intracavitary,
intracelial, intracerebellar, intracerebroventricular, intracolic,
intracervical, intragastric, intrahepatic, intramyocardial,
intraosteal, intrapelvic, intrapericardiac, intraperitoneal,
intrapleural, intraprostatic, intrapulmonary, intrarectal,
intrarenal, intraretinal, intraspinal, intrasynovial,
intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal,
buccal, sublingual, intranasal, and transdermal.
112. A method of treating a patient suffering from a disorder in
which DLL4 is detrimental comprising the step of administering a
DLL4 binding protein as described in claim 2 before, concurrent
with, or after the administration of a therapeutically effective
amount of a second agent, wherein the second agent is selected from
the group consisting of an antibody or fragment thereof capable of
binding human VEGFR2; methotrexate; an antibody or fragment thereof
capable of binding human TNF; a corticosteroid; a cyclosporine; a
rapamycin; FK506; a non-steroidal anti-inflammatory agent (NSAID);
a radiotherapeutic agent; an antineoplastic agent; a
chemotherapeutic agent; a DNA alkylating agent; cisplatin;
carboplatin; an anti-tubulin agent; paclitaxel; docetaxel; taxol;
doxorubicin; gemcitabine; gemzar; an anthracycline; adriamycin; a
topoisomerase I inhibitor; a topoisomerase II inhibitor;
5-fluorouracil (5-FU); leucovorin; irinotecan; a receptor tyrosine
kinase inhibitor; erlotinib; gefitinib; a COX-2 inhibitor;
celecoxib; a kinase inhibitor; an angiogenesis inhibitor; an
anti-VEGF antibody; aflibercept; a co-stimulation molecule blocker;
an anti-B7.1 antibody; an anti-B7.2 antibody; a CTLA4-Ig; an
anti-CD20 antibody; an adhesion molecule blocker; an anti-LFA-1
antibody; an anti-E selectin antibody; and anti-L selectin
antibody; a small molecule inhibitor; an anti-cytokine antibody or
functional fragment thereof; an anti-IL-18 antibody; anti-TNF
antibody; an anti-IL-6 antibody; an anti-cytokine receptor
antibody; a detectable label or reporter; a TNF antagonist; an
antirheumatic; a muscle relaxant; a narcotic; an analgesic; an
anesthetic; a sedative; a local anesthetic; a neuromuscular
blocker; an antimicrobial agent; an antipsoriatic drug; a
corticosteroid; an anabolic steroid; an erythropoietin; an
immunization; an immunoglobulin; an immunosuppressive agent; a
growth hormone; a hormone replacement drug; a radiopharmaceutical
drug; an antidepressant; an antipsychotic drug; a stimulant; an
asthma medication; a beta agonist; an inhaled steroid; an
epinephrine; an epinephrine analog; a cytokine; and a cytokine
antagonist.
113. A binding protein according to claim 2, wherein said binding
protein is an antibody.
114. The antibody according to claim 113, wherein the antibody is
selected from the group consisting of a monoclonal antibody, a
full-length tetrameric immunoglobulin, an IgG molecule, an
IgG.sub.1 molecule, a chimeric antibody, a CDR-grafted antibody, a
humanized antibody, and an affinity matured antibody.
115. The antibody according to claim 114, wherein the antibody is a
monoclonal antibody.
116. The binding protein according to claim 36, wherein said two
variable domains have the amino acid sequences selected from the
group consisting of: SEQ ID NO:188 and SEQ ID NO:197, SEQ ID NO:190
and SEQ ID NO:197, and SEQ ID NO:191 and SEQ ID NO:197.
117. The binding protein according to claim 36, wherein said two
variable domains have the amino acid sequences of SEQ ID NO:181 and
SEQ ID NO:185.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of co-pending U.S. patent
application Ser. No. 14/798,849, filed Jul. 14, 2015, which is a
Division of U.S. patent application Ser. No. 13/037,932, filed Mar.
1, 2011, now U.S. Pat. No. 9,115,195, issued Aug. 25, 2015, which
claims the benefit of priority to U.S. Provisional Application No.
61/309,494, filed Mar. 2, 2010. The contents of each of the above
priority documents are incorporated by reference herein.
FIELD OF THE INVENTION
[0002] The present invention relates to the development and use of
DLL4 binding proteins and uses thereof in the inhibition,
prevention, and/or treatment of cancers, tumors, other
angiogenesis-dependent diseases, angiogenesis-independent diseases,
and macular degeneration and age-related macular degeneration
diseases characterized by aberrant DLL4 expression or activity.
BACKGROUND OF THE INVENTION
[0003] Cell-to-cell communication is required for many biological
processes such as differentiation, proliferation, and homeostasis.
One system utilized by a wide range of eukaryotes is the
Notch-signaling pathway. This pathway, especially the Notch
receptor, is also critical for functional tumor angiogenesis. Thus,
inhibition of Notch receptor function, blockage of the Notch
receptor, and/or blockage of the Notch-signaling pathway are
potential strategies for anti-cancer compositions and therapies.
Small molecule inhibitors of the Notch receptor have proven to be
toxic because they suppress wild type (normal) tissue expression of
Notch receptors throughout the body. Thus, different members of the
Notch-signaling pathway should be considered as potential targets
for therapeutics.
[0004] A vasculature ligand for the Notch receptor is Delta 4 or
Delta-like 4 (DLL4). Largely expressed in the vasculature, DLL4 is
critical for vascular development (Yan et al., Clin. Cancer Res.,
13(24): 7243-7246 (2007); Shutter et al., Genes Dev., 14(11):
1313-1318 (2000); Gale et al., Proc. Natl. Acad. Sci. USA, 101(45):
15949-15954 (2004); Krebs et al., Genes Dev., 14(11): 1343-1352
(2000)). Mice heterozygous for DLL4 are embryonically lethal due to
major defects in vascular development (Gale et al., Proc. Natl.
Acad. Sci. USA, 101(45): 15949-15954 (2004); Duarte et al., Genes
Dev., 18(20): 2474-2478 (2004); Krebs et al., Genes Dev., 18(20):
2469-2473 (2004)). The expression of DLL4 can be induced by VEGF
(Liu et al., Mol. Cell Biol., 23(1): 14-25 (2003); Lobov et al.,
Proc. Natl. Acad. Sci. USA, 104(9): 3219-3224 (2007)). In sum, DLL4
can negatively regulate VEGF signaling, in part through repressing
VEGFR2 and inducing VEGFR1 (Harrington et al., Microvasc. Res.,
75(2): 144-154 (2008); Suchting et al., Proc. Natl. Acad. Sci. USA,
104(9): 3225-3230 (2007)). Exquisite coordination between DLL4 and
VEGF is essential for functional angiogenesis.
[0005] In addition to its physiological role, DLL4 is up-regulated
in tumor blood vessels (Gale et al., Proc. Natl. Acad. Sci. USA,
101(45): 15949-15954 (2004); Mailhos et al., Differentiation,
69(2-3): 135-144 (2001); Patel et al., Cancer Res., 65(19):
8690-8697 (2005); Patel et al., Clin. Cancer Res., 12(16):
4836-4844 (2006); Noguera-Troise et al., Nature, 444(7122):
1032-1037 (2006)). Blockade of DLL4 potently inhibited primary
tumor growth in multiple models (Noguera-Troise et al., Nature,
444(7122): 1032-1037 (2006); Ridgway et al., Nature, 444(7122):
1083-1087 (2006); Scehnet et al., Blood, 109(11): 4753-4760
(2007)). The inhibition of DLL4 was even effective against tumors
that are resistant to anti-VEGF therapy. The combinatorial
inhibition of both DLL4 and VEGF provided an enhanced anti-tumor
activity. Interestingly, unlike VEGF inhibition that reduces tumor
vessel formation, DLL4 blockade leads to an increase in tumor
vasculature density wherein the vessels are abnormal, cannot
support efficient blood transport, and are effectively
nonfunctional. Thus, DLL4 provides a potential target for cancer
treatment.
[0006] There is a need in the art for therapeutic agents capable of
targeting the DLL4-Notch pathway and thereby inhibiting, or even
preventing, tumor angiogenesis and growth.
SUMMARY OF THE INVENTION
[0007] The invention provides proteins that bind human DLL4. DLL4
binding proteins of the invention include, but are not limited to,
rat monoclonal antibodies, chimeric antibodies, CDR-grafted
antibodies, humanized antibodies, primate-ized antibodies, affinity
matured antibodies, and fragments thereof that are capable of
binding human DLL4. Preferably, a binding protein described herein
binds human DLL4 with high affinity. More preferably, a binding
protein according to the invention is capable of neutralizing human
DLL4. The invention also provides methods of making and using DLL4
binding proteins.
[0008] One aspect of the invention provides a binding protein
capable of binding human DLL4, wherein the binding protein
comprises at least one amino acid sequence selected from the group
of amino acid sequences consisting of SEQ ID NO:157, SEQ ID NO:158,
SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID
NO:163, and SEQ ID NO:164.
[0009] An aspect of this invention pertains to a binding protein
comprising an antigen binding domain wherein the binding protein is
capable of binding human DLL4, said antigen binding domain
comprising at least one or more (i.e., two, three, four, five, or
six) CDRs wherein:
[0010] CDR-H1 is selected from the group consisting of: [0011]
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5 (SEQ ID NO:151), wherein;
[0012] X.sub.1 is N, H, or Y; [0013] X.sub.2 is F; [0014] X.sub.3
is P; [0015] X.sub.4 is M; [0016] X.sub.5 is A or 5; [0017]
residues 31-35 of SEQ ID NO:157 (CDR-H1 38H12); [0018] residues
31-35 of SEQ ID NO:161 (CDR-H1 37D10); [0019] residues 31-35 of SEQ
ID NO:163 (CDR-H1 32C7); [0020] residues 31-35 of SEQ ID NO:165
(CDR-H1 14G1); [0021] residues 31-35 of SEQ ID NO:167 (CDR-H1
14A11); [0022] residues 31-35 of SEQ ID NO:169 (CDR-H1 15D6);
[0023] residues 31-35 of SEQ ID NO:171 (CDR-H1 VH.1 1A11); [0024]
residues 31-35 of SEQ ID NO:172 (CDR-H1 VH.1a 1A11); [0025]
residues 31-35 of SEQ ID NO:173 (CDR-H1 VH.1b 1A11); [0026]
residues 31-35 of SEQ ID NO:174 (CDR-H1 VH.2a 1A11); [0027]
residues 31-35 of SEQ ID NO:179 (CDR-H1 VH.1 38H12); [0028]
residues 31-35 of SEQ ID NO:180 (CDR-H1 VH.1A 38H12); [0029]
residues 31-35 of SEQ ID NO:181 (CDR-H1 VH.1b 38H12); [0030]
residues 31-35 of SEQ ID NO:182 (CDR-H1 VH.2a 38H12); [0031]
residues 31-35 of SEQ ID NO:187 (CDR-H1 h1A11VH.1); [0032] residues
31-35 of SEQ ID NO:188 (CDR-H1 h1A11.A6); [0033] residues 31-35 of
SEQ ID NO:189 (CDR-H1 h1A11.A8); [0034] residues 31-35 of SEQ ID
NO:190 (CDR-H1 h1A11.C6); [0035] residues 31-35 of SEQ ID NO:191
(CDR-H1 h1A11.A11); [0036] residues 31-35 of SEQ ID NO:192 (CDR-H1
h1A11.B5); [0037] residues 31-35 of SEQ ID NO:193 (CDR-H1
h1A11.E12); [0038] residues 31-35 of SEQ ID NO:194 (CDR-H1
h1A11.G3); [0039] residues 31-35 of SEQ ID NO:195 (CDR-H1
h1A11.F5); and [0040] residues 31-35 of SEQ ID NO:196 (CDR-H1
h1A11.H2);
[0041] CDR-H2 is selected from the group consisting of: [0042]
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X-
.sub.10-X.sub.11-X.sub.12-X.sub.13-X.sub.14-X.sub.15-X.sub.16-X.sub.17
(SEQ ID NO:152), wherein; [0043] X.sub.1 is T or S; [0044] X.sub.2
is I; [0045] X.sub.3 is S; [0046] X.sub.4 is S or G; [0047] X.sub.5
is S; [0048] X.sub.6 is D; [0049] X.sub.7 is G, A, D, S, or E;
[0050] X.sub.8 is T or W; [0051] X.sub.9 is T, P, or A; [0052]
X.sub.10 is Y, S, T, or N; [0053] X.sub.11 is Y or I; [0054]
X.sub.12 is R or G; [0055] X.sub.13 is D; [0056] X.sub.14 is S;
[0057] X.sub.15 is V; [0058] X.sub.16 is K; and [0059] X.sub.17 is
G; [0060] residues 50-66 of SEQ ID NO:157 (CDR-H2 38H12); [0061]
residues 50-68 of SEQ ID NO:161 (CDR-H2 37D10); [0062] residues
50-66 of SEQ ID NO:163 (CDR-H2 32C7); [0063] residues 50-66 of SEQ
ID NO:165 (CDR-H2 14G1); [0064] residues 50-66 of SEQ ID NO:167
(CDR-H2 14A11); [0065] residues 50-66 of SEQ ID NO:169 (CDR-H2
15D6); [0066] residues 50-66 of SEQ ID NO:171 (CDR-H2 VH.1 1A11);
[0067] residues 50-66 of SEQ ID NO:172 (CDR-H2 VH.1a 1A11); [0068]
residues 50-66 of SEQ ID NO:173 (CDR-H2 VH.1b 1A11); [0069]
residues 50-66 of SEQ ID NO:174 (CDR-H2 VH.2a 1A11); [0070]
residues 50-66 of SEQ ID NO:179 (CDR-H2 VH.1 38H12); [0071]
residues 50-66 of SEQ ID NO:180 (CDR-H2 VH.1A 38H12); [0072]
residues 50-66 of SEQ ID NO:181 (CDR-H2 VH.1b 38H12); [0073]
residues 31-35 of SEQ ID NO:182 (CDR-H1 VH.2a 38H12); [0074]
residues 50-66 of SEQ ID NO:187 (CDR-H2 h1A11VH.1); [0075] residues
50-66 of SEQ ID NO:188 (CDR-H2 h1A11.A6); [0076] residues 50-66 of
SEQ ID NO:189 (CDR-H2 h1A11.A8); [0077] residues 50-66 of SEQ ID
NO:190 (CDR-H2 h1A11.C6); [0078] residues 50-66 of SEQ ID NO:191
(CDR-H2 h1A11.A11); [0079] residues 50-66 of SEQ ID NO:192 (CDR-H2
h1A11.B5); [0080] residues 50-66 of SEQ ID NO:193 (CDR-H2
h1A11.E12); [0081] residues 50-66 of SEQ ID NO:194 (CDR-H2
h1A11.G3); [0082] residues 50-66 of SEQ ID NO:195 (CDR-H2
h1A11.F5); and [0083] residues 50-66 of SEQ ID NO:196 (CDR-H2
h1A11.H2); CDR-H3 is selected from the group consisting of: [0084]
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9
(SEQ ID NO:153), wherein; [0085] X.sub.1 is G; [0086] X.sub.2 is Y;
[0087] X.sub.3 is Y; [0088] X.sub.4 is N; [0089] X.sub.5 is S;
[0090] X.sub.6 is P; [0091] X.sub.7 is F; [0092] X.sub.8 is A; and
[0093] X.sub.9 is Y, F, or S; [0094] residues 99-107 of SEQ ID
NO:157 (CDR-H3 38H12); [0095] residues 101-111 of SEQ ID NO:161
(CDR-H3 37D10); [0096] residues 99-105 of SEQ ID NO:163 (CDR-H3
32C7); [0097] residues 99-105 of SEQ ID NO:165 (CDR-H3 14G1);
[0098] residues 99-110 of SEQ ID NO:167 (CDR-H3 14A11); [0099]
residues 99-110 of SEQ ID NO:169 (CDR-H3 15D6); [0100] residues
99-107 of SEQ ID NO:171 (CDR-H3 VH.1 1A11); [0101] residues 99-107
of SEQ ID NO:172 (CDR-H3 VH.1a 1A11); [0102] residues 99-107 of SEQ
ID NO:173 (CDR-H3 VH.1b 1A11); [0103] residues 99-107 of SEQ ID
NO:174 (CDR-H3 VH.2a 1A11); [0104] residues 99-107 of SEQ ID NO:179
(CDR-H3 VH.1 38H12); [0105] residues 99-107 of SEQ ID NO:180
(CDR-H3 VH.1A 38H12); [0106] residues 99-107 of SEQ ID NO:181
(CDR-H2 VH.1b 38H12); [0107] residues 99-107 of SEQ ID NO:182
(CDR-H1 VH.2a 38H12); [0108] residues 99-107 of SEQ ID NO:187
(CDR-H3 h1A11VH.1); [0109] residues 99-107 of SEQ ID NO:188 (CDR-H3
h1A11.A6); [0110] residues 99-107 of SEQ ID NO:189 (CDR-H3
h1A11.A8); [0111] residues 99-107 of SEQ ID NO:190 (CDR-H3
h1A11.C6); [0112] residues 99-107 of SEQ ID NO:191 (CDR-H3
h1A11.A11); [0113] residues 99-107 of SEQ ID NO:192 (CDR-H3
h1A11.B5); [0114] residues 99-107 of SEQ ID NO:193 (CDR-H3
h1A11.E12); [0115] residues 99-107 of SEQ ID NO:194 (CDR-H3
h1A11.G3); [0116] residues 99-107 of SEQ ID NO:195 (CDR-H3
h1A11.F5); and [0117] residues 99-107 of SEQ ID NO:196 (CDR-H3
h1A11.H2);
[0118] CDR-L1 is selected from the group consisting of: [0119]
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X-
.sub.10-X.sub.11 (SEQ ID NO:154), wherein; [0120] X.sub.1 is R;
[0121] X.sub.2 is A; [0122] X.sub.3 is S; [0123] X.sub.4 is E or Q;
[0124] X.sub.5 is D or E; [0125] X.sub.6 is I; [0126] X.sub.7 is Y
or W; [0127] X.sub.8 is S, I, Y, N, or R; [0128] X.sub.9 is N;
[0129] X.sub.10 is L; and [0130] X.sub.11 is A; [0131] residues
24-34 of SEQ ID NO:158 (CDR-L1 38H12); [0132] residues 24-34 of SEQ
ID NO:162 (CDR-L1 37D10); [0133] residues 24-34 of SEQ ID NO:164
(CDR-L1 32C7); [0134] residues 24-34 of SEQ ID NO:166 (CDR-L1
14G1); [0135] residues 23-37 of SEQ ID NO:168 (CDR-L1 14A11);
[0136] residues 23-37 of SEQ ID NO:170 (CDR-L1 15D6); [0137]
residues 24-34 of SEQ ID NO:175 (CDR-L1 VL.1 1A11); [0138] residues
24-34 of SEQ ID NO:176 (CDR-L1 VL.1a 1A11); [0139] residues 24-34
of SEQ ID NO:177 (CDR-L1 VL.1b 1A11); [0140] residues 24-34 of SEQ
ID NO:178 (CDR-L1 VL.2a 1A11); [0141] residues 24-34 of SEQ ID
NO:183 (CDR-L1 VL.1 38H12); [0142] residues 24-34 of SEQ ID NO:184
(CDR-L1 VL.1a 38H12); [0143] residues 24-34 of SEQ ID NO:185
(CDR-L1 VL.1b 38H12); [0144] residues 24-34 of SEQ ID NO:186
(CDR-L1 VL.2a 38H12); [0145] residues 24-34 of SEQ ID NO:197
(CDR-L1 h1A11VL.1); [0146] residues 24-34 of SEQ ID NO:198 (CDR-L1
h1A11.A2); [0147] residues 24-34 of SEQ ID NO:199 (CDR-L1
h1A11.A12); [0148] residues 24-34 of SEQ ID NO:200 (CDR-L1
h1A11.A7); [0149] residues 24-34 of SEQ ID NO:201 (CDR-L1
h1A11.B4); [0150] residues 24-34 of SEQ ID NO:202 (CDR-L1
h1A11.B5); and [0151] residues 24-34 of SEQ ID NO:203 (CDR-L1
h1A11.E12);
[0152] CDR-L2 is selected from group consisting of: [0153]
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7 (SEQ ID
NO:155), wherein; [0154] X.sub.1 is D; [0155] X.sub.2 is T; [0156]
X.sub.3 is N or S; [0157] X.sub.4 is N, D, S, I, Y, or V; [0158]
X.sub.5 is L; [0159] X.sub.6 is A; and [0160] X.sub.7 is D; [0161]
residues 50-56 of SEQ ID NO:158 (CDR-L2 38H12); [0162] residues
50-56 of SEQ ID NO:162 (CDR-L2 37D10); [0163] residues 50-56 of SEQ
ID NO:164 (CDR-L2 32C7); [0164] residues 50-56 of SEQ ID NO:166
(CDR-L2 14G1); [0165] residues 53-59 of SEQ ID NO:168 (CDR-L2
14A11); [0166] residues 53-59 of SEQ ID NO:170 (CDR-L2 15D6);
[0167] residues 50-56 of SEQ ID NO:175 (CDR-L2 VL.1 1A11); [0168]
residues 50-56 of SEQ ID NO:176 (CDR-L2 VL.1a 1A11); [0169]
residues 50-56 of SEQ ID NO:177 (CDR-L2 VL.1b 1A11); [0170]
residues 50-56 of SEQ ID NO:178 (CDR-L2 VL.2a 1A11); [0171]
residues 50-56 of SEQ ID NO:183 (CDR-L2 VL.1 38H12); [0172]
residues 50-56 of SEQ ID NO:184 (CDR-L2 VL.1a 38H12); [0173]
residues 50-56 of SEQ ID NO:185 (CDR-L2 VL.1b 38H12); [0174]
residues 50-56 of SEQ ID NO:186 (CDR-L2 VL.2a 38H12); [0175]
residues 50-56 of SEQ ID NO:197 (CDR-L2 h1A11VL.1); [0176] residues
50-56 of SEQ ID NO:198 (CDR-L2 h1A11.A2); [0177] residues 50-56 of
SEQ ID NO:199 (CDR-L2 h1A11.A12); [0178] residues 50-56 of SEQ ID
NO:200 (CDR-L2 h1A11.A7); [0179] residues 50-56 of SEQ ID NO:201
(CDR-L2 h1A11.B4); [0180] residues 50-56 of SEQ ID NO:202 (CDR-L2
h1A11.B5); and [0181] residues 50-56 of SEQ ID NO:203 (CDR-L2
h1A11.E12);
[0182] and
[0183] CDR-L3 is selected from the group consisting of: [0184]
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9
(SEQ ID NO:156), wherein; [0185] X.sub.1 is Q; [0186] X.sub.2 is Q;
[0187] X.sub.3 is Y; [0188] X.sub.4 is N, D, or T; [0189] X.sub.5
is N, Y, or W; [0190] X.sub.6 is Y or V; [0191] X.sub.7 is P;
[0192] X.sub.8 is P; and [0193] X.sub.9 is T. [0194] residues 89-97
of SEQ ID NO:158 (CDR-L3 38H12); [0195] residues 89-97 of SEQ ID
NO:162 (CDR-L3 37D10); [0196] residues 89-97 of SEQ ID NO:164
(CDR-L3 32C7); [0197] residues 89-98 of SEQ ID NO:166 (CDR-L3
14G1); [0198] residues 92-100 of SEQ ID NO:168 (CDR-L3 14A11);
[0199] residues 92-100 of SEQ ID NO:170 (CDR-L3 15D6); [0200]
residues 89-97 of SEQ ID NO:175 (CDR-L3 VL.1 1A11); [0201] residues
89-97 of SEQ ID NO:176 (CDR-L3 VL.1a 1A11); [0202] residues 89-97
of SEQ ID NO:177 (CDR-L3 VL.1b 1A11); [0203] residues 89-97 of SEQ
ID NO:178 (CDR-L3 VL.2a 1A11); [0204] residues 89-97 of SEQ ID
NO:183 (CDR-L3 VL.1 38H12); [0205] residues 89-97 of SEQ ID NO:184
(CDR-L3 VL.1a 38H12); [0206] residues 89-97 of SEQ ID NO:185
(CDR-L3 VL.1b 38H12); [0207] residues 89-97 of SEQ ID NO:186
(CDR-L3 VL.2a 38H12); [0208] residues 89-97 of SEQ ID NO:197
(CDR-L3 h1A11VL.1); [0209] residues 89-97 of SEQ ID NO:198 (CDR-L3
h1A11.A2); [0210] residues 89-97 of SEQ ID NO:199 (CDR-L3
h1A11.A12); [0211] residues 89-97 of SEQ ID NO:200 (CDR-L3
h1A11.A7); [0212] residues 89-97 of SEQ ID NO:201 (CDR-L3
h1A11.B4); [0213] residues 89-97 of SEQ ID NO:202 (CDR-L3
h1A11.B5); [0214] residues 89-97 of SEQ ID NO:203 (CDR-L3
h1A11.E12). [0215] Preferably, a DLL4 binding protein of the
invention comprises at least one CDR comprising an amino acid
sequence selected from the group consisting of: [0216] residues
31-35 of SEQ ID NO:157 (CDR-H1 38H12); residues 50-66 of SEQ ID
NO:157 (CDR-H2 38H12); residues 99-107 of SEQ ID NO:157 (CDR-H3
38H12); residues 24-34 of SEQ ID NO:158 (CDR-L1 38H12); residues
50-56 of SEQ ID NO:158 (CDR-L2 38H12); residues 89-97 of SEQ ID
NO:158 (CDR-L3 38H12); residues 31-35 of SEQ ID NO:159 (CDR-H1
1A11); residues 50-66 of SEQ ID NO:159 (CDR-H2 1A11); residues
99-107 of SEQ ID NO:159 (CDR-H3 1A11); residues 24-34 of SEQ ID
NO:160 (CDR-L1 1A11); residues 50-56 of SEQ ID NO:160 (CDR-L2
1A11); residues 89-97 of SEQ ID NO:160 (CDR-L3 1A11); residues
31-35 of SEQ ID NO:161 (CDR-H1 37D10); residues 50-68 of SEQ ID
NO:161 (CDR-H2 37D10); residues 101-111 of SEQ ID NO:161 (CDR-H3
37D10); [0217] residues 24-34 of SEQ ID NO:162 (CDR-L1 37D10);
residues 50-56 of SEQ ID NO:162 (CDR-L2 37D10); residues 89-97 of
SEQ ID NO:162 (CDR-L3 37D10); residues 31-35 of SEQ ID NO:163
(CDR-H1 32C7); residues 50-66 of SEQ ID NO:163 (CDR-H2 32C7);
residues 99-105 of SEQ ID NO:163 (CDR-H3 32C7); residues 24-34 of
SEQ ID NO:164 (CDR-L1 32C7); residues 50-56 of SEQ ID NO:164
(CDR-L2 32C7); residues 89-98 of SEQ ID NO:164 (CDR-L3 32C7);
residues 31-35 of SEQ ID NO:165 (CDR-H1 14G1); residues 50-66 of
SEQ ID NO:165 (CDR-H2 14G1); residues 99-105 of SEQ ID NO:165
(CDR-H3 14G1); residues 24-34 of SEQ ID NO:166 (CDR-L1 14G1);
residues 50-56 of SEQ ID NO:166 (CDR-L2 14G1); residues 89-98 of
SEQ ID NO:166 (CDR-L3 14G1); residues 31-35 of SEQ ID NO:167
(CDR-H1 14A11); residues 50-66 of SEQ ID NO:167 (CDR-H2 14A11);
residues 99-110 of SEQ ID NO:167 (CDR-H3 14A11); residues 23-37 of
SEQ ID NO:168 (CDR-L1 14A11); residues 53-59 of SEQ ID NO:168
(CDR-L2 14A11); residues 92-100 of SEQ ID NO:168 (CDR-L3 14A11);
residues 31-35 of SEQ ID NO:169 (CDR-H1 15D6); residues 50-66 of
SEQ ID NO:169 (CDR-H2 15D6); residues 99-110 of SEQ ID NO:169
(CDR-H3 15D6); residues 23-37 of SEQ ID NO:170 (CDR-L1 15D6);
residues 53-59 of SEQ ID NO:170 (CDR-L2 15D6); residues 92-100 of
SEQ ID NO:170 (CDR-L3 15D6); residues 31-35 of SEQ ID NO:171
(CDR-H1 VH.1 1A11); residues 50-66 of SEQ ID NO:171 (CDR-H2 VH.1
1A11); residues 99-107 of SEQ ID NO:171 (CDR-H3 VH.1 1A11);
residues 31-35 of SEQ ID NO:172 (CDR-H1 VH.1a 1A11); residues 50-66
of SEQ ID NO:172 (CDR-H2 VH.1a 1A11); residues 99-107 of SEQ ID
NO:172 (CDR-H3 VH.1a 1A11); residues 31-35 of SEQ ID NO:173 (CDR-H1
VH.1b 1A11); residues 50-66 of SEQ ID NO:173 (CDR-H2 VH.1b 1A11);
residues 99-107 of SEQ ID NO:173 (CDR-H3 VH.1b 1A11); residues
31-35 of SEQ ID NO:174 (CDR-H1 VH.2a 1A11); residues 50-66 of SEQ
ID NO:174 (CDR-H2 VH.2a 1A11); residues 99-107 of SEQ ID NO:174
(CDR-H3 VH.2a 1A11); residues 24-34 of SEQ ID NO:175 (CDR-L1 VL.1
1A11); residues 50-56 of SEQ ID NO:175 (CDR-L2 VL.1 1A11); residues
89-97 of SEQ ID NO:175 (CDR-L3 VL.1 1A11); residues 24-34 of SEQ ID
NO:176 (CDR-L1 VL.1a 1A11); residues 50-56 of SEQ ID NO:176 (CDR-L2
VL.1a 1A11); residues 89-97 of SEQ ID NO:176 (CDR-L3 VL.1a 1A11);
residues 24-34 of SEQ ID NO:177 (CDR-L1 VL.1b 1A11); residues 50-56
of SEQ ID NO:177 (CDR-L2 VL.1b 1A11); residues 89-97 of SEQ ID
NO:177 (CDR-L3 VL.1b 1A11); residues 24-34 of SEQ ID NO:178 (CDR-L1
VL.2a 1A11); residues 50-56 of SEQ ID NO:178 (CDR-L2 VL.2a 1A11);
residues 89-97 of SEQ ID NO:178 (CDR-L3 VL.2a 1A11); residues 31-35
of SEQ ID NO:179 (CDR-H1 VH.1 38H12); residues 50-66 of SEQ ID
NO:179 (CDR-H2 VH.1 38H12); residues 99-107 of SEQ ID NO:179
(CDR-H3 VH.1 38H12); residues 31-35 of SEQ ID NO:180 (CDR-H1 VH.1A
38H12); residues 50-66 of SEQ ID NO:180 (CDR-H2 VH.1A 38H12);
residues 99-107 of SEQ ID NO:180 (CDR-H3 VH.1A 38H12); residues
31-35 of SEQ ID NO:181 (CDR-H1 VH.1b 38H12); residues 50-66 of SEQ
ID NO:181 (CDR-H2 VH.1b 38H12); residues 99-107 of SEQ ID NO:181
(CDR-H3 VH.1b 38H12); residues 31-35 of SEQ ID NO:182 (CDR-H1 VH.2a
38H12); residues 50-66 of SEQ ID NO:182 (CDR-H2 VH.2a 38H12);
residues 99-107 of SEQ ID NO:182 (CDR-H3 VH.2a 38H12); residues
24-34 of SEQ ID NO:183 (CDR-L1 VL.1 38H12); residues 50-56 of SEQ
ID NO:183 (CDR-L2 VL.1 38H12); residues 89-97 of SEQ ID NO:183
(CDR-L3 VL.1 38H12); residues 24-34 of SEQ ID NO:184 (CDR-L1 VL.1a
38H12); residues 50-56 of SEQ ID NO:184 (CDR-L2 VL.1a 38H12);
residues 89-97 of SEQ ID NO:184 (CDR-L3 VL.1a 38H12); residues
24-34 of SEQ ID NO:185 (CDR-L1 VL.1b 38H12); residues 50-56 of SEQ
ID NO:185 (CDR-L2 VL.1b 38H12); residues 89-97 of SEQ ID NO:185
(CDR-L3 VL.1b 38H12); residues 24-34 of SEQ ID NO:186 (CDR-L1 VL.2a
38H12); residues 50-56 of SEQ ID NO:186 (CDR-L2 VL.2a 38H12);
residues 89-97 of SEQ ID NO:186 (CDR-L3 VL.2a 38H12); residues
31-35 of SEQ ID NO:187 (CDR-H1 h1A11VH.1), residues 50-66 of SEQ ID
NO:187 (CDR-H2 h1A11VH.1); residues 99-107 of SEQ ID NO:187 (CDR-H3
h1A11VH.1); residues 31-35 of SEQ ID NO:188 (CDR-H1 h1A11.A6),
residues 50-66 of SEQ ID NO:188 (CDR-H2 h1A11.A6); residues 99-107
of SEQ ID NO:188 (CDR-H3 h1A11.A6); residues 31-35 of SEQ ID NO:189
(CDR-H1 h1A11.A8), residues 50-66 of SEQ ID NO:189 (CDR-H2
h1A11.A8); residues 99-107 of SEQ ID NO:189 (CDR-H3 h1A11.A8);
residues 31-35 of SEQ ID NO:190 (CDR-H1 h1A11.C6), residues 50-66
of SEQ ID NO:190 (CDR-H2 h1A11.C6); residues 99-107 of SEQ ID
NO:190 (CDR-H3 h1A11.C6); residues 31-35 of SEQ ID NO:191 (CDR-H1
h1A11.A11), residues 50-66 of SEQ ID NO:191 (CDR-H2 h1A11.A11);
residues 99-107 of SEQ ID NO:191 (CDR-H3 h1A11.A11); residues 31-35
of SEQ ID NO:192 (CDR-H1 h1A11.B5), residues 50-66 of SEQ ID NO:192
(CDR-H2 h1A11.B5); residues 99-107 of SEQ ID NO:192 (CDR-H3
h1A11.B5); residues 31-35 of SEQ ID NO:193 (CDR-H1 h1A11.E12),
residues 50-66 of SEQ ID NO:193 (CDR-H2 h1A11.E12); residues 99-107
of SEQ ID NO:193 (CDR-H3 h1A11.E12); residues 31-35 of SEQ ID
NO:194 (CDR-H1 h1A11.G3), residues 50-66 of SEQ ID NO:194 (CDR-H2
h1A11.G3); residues 99-107 of SEQ ID NO:194 (CDR-H3 h1A11.G3);
residues 31-35 of SEQ ID NO:195 (CDR-H1 h1A11.F5), residues 50-66
of SEQ ID NO:195 (CDR-H2 h1A11.F5); residues 99-107 of SEQ ID
NO:195 (CDR-H3 h1A11.F5); residues 31-35 of SEQ ID NO:196 (CDR-H1
h1A11.H2), residues 50-66 of SEQ ID NO:196 (CDR-H2 h1A11.H2);
residues 99-107 of SEQ ID NO:196 (CDR-H3 h1A11.H2); residues 24-34
of SEQ ID NO:197 (CDR-L1 h1A11VL.1), residues 50-56 of SEQ ID
NO:197 (CDR-L2 h1A11VL.1); residues 89-97 of SEQ ID NO:197 (CDR-L3
h1A11VL.1); residues 24-34 of SEQ ID NO:198 (CDR-L1 h1A11.A2),
residues 50-56 of SEQ ID NO:198 (CDR-L2 h1A11.A2); residues 89-97
of SEQ ID NO:198 (CDR-L3 h1A11.A2); residues 24-34 of SEQ ID NO:199
(CDR-L1 h1A11.A12), residues 50-56 of SEQ ID NO:199 (CDR-L2
h1A11.A12); residues 89-97 of SEQ ID NO:199 (CDR-L3 h1A11.A12);
residues 24-34 of SEQ ID NO:200 (CDR-L1 h1A11.A7), residues 50-56
of SEQ ID NO:200 (CDR-L2 h1A11.A7); residues 89-97 of SEQ ID NO:200
(CDR-L3 h1A11.A7); residues 24-34 of SEQ ID NO:201 (CDR-L1
h1A11.B4), residues 50-56 of SEQ ID NO:201 (CDR-L2 h1A11.B4);
residues 89-97 of SEQ ID NO:201 (CDR-L3 h1A11.B4); residues 24-34
of SEQ ID NO:202 (CDR-L1 h1A11.B5), residues 50-56 of SEQ ID NO:202
(CDR-L2 h1A11.B5); residues 89-97 of SEQ ID NO:202 (CDR-L3
h1A11.B5); residues 24-34 of SEQ ID NO:203 (CDR-L1 h1A11.E12),
residues 50-56 of SEQ ID NO:203 (CDR-L2 h1A11.E12); and residues
89-97 of SEQ ID NO:203 (CDR-L3 h1A11.E12).
[0218] In an embodiment, a DLL4 binding protein of the invention
comprises at least three CDRs described herein (above or below). In
a non-limiting example, a DLL4 binding protein of the invention
comprises three CDRs described herein, wherein the three CDRs are a
CDR-H1, a CDR-H2, and a CDR-H3 as described herein. In another
non-limiting example, a DLL4 binding protein of the invention
comprising three CDRs described herein, wherein the three CDRs are
a CDR-L1, a CDR-L2, and a CDR-L3 as described herein.
[0219] In an embodiment, a DLL4 binding protein of the invention
comprises one or more CDRs described herein (above or below), such
as one, two, three, four, five, or six CDRs described herein. In a
preferred embodiment, a DLL4 binding protein according to the
invention comprises six CDRs described herein, e.g., a CDR-H1, a
CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2, and a CDR-L3 as described
herein.
[0220] In another embodiment, a DLL4 binding protein of the
invention comprises three CDRs selected from a set of variable
domain CDRs, wherein the set of variable domain CDRs is selected
from the group of variable domain CDR sets consisting of:
[0221] VH 38H12 CDR Set
[0222] CDR-H1: residues 31-35 of SEQ ID NO:157
[0223] CDR-H2: residues 50-66 of SEQ ID NO:157
[0224] CDR-H3 residues 99-107 of SEQ ID NO:157
[0225] VL 38H12 CDR Set
[0226] CDR-L1: residues 24-34 of SEQ ID NO:158
[0227] CDR-L2: residues 50-56 of SEQ ID NO:158
[0228] CDR-L3: residues 89-97 of SEQ ID NO:158
[0229] VH 1A11 CDR Set
[0230] CDR-H1: residues 31-35 of SEQ ID NO:159
[0231] CDR-H2: residues 50-66 of SEQ ID NO:159
[0232] CDR-H3: residues 99-107 of SEQ ID NO:159
[0233] VL 1A11 CDR Set
[0234] CDR-L1: residues 24-34 of SEQ ID NO:160
[0235] CDR-L2: residues 50-56 of SEQ ID NO:160
[0236] CDR-L3: residues 89-97 of SEQ ID NO:160
[0237] VH 37D10 CDR Set
[0238] CDR-H1: residues 31-35 of SEQ ID NO:161
[0239] CDR-H2: residues 50-68 of SEQ ID NO:161
[0240] CDR-H3: residues 101-111 of SEQ ID NO:161
[0241] VL 37D10 CDR Set
[0242] CDR-L1: residues 24-34 of SEQ ID NO:162
[0243] CDR-L2: residues 50-56 of SEQ ID NO:162
[0244] CDR-L3: residues 89-97 of SEQ ID NO:162
[0245] VH 32C7 CDR Set
[0246] CDR-H1: residues 31-35 of SEQ ID NO:163
[0247] CDR-H2: residues 50-66 of SEQ ID NO:163
[0248] CDR-H3: residues 99-105 of SEQ ID NO:163
[0249] VL 32C7 CDR Set
[0250] CDR-L1: residues 24-34 of SEQ ID NO:164
[0251] CDR-L2: residues 50-56 of SEQ ID NO:164
[0252] CDR-L3: residues 89-98 of SEQ ID NO:164
[0253] VH 14G1 CDR Set
[0254] CDR-H1: residues 31-35 of SEQ ID NO:165
[0255] CDR-H2: residues 50-66 of SEQ ID NO:165
[0256] CDR-H3: residues 99-105 of SEQ ID NO:165
[0257] VL 14G1 CDR Set
[0258] CDR-L1: residues 24-34 of SEQ ID NO:166
[0259] CDR-L2: residues 50-56 of SEQ ID NO:166
[0260] CDR-L3: residues 89-97 of SEQ ID NO:166
[0261] VH 14A11 CDR Set
[0262] CDR-H1: residues 31-35 of SEQ ID NO:167
[0263] CDR-H2: residues 50-66 of SEQ ID NO:167
[0264] CDR-H3: residues 99-110 of SEQ ID NO:167
[0265] VL 14A11 CDR Set
[0266] CDR-L1: residues 23-37 of SEQ ID NO:168
[0267] CDR-L2: residues 53-59 of SEQ ID NO:168
[0268] CDR-L3: residues 92-100 of SEQ ID NO:168
[0269] VH 15D6 CDR Set
[0270] CDR-H1: residues 31-35 of SEQ ID NO:169
[0271] CDR-H2: residues 50-66 of SEQ ID NO:169
[0272] CDR-H3: residues 99-110 of SEQ ID NO:169
[0273] VL 15D6 CDR Set
[0274] CDR-L1: residues 23-37 of SEQ ID NO:170
[0275] CDR-L2: residues 53-59 of SEQ ID NO:170
[0276] CDR-L3: residues 92-100 of SEQ ID NO:170
[0277] VH VH.1 1A11 CDR Set
[0278] CDR-H1: residues 31-35 of SEQ ID NO:171
[0279] CDR-H2: residues 50-66 of SEQ ID NO:171
[0280] CDR-H3: residues 99-107 of SEQ ID NO:171
[0281] VH VH.1a 1A11 CDR Set
[0282] CDR-H1: residues 31-35 of SEQ ID NO:172
[0283] CDR-H2: residues 50-66 of SEQ ID NO:172
[0284] CDR-H3: residues 99-107 of SEQ ID NO:172
[0285] VH VH.1b 1A11 CDR Set
[0286] CDR-H1: residues 31-35 of SEQ ID NO:173
[0287] CDR-H2: residues 50-66 of SEQ ID NO:173
[0288] CDR-H3: residues 99-107 of SEQ ID NO:173
[0289] VH VH.2a 1A11 CDR Set
[0290] CDR-H1: residues 31-35 of SEQ ID NO:174
[0291] CDR-H2: residues 50-66 of SEQ ID NO:174
[0292] CDR-H3: residues 99-107 of SEQ ID NO:174
[0293] VL VL.1 1A11 CDR Set
[0294] CDR-L1: residues 24-34 of SEQ ID NO:175
[0295] CDR-L2: residues 50-56 of SEQ ID NO:175
[0296] CDR-L3: residues 89-97 of SEQ ID NO:175
[0297] VL VL.1a 1A11 CDR Set
[0298] CDR-L1: residues 24-34 of SEQ ID NO:176
[0299] CDR-L2: residues 50-56 of SEQ ID NO:176
[0300] CDR-L3: residues 89-97 of SEQ ID NO:176
[0301] VL VL.1b 1A11 CDR Set
[0302] CDR-L1: residues 24-34 of SEQ ID NO:177
[0303] CDR-L2: residues 50-56 of SEQ ID NO:177
[0304] CDR-L3: residues 89-97 of SEQ ID NO:177
[0305] VL VL.2a 1A11 CDR Set
[0306] CDR-L1: residues 24-34 of SEQ ID NO:178
[0307] CDR-L2: residues 50-56 of SEQ ID NO:178
[0308] CDR-L3: residues 89-97 of SEQ ID NO:178
[0309] VH VH.1 38H12 CDR Set
[0310] CDR-H1: residues 31-35 of SEQ ID NO:179
[0311] CDR-H2: residues 50-66 of SEQ ID NO:179
[0312] CDR-H3: residues 99-107 of SEQ ID NO:179
[0313] VH VH.1a 38H12 CDR Set
[0314] CDR-H1: residues 31-35 of SEQ ID NO:180
[0315] CDR-H2: residues 50-66 of SEQ ID NO:180
[0316] CDR-H3: residues 99-107 of SEQ ID NO:180
[0317] VH VH.1b 38H12 CDR Set
[0318] CDR-H1: residues 31-35 of SEQ ID NO:181
[0319] CDR-H2: residues 50-66 of SEQ ID NO:181
[0320] CDR-H3: residues 99-107 of SEQ ID NO:181
[0321] VH VH.2a 38H12 CDR Set
[0322] CDR-H1: residues 31-35 of SEQ ID NO:182
[0323] CDR-H2: residues 50-66 of SEQ ID NO:182
[0324] CDR-H3: residues 99-107 of SEQ ID NO:182
[0325] VL VL.1 38H12 CDR Set
[0326] CDR-L1: residues 24-34 of SEQ ID NO:183
[0327] CDR-L2: residues 50-56 of SEQ ID NO:183
[0328] CDR-L3: residues 89-97 of SEQ ID NO:183
[0329] VL VL.1a 38H12 CDR Set
[0330] CDR-L1: residues 24-34 of SEQ ID NO:184
[0331] CDR-L2: residues 50-56 of SEQ ID NO:184
[0332] CDR-L3: residues 89-97 of SEQ ID NO:184
[0333] VL VL.1b 38H12 CDR Set
[0334] CDR-L1: residues 24-34 of SEQ ID NO:185
[0335] CDR-L2: residues 50-56 of SEQ ID NO:185
[0336] CDR-L3: residues 89-97 of SEQ ID NO:185
[0337] VL VL.2a 38H12 CDR Set
[0338] CDR-L1: residues 24-34 of SEQ ID NO:186
[0339] CDR-L2: residues 50-56 of SEQ ID NO:186
[0340] CDR-L3: residues 89-97 of SEQ ID NO:186
[0341] VH hA11VH.1 CDR Set
[0342] CDR-H1: residues 31-35 of SEQ ID NO:187
[0343] CDR-H2: residues 50-66 of SEQ ID NO:187
[0344] CDR-H3: residues 99-107 of SEQ ID NO:187
[0345] VH hA11.A6 CDR Set
[0346] CDR-H1: residues 31-35 of SEQ ID NO:188
[0347] CDR-H2: residues 50-66 of SEQ ID NO:188
[0348] CDR-H3: residues 99-107 of SEQ ID NO:188
[0349] VH hA11.A8 CDR Set
[0350] CDR-H1: residues 31-35 of SEQ ID NO:189
[0351] CDR-H2: residues 50-66 of SEQ ID NO:189
[0352] CDR-H3: residues 99-107 of SEQ ID NO:189
[0353] VH hA11.C6 CDR Set
[0354] CDR-H1: residues 31-35 of SEQ ID NO:190
[0355] CDR-H2: residues 50-66 of SEQ ID NO:190
[0356] CDR-H3: residues 99-107 of SEQ ID NO:190
[0357] VH hA11.A11 CDR Set
[0358] CDR-H1: residues 31-35 of SEQ ID NO:191
[0359] CDR-H2: residues 50-66 of SEQ ID NO:191
[0360] CDR-H3: residues 99-107 of SEQ ID NO:191
[0361] VH hA11.B5 CDR Set
[0362] CDR-H1: residues 31-35 of SEQ ID NO:192
[0363] CDR-H2: residues 50-66 of SEQ ID NO:192
[0364] CDR-H3: residues 99-107 of SEQ ID NO:192
[0365] VH hA11.E12 CDR Set
[0366] CDR-H1: residues 31-35 of SEQ ID NO:193
[0367] CDR-H2: residues 50-66 of SEQ ID NO:193
[0368] CDR-H3: residues 99-107 of SEQ ID NO:193
[0369] VH hA11.G3 CDR Set
[0370] CDR-H1: residues 31-35 of SEQ ID NO:194
[0371] CDR-H2: residues 50-66 of SEQ ID NO:194
[0372] CDR-H3: residues 99-107 of SEQ ID NO:194
[0373] VH hA11.F5 CDR Set
[0374] CDR-H1: residues 31-35 of SEQ ID NO:195
[0375] CDR-H2: residues 50-66 of SEQ ID NO:195
[0376] CDR-H3: residues 99-107 of SEQ ID NO:195
[0377] VH hA11.H2 CDR Set
[0378] CDR-H1: residues 31-35 of SEQ ID NO:196
[0379] CDR-H2: residues 50-66 of SEQ ID NO:196
[0380] CDR-H3: residues 99-107 of SEQ ID NO:196
[0381] VL h1A11VL.1 CDR Set
[0382] CDR-L1: residues 24-34 of SEQ ID NO:197
[0383] CDR-L2: residues 50-56 of SEQ ID NO:197
[0384] CDR-L3: residues 89-97 of SEQ ID NO:197
[0385] VL h1A11.A2 CDR Set
[0386] CDR-L1: residues 24-34 of SEQ ID NO:198
[0387] CDR-L2: residues 50-56 of SEQ ID NO:198
[0388] CDR-L3: residues 89-97 of SEQ ID NO:198
[0389] VL h1A11.A12 CDR Set
[0390] CDR-L1: residues 24-34 of SEQ ID NO:199
[0391] CDR-L2: residues 50-56 of SEQ ID NO:199
[0392] CDR-L3: residues 89-97 of SEQ ID NO:199
[0393] VL h1A11.A7 CDR Set
[0394] CDR-L1: residues 24-34 of SEQ ID NO:200
[0395] CDR-L2: residues 50-56 of SEQ ID NO:200
[0396] CDR-L3: residues 89-97 of SEQ ID NO:200
[0397] VL h1A11.B4 CDR Set
[0398] CDR-L1: residues 24-34 of SEQ ID NO:201
[0399] CDR-L2: residues 50-56 of SEQ ID NO:201
[0400] CDR-L3: residues 89-97 of SEQ ID NO:201
[0401] VL h1A11.B5 CDR Set
[0402] CDR-L1: residues 24-34 of SEQ ID NO:202
[0403] CDR-L2: residues 50-56 of SEQ ID NO:202
[0404] CDR-L3: residues 89-97 of SEQ ID NO:202
[0405] and
[0406] VL h1A11.E12 CDR Set
[0407] CDR-L1: residues 24-34 of SEQ ID NO:203
[0408] CDR-L2: residues 50-56 of SEQ ID NO:203
[0409] CDR-L3: residues 89-97 of SEQ ID NO: 203
[0410] In an embodiment, a DLL4 binding protein comprises CDRs from
at least two sets of variable domain CDRs in the group above.
[0411] In another embodiment, a DLL4 binding protein of the
invention comprises three CDRs selected from any VH set of three
CDRs in the group above and three CDRs selected from any VL set of
three CDRs in the group above.
[0412] In still another embodiment, a DLL4 binding protein of the
invention comprises a VH set of three CDRs as described above and a
VL set of three CDRs as described above from a pair of VH and VL
sets of CDRs selected from the group consisting of:
[0413] VH 38H12 CDR Set and VL 38H12 CDR Set,
[0414] VH 1A11 CDR Set and VL 1A11 CDR Set,
[0415] VH 37D10 CDR Set and VL 37D10 CDR Set,
[0416] VH 32C7 CDR Set and VL 32C7 CDR Set,
[0417] VH 14G1 Set and VL 14G1 CDR Set,
[0418] VH 14A11 CDR Set and VL 14A11 CDR Set,
[0419] VH 15D6 CDR Set and VL 15D6 CDR Set,
[0420] VH VH.1 1A11 CDR Set and VL VL.1 1A11 CDR Set,
[0421] VH VH.1 1A11 CDR Set and VL VL.1a 1A11 CDR Set,
[0422] VH VH.1 1A11 CDR Set and VL VL.1b 1A11 CDR Set,
[0423] VH VH.1 1A11 CDR Set and VL VL.2a 1A11 CDR Set,
[0424] VH VH.1a 1A11 CDR Set and VL VL.1 1A11 CDR Set,
[0425] VH VH.1a 1A11 CDR Set and VL VL.1a 1A11 CDR Set,
[0426] VH VH.1a 1A11 CDR Set and VL VL.1b 1A11 CDR Set,
[0427] VH VH.1a 1A11 CDR Set and VL VL.2a 1A11 CDR Set,
[0428] VH VH.1b 1A11 CDR Set and VL VL.1 1A11 CDR Set,
[0429] VH VH.1b 1A11 CDR Set and VL VL.1a 1A11 CDR Set,
[0430] VH VH.1b 1A11 CDR Set and VL VL.1b 1A11 CDR Set,
[0431] VH VH.1b 1A11 CDR Set and VL VL.2a 1A11 CDR Set,
[0432] VH VH.2a 1A11 CDR Set and VL VL.1 1A11 CDR Set,
[0433] VH VH.2a 1A11 CDR Set and VL VL.1a 1A11 CDR Set,
[0434] VH VH.2a 1A11 CDR Set and VL VL.1b 1A11 CDR Set,
[0435] VH VH.2a 1A11 CDR Set and VL VL.2a 1A11 CDR Set,
[0436] VH VH.1 38H12 CDR Set and VL VL.1 38H12 CDR Set,
[0437] VH VH.1 38H12 CDR Set and VL VL.1a 38H12 CDR Set,
[0438] VH VH.1 38H12 CDR Set and VL VL.1b 38H12 CDR Set,
[0439] VH VH.1 38H12 CDR Set and VL VL.2a 38H12 CDR Set,
[0440] VH VH.1a 38H12 CDR Set and VL VL.1 38H12 CDR Set,
[0441] VH VH.1a 38H12 CDR Set and VL VL.1a 38H12 CDR Set,
[0442] VH VH.1a 38H12 CDR Set and VL VL.1b 38H12 CDR Set,
[0443] VH VH.1a 38H12 CDR Set and VL VL.2a 38H12 CDR Set,
[0444] VH VH.1b 38H12 CDR Set and VL VL.1 38H12 CDR Set,
[0445] VH VH.1b 38H12 CDR Set and VL VL.1a 38H12 CDR Set,
[0446] VH VH.1b 38H12 CDR Set and VL VL.1b 38H12 CDR Set,
[0447] VH VH.1b 38H12 CDR Set and VL VL.2a 38H12 CDR Set,
[0448] VH VH.2a 38H12 CDR Set and VL VL.1 38H12 CDR Set,
[0449] VH VH.2a 38H12 CDR Set and VL VL.1a 38H12 CDR Set,
[0450] VH VH.2a 38H12 CDR Set and VL VL.1b 38H12 CDR Set,
[0451] VH VH.2a 38H12 CDR Set and VL VL.2a 38H12 CDR Set,
[0452] VH h1A11.A6 CDR Set and VL h1A11VL.1 CDR Set,
[0453] VH h1A11.C6 CDR Set and VL h1A11VL.1 CDR Set,
[0454] VH h1A11.A11 CDR Set and VL h1A11VL.1 CDR Set,
[0455] VH h1A11.A8 CDR Set and VL h1A11VL.1 CDR Set,
[0456] VH h1A11VH.1 CDR Set and VL h1A11.B4 CDR Set,
[0457] VH h1A11VH.1 CDR Set and VL h1A11.A7 CDR Set,
[0458] VH h1A11VH.1 CDR Set and VL h1A11.A12 CDR Set,
[0459] VH h1A11VH.1 CDR Set and VL h1A11.A2 CDR Set,
[0460] VH h1A11.B5 CDR Set and VL h1A11.B5 CDR Set,
[0461] VH h1A11.E12 CDR Set and VL h1A11.E12 CDR Set,
[0462] VH h1A11.G3 CDR Set and VL h1A11.E12 CDR Set,
[0463] VH h1A11.F5 CDR Set and VL h1A11.E12 CDR Set, and
[0464] VH h1A11.H2 CDR Set and VL h1A11.E12 CDR Set.
[0465] In a preferred embodiment, a DLL4 binding protein possess a
DLL4 antigen binding domain (or binding site) comprising six CDRs,
wherein a CDR-H1, CDR-H2, and CDR-H3 are located in a heavy chain
variable region (VH) and a CDR-L1, CDR-L2, and CDR-L3 are located
in a light chain variable region (VL), and wherein association of
the VH and VL regions form a functional DLL4 antigen binding domain
of the DLL4 binding protein. In a further non-limiting example of
this embodiment, a DLL4 binding protein that possesses two DLL4
antigen binding domains comprises two sets of VH and VL regions and
therefore comprises twelve CDRs.
[0466] In another embodiment, a DLL4 binding protein possesses a
DLL4 antigen binding domain comprising six CDRs, wherein a CDR-H1,
CDR-H2, and CDR-H3 are located in a heavy chain variable region
(VH) and a CDR-L1, CDR-L2, and CDR-L3 are located in a light chain
variable region (VL) and wherein the remaining sequences in each
variable region constitute a framework (FR) region such that each
CDR is positioned between two FR region sequences for a total of
four FR sequences, i.e., FR1, FR2, FR3, and FR4. In this
embodiment, the arrangement of FR and CDR sequences in a variable
region is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. In this embodiment, a
binding domain formed by association of a VH region and a VL region
comprises eight FR sequences and six CDRs.
[0467] DLL4 binding proteins of the invention include CDR-grafted
antibodies, wherein one or more CDRs of VH and/or VL regions of an
antibody of one species (donor species) are grafted into and
replace the corresponding CDRs of the VH and/or VL of an antibody
of another (acceptor) species using recombinant techniques
available in the art. An example of a donor species is a rat
anti-human DLL4 monoclonal antibody described herein and an example
of an acceptor species is a human immunoglobulin gamma (IgG)
molecule, wherein the human FR sequences of the VH and VL regions
of the human IgG molecule are human acceptor framework sequences
that receive the grafted in CDRs from the donor rat monoclonal
antibody. The human acceptor framework sequences of the resulting
CDR-grafted antibody may be further mutated to improve one or more
properties of the CDR-grafted antibody. By way of non-limiting
examples, one or more residues of one or more FR sequences of a
CDR-grafted antibody may be mutated to improve DLL4 binding
affinity or to lower immunogenicity of the CDR-grafted antibody in
a human subject.
[0468] In an embodiment of the invention, a DLL4 binding protein
comprising one or more CDRs described above further comprises a
human acceptor framework sequence. Preferably, a DLL4 binding
protein comprises one or more (e.g., one, two, three, four, five,
six, seven, or eight) human acceptor framework sequences.
[0469] A human acceptor framework sequence present in a DLL4
binding protein of the invention may comprise one or more amino
acid residues that have been back mutated to one or more
corresponding amino acid residues present in a rat monoclonal
antibody that binds DLL4 and/or that have been mutated to one or
more amino acid residues that reduce or eliminate a site(s) for an
undesirable reaction, for example, to reduce or eliminate a site
for undesired glycosylation and/or a site for undesired N-terminal
pyroglutamate formation and/or a site for potentially reduced
immunogenicity risk.
[0470] In an embodiment, a DLL4 binding protein comprising one or
more CDRs described above further comprises one or more (e.g., any
one, two, three, four, five, six, seven, or eight) of the human
acceptor framework sequences selected from the group of human
acceptor framework sequences in Tables 3 and 4, below. One or more
human acceptor framework sequences from Tables 3 and 4 present in a
DLL4 binding protein of the invention may further comprise one or
more amino acid residues that have been back mutated to one or more
corresponding amino acid residues present in a rat monoclonal
antibody that binds DLL4 and/or that have been mutated to one or
more amino acids that reduce or eliminate a site(s) for an
undesirable reaction, for example, to reduce or eliminate a site
for undesired glycosylation and/or a site for undesired N-terminal
pyroglutamate formation and/or a site for potentially reduced
immunogenicity risk.
[0471] In another embodiment, the invention provides a DLL4 binding
protein comprising one or more CDRs described above, wherein the
binding protein also comprises one or more (e.g., any one, two,
three, four, five, six, seven, or eight per binding domain) of the
human acceptor framework sequences selected from any framework
sequence present in a variable region sequence selected from the
group consisting of:
TABLE-US-00001 SEQ ID NO: 171 VH.1 1A11 SEQ ID NO: 172 VH.1a 1A11
SEQ ID NO: 173 VH.1b 1A11 SEQ ID NO: 174 VH.2a 1A11 SEQ ID NO: 175
VL.1 1A11 SEQ ID NO: 176 VL.1a 1A11 SEQ ID NO: 177 VL.1b 1A11 SEQ
ID NO: 178 VL.2a 1A11 SEQ ID NO: 179 VH.1 38H12 SEQ ID NO: 180
VH.1a 38H12 SEQ ID NO: 181 VH.1b 38H12 SEQ ID NO: 182 VH.2a 38H12
SEQ ID NO: 183 VL.1 38H12 SEQ ID NO: 184 VL.1a SEQ ID NO: 185 VL.1b
SEQ ID NO: 186 VL.2a SEQ ID NO: 187 VH h1A11VH.1 SEQ ID NO: 188 VH
h1A11.A6 SEQ ID NO: 189 VH h1A11.A8 SEQ ID NO: 190 VH h1A11.C6 SEQ
ID NO: 191 VH h1A11.A11 SEQ ID NO: 192 VH h1A11.B5 SEQ ID NO: 193
VH h1A11.E12 SEQ ID NO: 194 VH h1A11.G3 SEQ ID NO: 195 VH h1A11.F5
SEQ ID NO: 196 VH h1A11.H2 SEQ ID NO: 197 VL h1A11VL.1 SEQ ID NO:
198 VL h1A11.A2 SEQ ID NO: 199 VL h1A11.A12 SEQ ID NO: 200 VL
h1A11.A7 SEQ ID NO: 201 VL h1A11.B4 SEQ ID NO: 202 VL h1A11.B5 SEQ
ID NO: 203 VL h1A11.E12
[0472] In yet another embodiment of the invention, a DLL4 binding
protein further comprises one or more (e.g., any one, two, three,
four, five, six, seven, or eight) acceptor framework sequences
selected from the group consisting of: [0473] heavy chain
framework-1 (H-FR1):
E-V-Q-L-V-E-S-G-G-G-L-V-Q-P-G-G-S-L-R-L-S-C-A-A-S-G-F-T-F-X.sub.30
(SEQ ID NO:143), wherein X.sub.30 is S, R, or G; [0474] heavy chain
framework-2 (H-FR2): W-V-R-Q-A-P-G-K-G-L-E-W-V-A (SEQ ID NO:144);
[0475] heavy chain framework-3 (H-FR3):
R-F-T-I-S-R-D-N-A-K-X.sub.11-S-L-Y-L-Q-M-N-S-L-R-A-E-D-T-A-V-Y-Y-C-X.sub.-
31-R (SEQ ID NO:145), wherein; [0476] X.sub.11 is N or S; and
[0477] X.sub.31 is A or S; [0478] heavy chain framework-4 (H-FR4):
W-G-Q-G-T-L-V-T-V-S-S(SEQ ID NO:146); [0479] light chain
framework-1 (L-FR1):
D-I-Q-M-T-Q-S-P-S-S-L-S-A-S-V-G-D-R-V-T-I-T-C(SEQ ID NO:147);
[0480] light chain framework-2 (L-FR2): (SEQ ID NO:148), wherein;
[0481] X.sub.9 is A or S; and [0482] X.sub.15 is F or Y; [0483]
light chain framework-3 (L-FR3):
G-V-P-S-R-F-S-G-S-G-S-G-T-D-X.sub.15-T-L-T-I-S-S-L-Q-P-E-D-F-A-T-Y-Y-C(SE-
Q ID NO:149), wherein; [0484] X.sub.15 is F or S; and [0485] light
chain framework-4 (L-FR4): F-G-Q-G-T-K-L-E-I-K (SEQ ID NO:150).
[0486] In another embodiment, a DLL4 binding protein comprising one
or more CDRs described above also comprises a human acceptor
framework sequence described above wherein the human acceptor
framework sequence comprises at least one framework region amino
acid substitution at a key residue, wherein the key residue is
selected from the group consisting of a residue adjacent to a CDR,
a glycosylation site residue, a rare residue, a residue capable of
interacting with human DLL4, a residue capable of interacting with
a CDR, a canonical residue, a contact residue between heavy chain
variable region and light chain variable region, a residue within a
Vernier zone, and a residue in a region that overlaps between a
Chothia-defined variable heavy chain CDR1 and a Kabat-defined first
heavy chain framework.
[0487] In another embodiment, a human acceptor framework sequence
of a DLL4 binding protein described herein comprises at least one
framework region amino acid substitution, wherein the amino acid
sequence of the framework is at least 65% identical to a sequence
of a human germline acceptor framework and comprises at least 70
amino acid residues identical to the human germline acceptor
framework. In another embodiment, a DLL4 binding protein of the
invention comprises a consensus human variable domain sequence.
[0488] In an embodiment, the invention provides a DLL4 binding
protein comprises a human acceptor framework sequence, wherein the
binding protein comprises at least one variable domain having an
amino acid sequence selected from the group consisting of:
TABLE-US-00002 SEQ ID NO: 171 VH VH.1 1A11 SEQ ID NO: 172 VH VH.1a
1A11 SEQ ID NO: 173 VH VH.1b 1A11 SEQ ID NO: 174 VH VH.2a 1A11 SEQ
ID NO: 175 VL VL.1 1A11 SEQ ID NO: 176 VL VL.1a 1A11 SEQ ID NO: 177
VL VL.1b SEQ ID NO: 178 VL VL.2a 1A11 SEQ ID NO: 179 VH VH.1 38H12
SEQ ID NO: 180 VH VH.1a 38H12 SEQ ID NO: 181 VH VH.1b 38H12 SEQ ID
NO: 182 VH VH.2a 38H12 SEQ ID NO: 183 VL VL.1 38H12 SEQ ID NO: 184
VL VL.1a 38H12 SEQ ID NO: 185 VL VL.1b 38H12 SEQ ID NO: 186 VL
VL.2a 38H12 SEQ ID NO: 187 VH h1A11VH.1 SEQ ID NO: 188 VH h1A11.A6
SEQ ID NO: 189 VH h1A11.A8 SEQ ID NO: 190 VH h1A11.C6 SEQ ID NO:
191 VH h1A11.A11 SEQ ID NO: 192 VH h1A11.B5 SEQ ID NO: 193 VH
h1A11.E12 SEQ ID NO: 194 VH h1A11.G3 SEQ ID NO: 195 VH h1A11.F5 SEQ
ID NO: 196 VH h1A11.H2 SEQ ID NO: 197 VL h1 A11 VL.1 SEQ ID NO: 198
VL h1A11.A2 SEQ ID NO: 199 VL h1A11.A12 SEQ ID NO: 200 VL h1A11.A7
SEQ ID NO: 201 VL h1A11.B4 SEQ ID NO: 202 VL h1A11.B5 SEQ ID NO:
203 VL h1A11.E12
[0489] In another embodiment, a DLL4 binding protein of the
invention comprises two or more variable domains described above.
In a preferred embodiment, a DLL4 binding protein of the invention
comprises two variable domains, wherein the two variable domains
have amino acid sequences selected from the group consisting
of:
TABLE-US-00003 SEQ ID NO: 171 and SEQ ID NO: 175 SEQ ID NO: 180 and
SEQ ID VH.1 and VL.1 1A11 (Table 12) NO: 186 VH.1a and VL.2a (Table
16) SEQ ID NO: 171 and SEQ ID NO: 176 SEQ ID NO: 181 and SEQ ID
VH.1 and VL.1a 1A11 (Table 12) NO: 183 VH.1b and VL.1 (Table 16)
SEQ ID NO: 171 and SEQ ID NO: 177 SEQ ID NO: 181 and SEQ ID VH.1
and VL.1b 1A11 (Table 12) NO: 184 VH.1b and VL.1a (Table 16) SEQ ID
NO: 171 and SEQ ID NO: 178 SEQ ID NO: 181 and SEQ ID VH.1 and VL.2a
1A11(Table 12) NO: 185 VH.1b and VL.1b (Table 16) SEQ ID NO: 172
and SEQ ID NO: 175 SEQ ID NO: 181 and SEQ ID VH.1a and VL.1 1A11
(Table 12) NO: 186 VH.1b and VL.2a (Table 16) SEQ ID NO: 172 and
SEQ ID NO: 176 SEQ ID NO: 182 and SEQ ID VH.1a and VL.1a 1A11
(Table 12) NO: 183 VH.2a and VL.1 (Table 16) SEQ ID NO: 172 and SEQ
ID NO: 177 SEQ ID NO: 182 and SEQ ID VH.1a and VL.1b 1A11 (Table
12) NO: 184 VH.2a and VL.1a (Table 16) SEQ ID NO: 172 and SEQ ID
NO: 178 SEQ ID NO: 182 and SEQ ID VH.1a and VL.2a 1A11 (Table 12)
NO: 185 VH.2a and VL.1b (Table 16) SEQ ID NO: 173 and SEQ ID NO:
175 SEQ ID NO: 182 and SEQ ID VH.1b and VL.1 1A11 (Table 12) NO:
186 VH.2a and VL.2a (Table 16) SEQ ID NO: 173 and SEQ ID NO: 176
SEQ ID NO: 188 and SEQ ID VH.1b and VL.1a 1A11 (Table 12) NO: 197
h1A11.A6 VH and h1A11VL.1 Tables 20/21 SEQ ID NO: 173 and SEQ ID
NO: 177 SEQ ID NO: 190 and SEQ ID VH.1b and VL.1b 1A11 (Table 12)
NO: 197 h1A11.C6 VH and h1A11VL.1 Tables 20/21 SEQ ID NO: 173 and
SEQ ID NO: 178 SEQ ID NO: 191 and SEQ ID VH.1b and VL.2a 1A11
(Table 12) NO: 197 h1A11.A11VH and h1A11VL.1 Tables 20/21 SEQ ID
NO: 174 and SEQ ID NO: 175 SEQ ID NO: 189 and SEQ ID VH.2a and VL.1
1A11 (Table 12) NO: 197 h1A11.A8 VH and h1A11 VL.1 Tables 20/21 SEQ
ID NO: 174 and SEQ ID NO: 176 SEQ ID NO: 1878 and SEQ ID VH.2a and
VL.1a 1A11 (Table 12) NO: 201 h1A11VH.1 and h1A11.B4 VL Tables
20/21 SEQ ID NO: 174 and SEQ ID NO: 177 SEQ ID NO: 187 and SEQ ID
VH.2a and VL.1b 1A11 (Table 12) NO: 200 h1A11VH.1 and h1All.A7 VL
Tables 20/21 SEQ ID NO: 174 and SEQ ID NO: 178 SEQ ID NO: 187 and
SEQ ID VH.2a and VL.2a 1A11 (Table 12) NO: 199 h1A11VH.1 and
h1All.A12 VL Tables 20/21 SEQ ID NO: 179 and SEQ ID NO: 183 SEQ ID
NO: 187 and SEQ ID VH.1 and VL.1 38H12 (Table 16) NO: 198 h1A11VH.1
VH and h1All.A2 VL Tables 20/21 SEQ ID NO: 179 and SEQ ID NO: 184
SEQ ID NO: 192 and SEQ ID VH.1 and VL.1a 38H12 (Table 16) NO: 202
h1A11.B5 VH and h1All.B5 VL Tables 20/21 SEQ ID NO: 179 and SEQ ID
NO: 185 SEQ ID NO: 193 and SEQ ID VH.1 and VL.1b 38H12 (Table 16)
NO: 203 h1A11.E12 VH and h1All.E12 VL Tables 20/21 SEQ ID NO: 179
and SEQ ID NO: 186 SEQ ID NO: 194 and SEQ ID VH.1 and VL.2a 38H12
(Table 16) NO: 203 h1A11.G3 VH and h1All.E12 VL Tables 20/21 SEQ ID
NO: 180 and SEQ ID NO: 183 SEQ ID NO: 195 and SEQ ID VH.1a and VL.1
38H12 (Table 16) NO: 203 h1A11.F5 VH and h1All.E12 VL Tables 20/21
SEQ ID NO: 180 and SEQ ID NO: 184 SEQ ID NO: 196 and SEQ ID VH.1a
and VL.1a 38H12 (Table 16) NO: 203 h1A11.H2 VH and h1All.E12 VL
Tables 20/21 SEQ ID NO: 180 and SEQ ID NO: 185 VH.1a and VL.1b
38H12 (Table 16)
[0490] In an embodiment, a DLL4 binding protein of the invention
comprises at least one variable domain having an amino acid
sequence selected from the group consisting of:
TABLE-US-00004 SEQ ID NO: 157 SEQ ID NO: 181 VH 38H12 VH VH.1b
38H12 SEQ ID NO: 158 SEQ ID NO: 182 VL 38H12 VH VH.2a 38H12 SEQ ID
NO: 159 SEQ ID NO: 182 VH 1A11 VL VL.1 38H12 SEQ ID NO: 160 SEQ ID
NO: 184 VL 1A11 VL VL.1a 38H12 SEQ ID NO: 161 SEQ ID NO: 185 VH
37D10 VL VL.1b 38H12 SEQ ID NO: 162 SEQ ID NO: 186 VL 37D10 VL
VL.2a 38H12 SEQ ID NO: 163 SEQ ID NO: 187 VH 32C7 VH h1A11VH.1 SEQ
ID NO: 164 SEQ ID NO: 188 VL 32C7 VH h1A11.A6 SEQ ID NO: 165 SEQ ID
NO: 189 VH 14G1 VH h1A11.A8 SEQ ID NO: 166 SEQ ID NO: 190 VL 14G1
VH h1A11.C6 SEQ ID NO: 167 SEQ ID NO: 191 VH 14A11 VH h1A11.A11 SEQ
ID NO: 168 SEQ ID NO: 192 VL 14A11 VH h1A11.B5 SEQ ID NO: 169 SEQ
ID NO: 193 VH 15D6 VH h1A11.E12 SEQ ID NO: 170 SEQ ID NO: 194 VL
15D6 VH h1A11.G3 SEQ ID NO: 171 SEQ ID NO: 195 VH VH.1 1A11 VH
h1A11.F5 SEQ ID NO: 172 SEQ ID NO: 196 VH VH.1a 1A11 VH h1A11.H2
SEQ ID NO: 173 SEQ ID NO: 197 VH VH.1b 1A11 VL h1A11VL.1 SEQ ID NO:
174 SEQ ID NO: 198 VH VH.2a 1A11 VL h1A11.A2 SEQ ID NO: 175 SEQ ID
NO: 199 VL VL.1 1A11 VL h1A11.Al2 SEQ ID NO: 176 SEQ ID NO: 200 VL
VL.1a 1A11 VL h1A11.A7 SEQ ID NO: 177 SEQ ID NO: 201 VL VL.1b VL
h1A11.B4 SEQ ID NO: 178 SEQ ID NO: 202 VL VL.2a 1A11 VL h1A11.B5
SEQ ID NO: 179 SEQ ID NO: 203 VH VH.1 38H12 VL h1A11.E12 SEQ ID NO:
180 VH VH.1a 38H12
[0491] In another embodiment, a DLL4 binding protein of the
invention comprises two variable domains, wherein the two variable
domains have the amino acid sequences selected from the group
consisting of:
TABLE-US-00005 SEQ ID NO: 157 and SEQ ID NO: 158 SEQ ID NO: 179 and
SEQ ID 38H12 NO: 186 VH.1 and VL.2a 38H12 (Table 16) SEQ ID NO: 159
and SEQ ID NO: 160 SEQ ID NO: 180 and SEQ ID 1A11 NO: 183 VH.1a and
VL.1 38H12 (Table 16) SEQ ID NO: 161 and SEQ ID NO: 162 SEQ ID NO:
180 and SEQ ID 37D10 NO: 184 VH.1a and VL.1a 38H12 (Table 16) SEQ
ID NO: 163 and SEQ ID NO: 164 SEQ ID NO: 180 and SEQ ID 32C7 NO:
185 VH.1a and VL.1b 38H12 (Table 16) SEQ ID NO: 165 and SEQ ID NO:
166 SEQ ID NO: 180 and SEQ ID 14G1 NO: 186 VH.1a and VL.2a (Table
16) SEQ ID NO: 167 and SEQ ID NO: 168 SEQ ID NO: 181 and SEQ ID
14A11 NO: 183 VH.1b and VL.1 (Table 16) SEQ ID NO: 169 and SEQ ID
NO: 170 SEQ ID NO: 181 and SEQ ID 15D6 NO: 184 VH.1b and VL.1a
(Table 16) SEQ ID NO: 171 and SEQ ID NO: 175 SEQ ID NO: 181 and SEQ
ID VH.1 and VL.1 1A11 (Table 11) NO: 185 VH.1b and VL.1b (Table 16)
SEQ ID NO: 171 and SEQ ID NO: 176 SEQ ID NO: 181 and SEQ ID VH.1
and VL.1a 1A11 (Table 11) NO: 186 VH.1b and VL.2a (Table 16) SEQ ID
NO: 171 and SEQ ID NO: 177 SEQ ID NO: 182 and SEQ ID VH.1 and VL.1b
1A11 (Table 11) NO: 183 VH.2a and VL.1 (Table 16) SEQ ID NO: 171
and SEQ ID NO: 178 SEQ ID NO: 182 and SEQ ID VH.1 and VL.2a (Table
11) NO: 184 VH.2a and VL.1a (Table 16) SEQ ID NO: 172 and SEQ ID
NO: 175 SEQ ID NO: 182 and SEQ ID VH.1a and VL.1 (Table 11) NO: 185
VH.2a and VL.1b (Table 16) SEQ ID NO: 172 and SEQ ID NO: 176 SEQ ID
NO: 182 and SEQ ID VH.1a and VL.1a (Table 11) NO: 186 VH.2a and
VL.2a (Table 16) SEQ ID NO: 172 and SEQ ID NO: 177 SEQ ID NO: 188
and SEQ ID VH.1a and VL.1b (Table 11) NO: 197 h1A11.A6 VH and
h1A11VL.1 Tables 20/21 SEQ ID NO: 172 and SEQ ID NO: 178 SEQ ID NO:
190 and SEQ ID VH.1a and VL.2a (Table 11) NO: 197 h1A11.C6 VH and
h1A11VL.1 Tables 20/21 SEQ ID NO: 173 and SEQ ID NO: 175 SEQ ID NO:
191 and SEQ ID VH.1b and VL.1 (Table 11) NO: 197 h1A11.All VH and
h1A11VL.1 Tables 20/21 SEQ ID NO: 173 and SEQ ID NO: 176 SEQ ID NO:
189 and SEQ ID VH.1b and VL.1a (Table 11) NO: 197 h1A11.A8 VH and
h1A11 VL.1 Tables 20/21 SEQ ID NO: 173 and SEQ ID NO: 177 SEQ ID
NO: 187 and SEQ ID VH.1b and VL.1b (Table 11) NO: 201 h1A11VH.1 and
h1A11.B4 VL Tables 20/21 SEQ ID NO: 173 and SEQ ID NO: 178 SEQ ID
NO: 187 and SEQ ID VH.1b and VL.2a (Table 11) NO: 200 h1A11VH.1 and
h1All.A7 VL Tables 20/21 SEQ ID NO: 174 and SEQ ID NO: 175 SEQ ID
NO: 187 and SEQ ID VH.2a and VL.1 (Table 11) NO: 199 h1A11VH.1 and
h1All.A12 VL Tables 20/21 SEQ ID NO: 174 and SEQ ID NO: 176 SEQ ID
NO: 187 and SEQ ID VH.2a and VL.1a (Table 11) NO: 198 h1A11VH.1 VH
and h1All.A2 VL Tables 20/21 SEQ ID NO: 174 and SEQ ID NO: 177 SEQ
ID NO: 192 and SEQ ID VH.2a and VL.1b (Table 11) NO: 202 h1A11.B5
VH and h1All.B5 VL Tables 20/21 SEQ ID NO: 174 and SEQ ID NO: 178
SEQ ID NO: 193 and SEQ ID VH.2a and VL.2a (Table 11) NO: 203
h1A11.E12 VH and h1All.E12 VL Tables 20/21 SEQ ID NO: 179 and SEQ
ID NO: 183 SEQ ID NO: 194 and SEQ ID VH.1 and VL.1 38H12 (Table 16)
NO: 203 h1A11.G3 VH and h1All.E12 VL Tables 20/21 SEQ ID NO: 179
and SEQ ID NO: 184 SEQ ID NO: 195 and SEQ ID VH.1 and VL.1a 38H12
(Table 16) NO: 203 h1A11.F5 VH and h1All1.E12 VL Tables 20/21 SEQ
ID NO: 179 and SEQ ID NO: 185 SEQ ID NO: 196 and SEQ ID VH.1 and
VL.1b 38H12 (Table 16) NO: 203 h1A11.H2 VH and h1All.E12 VL Tables
20/21
[0492] In an embodiment, a DLL4 binding protein of the invention
comprises two variable domains having the amino acid sequences
selected from the group consisting of: [0493] SEQ ID NO:188
(h1A11.A6 VH) and SEQ ID NO:197 (h1A11VL.1), [0494] SEQ ID NO:190
(h1A11.C6 VH) and SEQ ID NO:197 (h1A11VL.1), and [0495] SEQ ID
NO:191 (h1A11.A11 VH) and SEQ ID NO:197 (h1A11.VL.1).
[0496] In an embodiment, a DLL4 binding protein of the invention
comprises two variable domains having the amino acid sequences SEQ
ID NO:181 (VH.1b 38H12) and SEQ ID NO:185 (VL.1b 38H12).
[0497] According to the invention, variable heavy (VH) domains and
variable light (VL) domains of any of the DLL4 binding proteins
described herein may also be shuffled using recombinant techniques
available in the art to generate and select for additional DLL4
binding proteins that comprise various combinations of VH and VL
domains described herein.
[0498] In an embodiment, a DLL4 binding protein according to the
invention binds human DLL4 (hu DLL4) and at least one other species
of DLL4. More preferably, a DLL4 binding protein described herein
binds human DLL4 and a DLL4 selected from the group consisting of a
cynomolgus monkey DLL4 (cynomolgus DLL4, cyno DLL4), a mouse DLL4
(mu DLL4), a rat DLL4, and combinations thereof.
[0499] In another embodiment, a DLL4 binding protein described
herein is capable of blocking DLL4 interaction with a Notch
protein. Preferably, the Notch protein is selected from the group
consisting of Notch-1, Notch-2, Notch-3, Notch-4, and combinations
thereof.
[0500] In an embodiment, a DLL4 binding protein described herein is
capable of modulating, inhibiting, or neutralizing one or more
biological functions of human DLL4. More preferably, a DLL4 binding
protein of the invention is capable of modulating, inhibiting, or
neutralizing an activity of a DLL4 selected from the group
consisting of a human DLL4, a cynomolgus DLL4, a monkey DLL4, a rat
DLL4, and combinations thereof.
[0501] In a further embodiment, a DLL4 binding protein described
herein is capable of inhibiting VEGFR2 activity, VEGFR1 activity,
or both VEGFR2 and VEGFR1 activities.
[0502] In an embodiment, a DLL4 binding protein described herein is
capable of inhibiting normal angiogenesis.
[0503] In an embodiment, a DLL4 binding protein of the invention
has an on rate constant (K.sub.on) to DLL4 of at least about
10.sup.2M.sup.-1s.sup.-1; at least about 10.sup.3M.sup.-1s.sup.-1;
at least about 10.sup.4M.sup.-1s.sup.-1; at least about
10.sup.5M.sup.-1s.sup.-1; or at least about
10.sup.6M.sup.-1s.sup.-1, as measured by surface plasmon resonance.
Preferably, the binding protein of the invention has an on rate
constant (K.sub.on) to DLL4 between 10.sup.2M.sup.-1s.sup.-1 to
10.sup.3M.sup.-1s.sup.-1; between 10.sup.3M.sup.-1s.sup.-1 to
10.sup.4M.sup.-1s.sup.-1; between 10.sup.4M.sup.-1s.sup.-1 to
10.sup.5M.sup.-1s.sup.-1; or between 10.sup.5M.sup.-1s.sup.-1 to
10.sup.6M.sup.-1s.sup.-1, as measured by surface plasmon
resonance.
[0504] In another embodiment, a DLL4 binding protein of the
invention has an off rate constant (K.sub.off) for DLL4 of at most
about 10.sup.-3s.sup.-1; at most about 10.sup.-4s.sup.-1; at most
about 10.sup.-5s.sup.-1; or at most about 10.sup.-6s.sup.-1, as
measured by surface plasmon resonance. Preferably, the binding
protein of the invention has an off rate constant (K.sub.off) to
DLL4 of 10.sup.-3s.sup.-1 to 10.sup.-4s.sup.-1; of
10.sup.-4s.sup.-1 to 10.sup.-5s.sup.-1; or of 10.sup.-5s.sup.-1 to
10.sup.-6s.sup.-1, as measured by surface plasmon resonance.
[0505] In another embodiment, a DLL4 binding protein of the
invention has a dissociation constant (K.sub.D) to DLL4 of at most
about 10.sup.-7 M; at most about 10.sup.-8 M; at most about
10.sup.-9M; at most about 10.sup.-10 M; at most about 10.sup.-11M;
at most about 10.sup.-12 M; or at most 10.sup.-13M. Preferably, the
binding protein of the invention has a dissociation constant
(K.sub.D) to DLL4 of 10.sup.-7 M to 10.sup.-8 M; of 10.sup.-8 M to
10.sup.-9M; of 10.sup.-9 M to 10.sup.-10 M; of 10.sup.-10 to
10.sup.-11M; of 10.sup.-11M to 10.sup.-12 M; or of 10.sup.-12 to M
10.sup.-13M.
[0506] In an embodiment, the invention provides an antibody
construct comprising a DLL4 binding protein described above and a
linker polypeptide or an immunoglobulin constant domain. In a
preferred embodiment, an antibody construct according to the
invention is selected from the group consisting of: an
immunoglobulin molecule, a monoclonal antibody, a chimeric
antibody, a CDR-grafted antibody, a humanized antibody, a Fab, a
Fab', a F(ab').sub.2, an Fv, a disulfide linked Fv, a scFv, a
single domain antibody, a diabody, a multispecific antibody, a dual
specific antibody, and a bispecific antibody.
[0507] In a preferred embodiment, an antibody construct of the
invention comprises a heavy chain immunoglobulin constant domain
selected from the group consisting of a human IgM constant domain,
a human IgG1 constant domain, a human IgG2 constant domain, a human
IgG3 constant domain, a human IgG4 constant domain, a human IgE
constant domain, and a human IgA constant domain.
[0508] In another embodiment, an antibody construct of the
invention comprises an immunoglobulin constant region selected from
the group consisting of an immunoglobulin gamma-1 (IgG-1) heavy
chain constant region (such as SEQ ID NO:3), a mutant IgG-1 heavy
chain constant region (such as SEQ ID NO:4), an immunoglobulin
kappa light chain constant region (such as SEQ ID NO:5), an
immunoglobulin lambda light chain constant region (such as SEQ ID
NO:6), and combinations thereof.
[0509] In another embodiment, an antibody construct is
glycosylated. Preferably, the glycosylation is a human
glycosylation pattern.
[0510] In an embodiment, the invention provides an antibody
conjugate comprising an antibody construct described herein
conjugated to an agent. Preferably, the agent is selected from the
group consisting of: an imaging agent, a therapeutic agent, a
cytotoxic agent, and an immunoadhesion molecule. In a preferred
embodiment, an imaging agents is selected from the group consisting
of: a radiolabel, an enzyme, a fluorescent label, a luminescent
label, a bioluminescent label, a magnetic label, and biotin. More
preferably the imaging agent is a radiolabel selected from the
group consisting of: .sup.3H, .sup.14C, .sup.35S, .sup.90Y,
.sup.99Tc, .sup.111In, .sup.125I, .sup.131I, .sup.177Lu,
.sup.166Ho, and .sup.153Sm. In a preferred embodiment, the
therapeutic or cytotoxic agent is selected from the group
consisting of: an anti-metabolite, an alkylating agent, an
antibiotic, a growth factor, a cytokine, an anti-angiogenic agent,
an anti-mitotic agent, an anthracycline, toxin, and an apoptotic
agent.
[0511] In another embodiment, a DLL4 binding protein, an antibody
construct, or antibody conjugate described above exists as a
crystal. Preferably, the crystal is a carrier-free pharmaceutical
controlled release crystal. In another embodiment, such a
crystallized binding protein, crystallized antibody construct, or
crystallized antibody conjugate has a greater half life in vivo
than its soluble counterpart. In a preferred embodiment, a
crystallized binding protein, crystallized antibody construct, or
crystallized antibody conjugate retains the biological activity of
the soluble or non-crystal form of the binding protein, antibody
construct, or antibody conjugate after crystallization.
[0512] In an embodiment, the invention provides an isolated nucleic
acid encoding one or more amino acid sequences of a DLL4 binding
protein (including any antibody construct or antibody conjugate)
described herein
[0513] In a preferred embodiment, the invention provides an
isolated nucleic acid encoding a polypeptide selected from the
group consisting of: a polypeptide comprising a heavy chain
variable domain, wherein the heavy chain variable domain comprises
one or more of a CDR-H1, a CDR-H2, and a CDR-H3 as described above;
a polypeptide comprising a light chain variable domain, wherein the
light chain variable domain comprises one or more of a CDR-L1, a
CDR-L2, and a CDR-L3 as described above; and a combination of both
polypeptides.
[0514] One aspect of the invention pertains to an isolated nucleic
acid encoding a DLL4 binding protein, an antibody construct, a DLL4
binding antibody conjugate, or DLL4 binding portion thereof.
Particularly preferred is an isolated nucleic acid that encodes a
polypeptide selected from the group consisting of: a polypeptide
comprising a heavy chain variable domain, wherein the heavy chain
variable domain comprises a CDR-H1, a CDR-H2, or a CDR-H3 described
above; a polypeptide comprising a light chain variable domain,
wherein the light chain variable domain comprises a CDR-L1, a
CDR-L2, or a CDR-L3 as described above; or a combination of both
polypeptides.
[0515] A further embodiment provides a vector comprising an
isolated nucleic acid described herein. In a preferred embodiment,
the vector is selected from the group consisting of: pcDNA, pTT
(Durocher et al., Nucl. Acids Res., 30(2e9): 1-9 (2002)), pTT3 (pTT
with additional multiple cloning sites), pEFBOS (Mizushima et al.,
Nucl. Acids. Res., 18 (17): 5322 (1990)), pBV, pJV, and pBJ.
[0516] In another aspect of the invention there is provided a host
cell transformed with the vector described above. The host cell can
be a prokaryotic or eukaryotic cell. A preferred prokaryotic host
cell is Escherichia coli. Preferably, the eukaryotic cell is
selected from the group consisting of: a protist cell, an animal
cell, a plant cell, and a fungal cell. More preferably, the host
cell is a mammalian cell including, but not limited to, CHO and COS
cells. A preferred fungal cell is Saccharomyces cerevisiae. A
preferred insect cell is an Sf9 cell.
[0517] In another aspect of the invention there is provided a
method of producing a binding protein that binds human DLL4
comprising the step of culturing any one of the host cells
described above in a culture medium under conditions sufficient to
produce a binding protein that binds human DLL4.
[0518] One embodiment provides a composition for the release of a
DLL4 binding protein according to the invention wherein the
composition comprises a formulation that comprises a crystallized
DLL4 binding protein, a crystallized antibody construct, or a
crystallized antibody conjugate as described above and an
ingredient, and further at least one polymeric carrier. Preferably,
the polymeric carrier is a polymer selected from one or more of the
group consisting of: poly (acrylic acid), poly (cyanoacrylates),
poly (amino acids), poly (anhydrides), poly (depsipeptide), poly
(esters), poly (lactic acid), poly (lactic-co-glycolic acid) or
PLGA, poly (b-hydroxybutyrate), poly (caprolactone), poly
(dioxanone); poly (ethylene glycol), poly ((hydroxypropyl)
methacrylamide, poly [(organo)phosphazene], poly (ortho esters),
poly (vinyl alcohol), poly (vinylpyrrolidone), maleic
anhydride-alkyl vinyl ether copolymers, pluronic polyols, albumin,
alginate, cellulose and cellulose derivatives, collagen, fibrin,
gelatin, hyaluronic acid, oligosaccharides, glycaminoglycans,
sulfated polysaccharides, blends and copolymers thereof.
Preferably, the ingredient is selected from the group consisting of
albumin, sucrose, trehalose, lactitol, gelatin,
hydroxypropyl-P-cyclodextrin, methoxypolyethylene glycol and
polyethylene glycol.
[0519] Another embodiment provides a method for treating a mammal
comprising the step of administering to the mammal an effective
amount of a composition comprising a crystallized DLL4 binding
protein, a crystallized antibody construct, or a crystallized
antibody conjugates described above.
[0520] The invention also provides a pharmaceutical composition
comprising a DLL4 binding protein as described above (including an
antibody construct or an antibody conjugate as described above) and
a pharmaceutically acceptable carrier. In a further embodiment, the
pharmaceutical composition comprises at least one additional agent.
The additional agent may be a therapeutic agent for treating a
disorder in which DLL4 is detrimental. Preferably, a pharmaceutical
composition of the invention comprises an additional agent selected
from the group consisting of: a therapeutic agent; an imaging
agent; an antineoplastic agent; a chemotherapeutic agent; an
angiogenesis inhibitor; an anti-VEGF antibody; an anti-EGFR
antibody; an anti-cMet antibody; an anti-ErbB3 antibody; an
anti-HER2 antibody; an anti-CD20 antibody; a VEGF-trap molecule; a
kinase inhibitor; a co-stimulation molecule blocker; an anti-B7.2
antibody; a CTLA4-Ig; an adhesion molecule blocker; an anti-E
selectin antibody; an anti-L selectin antibody; an anti-cytokine
antibody or functional fragment thereof; an anti-IL-18 antibody; an
anti-TNF antibody; anti-IL-6 antibody; methotrexate; a
corticosteroid; a cyclosporin; a rapamycin; FK506; a DNA alkylating
agent; cisplatin; carboplatin; an anti-tubulin agent; paclitaxel;
docetaxel; doxorubicin; gemcitabine; gemzar; an anthracycline;
adriamycin; a topoisiomersase I inhibitor; a topoisomerase II
inhibitor; 5-fluorouracil (5-FU); leucovorin; irinotecan; a
receptor tyrosine kinase inhibitor; an apoptosis inhibitor; a
Bcl2/Bclx inhibitor; erlotinib; gefitinib; a COX-2 inhibitor;
celecoxib; cyclosporin; rapamycin; a detectable label or reporter
molecule; a TNF antagonist; an antirheumatic; a muscle relaxant; a
narcotic; an analgesic; an anesthetic; a sedative; a local
anesthetic; a neuromuscular blocker; an antimicrobial agent; an
antipsoriatic agent; a corticosteroid; an anabolic steroid; an
erythropoietin; an immunization; an immunoglobulin; an
immunosuppressive agent; a growth hormone; a hormone replacement
drug; a radiopharmaceutical drug; an antidepressant; an
antipsychotic drug; a stimulant; an asthma medication; a beta
agonist; an inhaled steroid; an epinephrine; an epinephrine analog
thereof; a cytokine; and a cytokine antagonist.
[0521] In another aspect, the invention provides a method for
inhibiting human DLL4 activity comprising contacting human DLL4
with a binding protein disclosed above such that human DLL4 is
inhibited or neutralized. In a related aspect, the invention
provides a method for inhibiting DLL4 activity in a human subject
suffering from a disorder in which DLL4 is detrimental, comprising
administering to the human subject a binding protein disclosed
above such that human DLL4 in the human subject is inhibited and
treatment is achieved. Preferably, the disorder is selected from
the group comprising primary and metastatic cancers, including
carcinomas of breast, colon, rectum, lung, oropharynx, hypopharynx,
esophagus, stomach, pancreas, liver, gallbladder and bile ducts,
small intestine, urinary tract (including kidney, bladder, and
urothelium), female genital tract (including cervix, uterus, and
ovaries as well as choriocarcinoma and gestational trophoblastic
disease), male genital tract (including prostate, seminal vesicles,
testes, and germ cell tumors), endocrine glands (including the
thyroid, adrenal, and pituitary glands), and skin, as well as
hemangiomas, melanomas, sarcomas (including those arising from bone
and soft tissues as well as Kaposi's sarcoma), tumors of the brain,
nerves, eyes, and meninges (including astrocytomas, gliomas,
glioblastomas, retinoblastomas, neuromas, neuroblastomas,
Schwannomas, and meningiomas), solid tumors arising from
hematopoietic malignancies such as leukemias and lymphomas (both
Hodgkin's and non-Hodgkin's lymphomas), tumor metastases, ocular
neovascularization (including diabetic blindness, retinopathies,
age-induced macular degeneration and rubeosis), edema, rheumatoid
arthritis, atherosclerotic plaques, refractory ascites, psoriasis,
pancreatitis, polycystic ovarian disease (POD), endometriosis,
uterine fibroids, benign prostate hypertrophy, T-cell acute
lymphoblastic leukemia (T-ALL), cerebral autosomal dominant
arteriopathy with subcortical infarcts and leukoencephalopathy
(CADASIL), multiple sclerosis (MS), tetralogy of Fallot (TOF),
Alagille syndrome (AS), macular degeneration and age-related
macular degeneration diseases, and other angiogenesis independent
and dependent diseases characterized by aberrant DLL4 expression or
activity.
[0522] In another aspect, the invention provides a method of
treating a patient suffering from a disorder in which human DLL4 is
detrimental comprising the step of administering any one of the
binding proteins disclosed above before, concurrent, or after the
administration of a therapeutically effective amount of a second
agent. In a preferred embodiment, the second agent is selected from
the group consisting of: a radiotherapeutic agent; an
antineoplastic agent; a chemotherapeutic agent; a DNA alkylating
agent; cisplatin; carboplatin; an anti-tubulin agent; paclitaxel;
docetaxel; taxol; doxorubicin; gemcitabine; gemzar; an
anthracycline; adriamycin; a topoisomerase I inhibitor; a
topoisomerase II inhibitor; 5-fluorouracil (5-FU); leucovorin;
irinotecan; a receptor tyrosine kinase inhibitor; an apoptosis
inhibitor; a Bcl2/Bclx inhibitor; erlotinib; gefitinib; a COX-2
inhibitor; celecoxib; a kinase inhibitor; an angiogenesis
inhibitor; an anti-VEGF antibody; anti-EGFR antibody; an anti-cMet
antibody; an anti-ErbB3 antibody; an anti-HER2 antibody; an
anti-CD20 antibody; VEGF-Trap (aflibercept); a co-stimulation
molecule blocker; an anti-B7.1 antibody; an anti-B7.2 antibody;
CTLA4-Ig; an adhesion molecule blocker; an anti-LFA-1 antibody; an
anti-E selectin antibody; an anti-L selectin antibody; a small
molecule inhibitor; an anti-cytokine antibody or functional
fragment thereof; an anti-IL-18 antibody; an anti-TNF antibody; an
anti-IL-6 antibody; an anti-cytokine receptor antibody;
methotrexate; cyclosporin; rapamycin; FK506; a detectable label or
reporter; a TNF antagonist; an antirheumatic; a muscle relaxant; a
narcotic; a non-steroid anti-inflammatory drug (NSAID); an
analgesic; an anesthetic; a sedative; a local anesthetic; a
neuromuscular blocker; an antimicrobial agent; an antipsoriatic
drug; a corticosteroid; an anabolic steroid; an erythropoietin; an
immunization; an immunoglobulin; an immunosuppressive agent; a
growth hormone; a hormone replacement drug; a radiopharmaceutical
drug; an antidepressant; an antipsychotic drug; a stimulant; an
asthma medication; a beta agonist; an inhaled steroid; an
epinephrine; an epinephrine analog; a cytokine; and a cytokine
antagonist.
[0523] In a preferred embodiment, the pharmaceutical compositions
disclosed above are administered to the subject by at least one
mode selected from the group consisting of: parenteral,
subcutaneous, intramuscular, intravenous, intraarterial,
intraarticular, intrabronchial, intraabdominal, intracapsular,
intracartilaginous, intracavitary, intracelial, intracerebellar,
intracerebroventricular, intracolic, intracervical, intragastric,
intrahepatic, intramyocardial, intraosteal, intrapelvic,
intrapericardiac, intraperitoneal, intrapleural, intraprostatic,
intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,
intrasynovial, intrathoracic, intrauterine, intravesical, bolus,
vaginal, rectal, buccal, sublingual, intranasal, and
transdermal.
[0524] Another aspect of the invention provides at least one DLL4
anti-idiotype antibody to at least one DLL4 binding protein of the
present invention. The anti-idiotype antibody includes any protein
or peptide containing molecule that comprises at least a portion of
an immunoglobulin molecule such as, but not limited to, at least
one complementarily determining region (CDR) of a heavy or light
chain or a ligand binding portion thereof, a heavy chain or light
chain variable region, a heavy chain or light chain constant
region, a framework region, and any portion thereof, that can be
incorporated into a binding protein of the present invention.
[0525] Any of a variety of immunodetection assay formats may be
adapted to employ a DLL4 binding protein of the invention to detect
or measure DLL4 in a mixture, solution, or biological sample. Such
immunodetection assay formats include but are not limited to
radioimmunoassay (RIA), immunoprecipitation, enzyme-linked
immunosorbent assay (ELISA), immunoblot (e.g., Western),
immunostrips (e.g., immunodipsticks) comprising a DLL4 binding
protein of the invention adsorbed or immobilized to substrate,
FACS, and the like. Detection of DLL4 using a DLL4 binding protein
of the invention may be conducted in vitro on a mixture, solution,
or in biological sample. A biological sample that may be contacted
with a binding protein of the invention to detect or measure DLL4
in the sample includes, but is not limited to, urine, saliva, oral
swab (buccal, lingual, or throat swab), dermal swab, dermal scrape,
rectal swab, vaginal swab, whole blood sample, plasma sample, serum
sample, tissue biopsy, and any other sample obtained from an
individual by a procedure known in the art. In another embodiment,
a DLL4 binding protein may be employed to detect DLL4 in vivo such
as various tomography and scanning methods, including but not
limited to X-ray computer assisted tomography (CT), magnetic
resonance imaging (MRI), and positron emission tomography
(PET).
DETAILED DESCRIPTION OF THE INVENTION
[0526] This invention pertains to DLL4 binding proteins,
particularly anti-DLL4 antibodies, or antigen-binding portions
thereof that bind DLL4. An amino acid sequence (SEQ ID NO:1) for
human DLL4 is shown in Table 1 along with a corresponding DLL4
nucleotide coding sequence (SEQ ID NO:2). Various aspects of the
invention relate to antibodies and antibody fragments, and
pharmaceutical compositions thereof, as well as nucleic acids,
recombinant expression vectors, and host cells for making such
antibodies and fragments. Methods of using the antibodies of the
invention to detect human DLL4 or murine DLL4, methods to inhibit
human or mouse DLL4 and/or human or mouse VEGFR2 or VEGFR1
activity, either in vitro or in vivo, and methods to regulate gene
expression are also encompassed by the invention.
TABLE-US-00006 TABLE 1 Amino acid and nucleotide coding sequences
for human DLL4. Sequence Sequence Kind of Sequence Identifier
123456789012345678901234567890 Human DLL4 Amino SEQ ID
MAAASRSASGWALLLLVALWQQR Acid Sequence NO: 1 AAGSGVF
QLQLQEFINERGVLASGRPCEPGCR TFFRV CLKHFQAVVSPGPCTFGTVSTPVLG TNSFA
VRDDSSGGGRNPLQLPFNFTWPGTF SLIIE AWHAPGDDLRPEALPPDALISKIAIQ GSLA
VGQNWLLDEQTSTLTRLRYSYRVIC SDNYY GDNCSRLCKKRNDHFGHYVCQPDG NLSCLP
GWTGEYCQQPICLSGCHEQNGYCS KPAECL CRPGWQGRLCNECIPHNGCRHGTC STPWQC
TCDEGWGGLFCDQDLNYCTHHSPC KNGATC SNSGQRSYTCTCRPGYTGVDCELEL SECDS
NPCRNGGSCKDQEDGYHCLCPPGY YGLHCE HSTLSCADSPCFNGGSCRERNQGAN YACEC
PPNFTGSNCEKKVDRCTSNPCANGG QCLNR GPSRMCRCRPGFTGTYCELHVSDCA RNPCA
HGGTCHDLENGLMCTCPAGFSGRR CEVRTS IDACASSPCFNRATCYTDLSTDTFVC NCPY
GFVGSRCEFPVGLPPSFPWVAVSLG VGLAV LLVLLGMVAVAVRQLRLRRPDDGS REAMNN
LSDFQKDNLIPAAQLKNTNQKKELE VDCGL DKSNCGKQQNHTLDYNLAPGPLGR GTMPGK
FPHSDKSLGEKAPLRLHSEKPECRIS AICS PRDSMYQSVCLISEERNECVIATEV Human
DLL4 SEQ ID atggcggcagcgtcccggagcgcctctggctgggcgc Nucleotide Coding
NO: 2 tactgctgctggtggcactttggcagcagcgcgcggcc Sequence
ggctccggcgtcttccagctgcagctgcaggagttcatc
aacgagcgcggcgtactggccagtgggcggccttgcg
agcccggctgccggactttcttccgcgtctgccttaagc
acttccaggcggtcgtctcgcccggaccctgcaccttcg
ggaccgtctccacgccggtattgggcaccaactccttcg
ctgtccgggacgacagtagcggcggggggcgcaacc
ctctccaactgcccttcaatttcacctggccgggtaccttc
tcgctcatcatcgaagcttggcacgcgccaggagacga
cctgcggccagaggccttgccaccagatgcactcatca
gcaagatcgccatccagggctccctagctgtgggtcag
aactggttattggatgagcaaaccagcaccctcacaag
gctgcgctactcttaccgggtcatctgcagtgacaactac
tatggagacaactgctcccgcctgtgcaagaagcgcaa
tgaccacttcggccactatgtgtgccagccagatggcaa
cttgtcctgcctgcccggttggactggggaatattgcca
acagcctatctgtctttcgggctgtcatgaacagaatggc
tactgcagcaagccagcagagtgcctctgccgcccag
gctggcagggccggctgtgtaacgaatgcatcccccac
aatggctgtcgccacggcacctgcagcactccctggca
atgtacttgtgatgagggctggggaggcctgttttgtgac
caagatctcaactactgcacccaccactccccatgcaag
aatggggcaacgtgctccaacagtgggcagcgaagct
acacctgcacctgtcgcccaggctacactggtgtggact
gtgagctggagctcagcgagtgtgacagcaacccctgt
cgcaatggaggcagctgtaaggaccaggaggatggct
accactgcctgtgtcctccgggctactatggcctgcattg
tgaacacagcaccttgagctgcgccgactccccctgctt
caatgggggctcctgccgggagcgcaaccagggggc
caactatgcttgtgaatgtccccccaacttcaccggctcc
aactgcgagaagaaagtggacaggtgcaccagcaacc
cctgtgccaacgggggacagtgcctgaaccgaggtcc
aagccgcatgtgccgctgccgtcctggattcacgggca
cctactgtgaactccacgtcagcgactgtgcccgtaacc
cttgcgcccacggtggcacttgccatgacctggagaat
gggctcatgtgcacctgccctgccggcttctctggccga
cgctgtgaggtgcggacatccatcgatgcctgtgcctcg
agtccctgcttcaacagggccacctgctacaccgacctc
tccacagacacctttgtgtgcaactgcccttatggctttgt
gggcagccgctgcgagttccccgtgggcttgccgccc
agcttcccctgggtggccgtctcgctgggtgtggggct
ggcagtgctgctggtactgctgggcatggtggcagtgg
ctgtgcggcagctgcggcttcgacggccggacgacgg
cagcagggaagccatgaacaacttgtcggacttccaga
aggacaacctgattcctgccgcccagcttaaaaacaca
aaccagaagaaggagctggaagtggactgtggcctgg
acaagtccaactgtggcaaacagcaaaaccacacattg
gactataatctggccccagggcccctggggcggggga
ccatgccaggaaagtttccccacagtgacaagagctta
ggagagaaggcgccactgcggttacacagtgaaaagc
cagagtgtcggatatcagcgatatgctcccccagggact
ccatgtaccagtctgtgtgtttgatatcagaggagagga
atgaatgtgtcattgccacggaggtataa
[0527] Unless otherwise defined herein, scientific and technical
terms used in connection with the present invention shall have the
meanings that are commonly understood by those of ordinary skill in
the art. The meaning and scope of the terms should be clear,
however, in the event of any latent ambiguity, definitions provided
herein take precedent over any dictionary or extrinsic definition.
Further, unless otherwise required by context, singular terms shall
include pluralities and plural terms shall include the singular. In
this application, the use of "or" means "and/or" unless stated
otherwise. Furthermore, the use of the term "including," as well as
other forms, such as "includes" and "included," is not limiting.
Also, terms such as "element" or "component" encompass both
elements and components comprising one unit and elements and
components that comprise more than one subunit unless specifically
stated otherwise.
[0528] Generally, nomenclatures used in connection with, and
techniques of, cell and tissue culture, molecular biology,
immunology, microbiology, genetics and protein and nucleic acid
chemistry and hybridization described herein are those well known
and commonly used in the art. The methods and techniques of the
present invention are generally performed according to conventional
methods well known in the art and as described in various general
and more specific references that are cited and discussed
throughout the present specification unless otherwise indicated.
Enzymatic reactions and purification techniques are performed
according to manufacturer's specifications, as commonly
accomplished in the art or as described herein. The nomenclatures
used in connection with, and the laboratory procedures and
techniques of, analytical chemistry, synthetic organic chemistry,
and medicinal and pharmaceutical chemistry described herein are
those well known and commonly used in the art. Standard techniques
are used for chemical syntheses, chemical analyses, pharmaceutical
preparation, formulation, and delivery, and treatment of
patients.
[0529] That the present invention may be more readily understood,
select terms are defined below.
[0530] The term "polypeptide" as used herein, refers to any
polymeric chain of amino acids. The terms "peptide" and "protein"
are used interchangeably with the term polypeptide and also refer
to a polymeric chain of amino acids. The term "polypeptide"
encompasses native or artificial proteins, protein fragments and
polypeptide analogs of a protein sequence. A polypeptide may be
monomeric or polymeric. Use of "polypeptide" herein is intended to
encompass polypeptide and fragments and variants (including
fragments of variants) thereof, unless otherwise stated. For an
antigenic polypeptide, a fragment of polypeptide optionally
contains at least one contiguous or nonlinear epitope of
polypeptide. The precise boundaries of the at least one epitope
fragment can be confirmed using ordinary skill in the art. The
fragment comprises at least about 5 contiguous amino acids, such as
at least about 10 contiguous amino acids, at least about 15
contiguous amino acids, or at least about 20 contiguous amino
acids. A variant of polypeptide is as described herein.
[0531] The term "isolated protein" or "isolated polypeptide" is a
protein or polypeptide that by virtue of its origin or source of
derivation is not associated with naturally associated components
that accompany it in its native state; is substantially free of
other proteins from the same species; is expressed by a cell from a
different species; or does not occur in nature. Thus, a polypeptide
that is chemically synthesized or synthesized in a cellular system
different from the cell from which it naturally originates will be
"isolated" from its naturally associated components. A protein may
also be rendered substantially free of naturally associated
components by isolation, using protein purification techniques well
known in the art.
[0532] The term "recovering" as used herein, refers to the process
of rendering a chemical species such as a polypeptide substantially
free of naturally associated components by isolation, e.g., using
protein purification techniques well known in the art.
[0533] The term "human DLL4" (abbreviated herein as "hDLL4" or
"huDLL4"), as used herein, includes several EGF-like domains and a
DSL domain that is required for receptor binding. The term includes
a protein comprising about 74-75 kDa. The structure and deduced DNA
and protein sequences of human DLL4 is described further in, for
example, Shutter et al., Genes & Dev., 4: 1313-1318 (2000). The
term "human DLL4" is intended to include recombinant human DLL4 (rh
DLL4), which can be prepared by standard recombinant expression
methods.
[0534] "Biological activity", as used herein with respect to DLL4,
refers to all inherent biological properties of DLL4. Biological
properties of DLL4 include, but are not limited to, binding a Notch
receptor, activating a Notch receptor, negatively regulating VEGF
signaling, repressing VEGFR2, and inducing VEGR1.
[0535] The terms "specific binding" or "specifically binding", as
used herein, in reference to the interaction of an antibody, a
protein, or a peptide with a second chemical species, means that
the interaction is dependent upon the presence of a particular
structure (e.g., an antigenic determinant or epitope) on the
chemical species; for example, an antibody recognizes and binds to
a specific protein structure rather than to proteins generally. If
an antibody is specific for epitope "A", the presence of a molecule
containing epitope A (or free, unlabeled A), in a reaction
containing labeled "A" and the antibody, will reduce the amount of
labeled A bound to the antibody.
[0536] A "binding protein" is a monomeric or multimeric protein
that binds to and forms a complex with a binding partner, which may
be a polypeptide, an antigen, a chemical compound or other
molecule, or a substrate of any kind. A binding protein
specifically binds a binding partner. Binding proteins include
antibodies and other molecules comprising one or more
antigen-binding domains that bind to an antigen molecule or a
particular site (epitope) on the antigen molecule. A binding
protein includes an antibody or any of its antigen-binding
fragments, and various forms and derivatives of antibodies known in
the art and described below. Accordingly, a binding protein
includes, but is not limited to, an antibody, a tetrameric
immunoglobulin, an IgG molecule, an IgG.sub.1 molecule, a
monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a
humanized antibody, an affinity matured antibody, and fragments of
any such antibodies that retain the ability to bind to an
antigen.
[0537] The term "antibody", as used herein, broadly refers to any
immunoglobulin (Ig) molecule comprised of four polypeptide chains,
two heavy (H) chains and two light (L) chains, or any functional
fragment, mutant, variant, or derivation thereof, which retains the
essential epitope binding features of an Ig molecule. Such mutant,
variant, or derivative antibody formats are known in the art.
Nonlimiting embodiments of which are discussed below.
[0538] In a full-length antibody, each heavy chain is comprised of
a heavy chain variable region (abbreviated herein as HCVR or VH)
and a heavy chain constant region. The heavy chain constant region
is comprised of three domains, CH1, CH2 and CH3. Each light chain
is comprised of a light chain variable region (abbreviated herein
as LCVR or VL) and a light chain constant region. The light chain
constant region is comprised of one domain, CL. The VH and VL
regions can be further subdivided into regions of hypervariability,
termed complementarity determining regions (CDR), interspersed with
regions that are more conserved, termed framework regions (FR).
Each VH and VL is composed of three CDRs and four FRs, arranged
from amino-terminus to carboxy-terminus in the following order:
FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Immunoglobulin molecules
can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class
(e.g., IgG 1, IgG2, IgG 3, IgG4, IgA1 and IgA2) or subclass.
[0539] The term "Fc region" is used to define the C-terminal region
of an immunoglobulin heavy chain, which may be generated by papain
digestion of an intact antibody. The Fc region may be a native
sequence Fc region or a variant Fc region. The Fc region of an
immunoglobulin generally comprises two constant domains, a CH2
domain and a CH3 domain, and optionally comprises a CH4 domain.
Replacements of amino acid residues in the Fc portion to alter
antibody effector function are known in the art (U.S. Pat. Nos.
5,648,260 and 5,624,821). The Fc portion of an antibody mediates
several important effector functions, e.g., cytokine induction,
ADCC, phagocytosis, complement dependent cytotoxicity (CDC), and
half-life/clearance rate of antibody and antigen-antibody
complexes. In some cases these effector functions are desirable for
a therapeutic antibody but in other cases might be unnecessary or
even deleterious, depending on the therapeutic objectives. Certain
human IgG isotypes, particularly IgG1 and IgG3, mediate ADCC and
CDC via binding to Fc.gamma.Rs and complement C1q, respectively.
Neonatal Fc receptors (FcRn) are the critical components
determining the circulating half-life of antibodies. In still
another embodiment, at least one amino acid residue is replaced in
the constant region of the antibody, for example, the Fc region of
the antibody, such that effector functions of the antibody are
altered. The dimerization of two identical heavy chains of an
immunoglobulin is mediated by the dimerization of CH3 domains and
is stabilized by the disulfide bonds within the hinge region (Huber
et al., Nature, 264: 415-420 (1976); Thies et al., J. Mol. Biol.,
293: 67-79 (1999)). Mutation of cysteine residues within the hinge
regions to prevent heavy chain-heavy chain disulfide bonds will
destabilize dimerization of CH3 domains. Residues responsible for
CH3 dimerization have been identified (Dall'Acqua, Biochem., 37:
9266-9273 (1998)). Therefore, it is possible to generate a
monovalent half-Ig. Interestingly, these monovalent half Ig
molecules have been found in nature for both IgG and IgA subclasses
(Seligman, Ann. Immunol., 129: 855-70 (1978); Biewenga et al.,
Clin. Exp. Immunol., 51: 395-400 (1983)). The stoichiometry of
FcRn: Ig Fc region has been determined to be 2:1 (West et al.,
Biochem., 39: 9698-9708 (2000)), and half Fc is sufficient for
mediating FcRn binding (Kim et al., Eur. J. Immunol., 24: 542-548
(1994)). Mutations to disrupt the dimerization of CH3 domain may
not have greater adverse effect on its FcRn binding as the residues
important for CH3 dimerization are located on the inner interface
of CH3 b sheet structure, whereas the region responsible for FcRn
binding is located on the outside interface of CH2-CH3 domains.
However, the half Ig molecule may have certain advantages in tissue
penetration due to its smaller size in comparison to that of a
regular antibody. In one embodiment, at least one amino acid
residue is replaced in the constant region of a binding protein of
the invention, for example the Fc region, such that the
dimerization of the heavy chains is disrupted, resulting in half Ig
molecules. The anti-inflammatory activity of IgG is completely
dependent on sialylation of the N-linked glycan of the IgG Fc
fragment. The precise glycan requirements for anti-inflammatory
activity has been determined, such that an appropriate IgG1 Fc
fragment can be created, thereby generating a fully recombinant,
sialylated IgG1 Fc with greatly enhanced potency (Anthony et al.,
Science, 320: 373-376 (2008)).
[0540] The term "antigen-binding portion" of an antibody (or simply
"antibody portion"), as used herein, refers to one or more
fragments of an antibody that retains the ability to bind
specifically to an antigen (i.e., to a particular epitope of an
antigen, such as an epitope of DLL4). It has been shown that the
antigen-binding function of an antibody can be performed by
fragments of a full-length antibody. Such antibody embodiments may
also be bispecific, dual specific, or multi-specific formats;
specifically binding to two or more different antigens (or two or
more different epitopes of the same antigen). Examples of binding
fragments encompassed within the term "antigen-binding portion" of
an antibody include (i) a Fab fragment, a monovalent fragment
consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab').sub.2
fragment, a bivalent fragment comprising two Fab fragments linked
by a disulfide bridge at the hinge region; (iii) an Fd fragment
consisting of the VH and CH1 domains; (iv) an Fv fragment
consisting of the VL and VH domains of a single arm of an antibody;
(v) a dAb fragment (Ward et al., Nature, 341:544-546 (1989); PCT
Publication No. WO 90/05144 A1), which comprises a single variable
domain; and (vi) an isolated complementarity determining region
(CDR). Furthermore, although the two domains of the Fv fragment, VL
and VH, are coded for by separate genes, they can be joined, using
recombinant methods, by a synthetic linker that enables them to be
made as a single protein chain in which the VL and VH regions pair
to form monovalent molecules (known as single chain Fv (scFv); see,
e.g., Bird et al., Science, 242: 423-426 (1988); Huston et al.,
Proc. Natl. Acad. Sci. USA, 85: 5879-5883 (1988)). Such single
chain antibodies are also intended to be encompassed within the
term "antigen-binding portion" of an antibody. Other forms of
single chain antibodies, such as diabodies, are also encompassed.
Diabodies are bivalent, bispecific antibodies in which VH and VL
domains are expressed on a single polypeptide chain, but using a
linker that is too short to allow for pairing between the two
domains on the same chain, thereby forcing the domains to pair with
complementary domains of another chain and creating two antigen
binding sites (see, e.g., Holliger et al., Proc. Natl. Acad. Sci.
USA, 90: 6444-6448 (1993); Poljak, R. J., Structure, 2: 1121-1123
(1994)). Such antibody binding portions are known in the art (see,
Kontermann and Dubel eds., Antibody Engineering (Springer-Verlag.
New York, 2001), p. 790 (ISBN 3-540-41354-5)). In addition, single
chain antibodies also include "linear antibodies" comprising a pair
of tandem Fv segments (VH-CH1-VH-CH1) which, together with
complementary light chain polypeptides, form a pair of antigen
binding regions (Zapata et al. Protein Eng., 8(10): 1057-1062
(1995); and U.S. Pat. No. 5,641,870).
[0541] The term "antibody construct" (or "DLL4 antibody construct")
as used herein refers to a polypeptide comprising one or more the
antigen binding portions of the invention linked to a linker
polypeptide or an immunoglobulin constant domain. Linker
polypeptides comprise two or more amino acid residues joined by
peptide bonds and are used to link one or more antigen binding
portions. Such linker polypeptides are well known in the art (see
e.g., Holliger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448
(1993); Poljak, R. J., Structure, 2: 1121-1123 (1994)). An
immunoglobulin constant domain refers to a heavy or light chain
constant domain. Human IgG heavy chain and light chain constant
domain amino acid sequences are known in the art and represented in
Table 2.
TABLE-US-00007 TABLE 2 Sequence of human IgG heavy chain constant
domain and light chain constant domain Sequence Sequence Protein
Identifier 12345678901234567890123456789012 Ig gamma-1 constant SEQ
ID ASTKGPSVFFLAPSSKSTSGGTAALGCL region NO: 3
VKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYIC
NVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISR
TPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLT
VLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTK
NQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDK
SRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK Ig gamma-1 constant SEQ ID
ASTKGPSVFPLAPSSKSTSGGTAALGCL region mutant NO: 4
VKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYIC
NVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPEAAGGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVL
TVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSREEMTK
NQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDK
SRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK Ig Kappa constant region SEQ ID
TVAAPSVFIFPPSDEQLKSGTASVVCLL NO: 5 NNFYPREAKVQWKVDNALQSGNSQES
VTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC Ig Lambda
constant SEQ ID QPKAAPSVTLFPPSSEELQANKATLVCL region NO: 6
ISDFYPGAVTVAWKADSSPVKAGVETT TPSKQSNNKYAASSYLSLTPEQWKSHR
SYSCQVTHEGSTVEKTVAPTECS
[0542] Still further, an antibody or antigen-binding portion
thereof may be part of a larger immunoadhesion molecule, formed by
covalent or noncovalent association of the antibody or antibody
portion with one or more other proteins or peptides. Examples of
such immunoadhesion molecules include use of the streptavidin core
region to make a tetrameric scFv molecule (Kipriyanov et al., Human
Antibodies and Hybridomas, 6: 93-101 (1995)) and use of a cysteine
residue, a marker peptide, and a C-terminal polyhistidine tag to
make bivalent and biotinylated scFv molecules (Kipriyanov et al.,
Mol. Immunol., 31: 1047-1058 (1994)). Antibody portions, such as
Fab and F(ab')2 fragments, can be prepared from whole antibodies
using conventional techniques, such as papain or pepsin digestion,
respectively, of whole antibodies. Moreover, antibodies, antibody
portions, and immunoadhesion molecules can be obtained using
standard recombinant DNA techniques, as described herein and known
in the art.
[0543] An "isolated antibody", as used herein, is intended to refer
to an antibody that is substantially free of other antibodies
having different antigenic specificities (e.g., an isolated
antibody that specifically binds hDLL4 is substantially free of
antibodies that specifically bind antigens other than hDLL4). An
isolated antibody that specifically binds hDLL4 may, however, have
cross-reactivity to other antigens, such as DLL4 molecules from
other species (e.g., muDLL4). Moreover, an isolated antibody may be
substantially free of other cellular material and/or chemicals.
[0544] The term "monoclonal antibody" and abbreviations "MAb" and
"mAb", as used herein, refers to an antibody obtained from a
population of substantially homogeneous antibodies, i.e., the
individual antibodies comprising the population are identical
except for possible naturally occurring mutations that may be
present in minor amounts. Monoclonal antibodies are highly
specific, being directed against a single antigen. Furthermore, in
contrast to polyclonal antibody preparations that typically include
different antibodies directed against different determinants
(epitopes), each mAb is directed against a single determinant on
the antigen. The modifier "monoclonal" is not to be construed as
requiring production of the antibody by any particular method.
[0545] The term "human antibody", as used herein, is intended to
include antibodies having variable and constant regions derived
from human germline immunoglobulin sequences. The human antibodies
of the invention may include amino acid residues not encoded by
human germline immunoglobulin sequences (e.g., mutations introduced
by random or site-specific mutagenesis in vitro or by somatic
mutation in vivo), for example in the CDRs and in particular CDR3.
However, the term "human antibody", as used herein, is not intended
to include antibodies in which CDR sequences derived from the
germline of another mammalian species, such as a mouse, have been
grafted onto human framework sequences.
[0546] The term "recombinant human antibody", as used herein, is
intended to include all human antibodies that are prepared,
expressed, created, or isolated by recombinant means, such as
antibodies expressed using a recombinant expression vector
transfected into a host cell, antibodies isolated from a
recombinant, combinatorial human antibody library (Hoogenboom,
Trends Biotechnol., 15:62-70 (1997); Azzazy and Highsmith, Clin.
Biochem., 35: 425-445 (2002); Gavilondo and Larrick, BioTechniques,
29: 128-145 (2000); Hoogenboom and Chames, Immunol. Today, 21:
371-378 (2000)), antibodies isolated from an animal (e.g., a mouse)
that is transgenic for human immunoglobulin genes (see, Taylor et
al., Nucl. Acids Res., 20: 6287-6295 (1992); Kellermann and Green,
Curr. Opin. Biotechnol., 13: 593-597 (2002); Little et al.,
Immunol. Today, 21: 364-370 (2000)) or antibodies prepared,
expressed, created or isolated by any other means that involves
splicing of human immunoglobulin gene sequences to other DNA
sequences. Such recombinant human antibodies have variable and
constant regions derived from human germline immunoglobulin
sequences. In certain embodiments, however, such recombinant human
antibodies are subjected to in vitro mutagenesis (or, when an
animal transgenic for human Ig sequences is used, in vivo somatic
mutagenesis) and thus the amino acid sequences of the VH and VL
regions of the recombinant antibodies are sequences that, while
derived from and related to human germline VH and VL sequences, may
not naturally exist within the human antibody germline repertoire
in vivo.
[0547] The term "chimeric antibody" refers to antibodies which
comprise heavy and light chain variable region sequences from one
species and constant region sequences from another species, such as
antibodies having murine heavy and light chain variable regions
linked to human constant regions.
[0548] As used herein, the term "CDR" refers to a complementarity
determining region within antibody variable sequences. There are
three CDRs in each of the variable regions of the heavy chain and
the light chain, which are designated "CDR1", "CDR2", and "CDR3",
for each of the variable regions. The term "CDR set" as used herein
refers to a group of three CDRs that occur in a single variable
region that binds the antigen. The exact boundaries of these CDRs
have been defined differently according to different systems. The
system described by Kabat (Kabat et al., Sequences of Proteins of
Immunological Interest (National Institutes of Health, Bethesda,
Md. (1987) and (1991)) not only provides an unambiguous residue
numbering system applicable to any variable region of an antibody,
but also provides precise residue boundaries defining the three
CDRs. These CDRs may be referred to as "Kabat CDRs". Chothia and
coworkers (Chothia and Lesk, J. Mol. Biol., 196: 901-917 (1987);
Chothia et al., Nature, 342: 877-883 (1989)) found that certain
sub-portions within Kabat CDRs adopt nearly identical peptide
backbone conformations, despite having great diversity at the level
of amino acid sequence. These sub-portions were designated as "L1",
"L2", and "L3", or "H1", "H2", and "H3", where the "L" and the "H"
designate the light chain and the heavy chain regions,
respectively. These regions may be referred to as "Chothia CDRs",
which have boundaries that overlap with Kabat CDRs. Other
boundaries defining CDRs overlapping with the Kabat CDRs have been
described by Padlan, FASEB J., 9: 133-139 (1995) and MacCallum, J.
Mol. Biol., 262(5): 732-745 (1996). Still other CDR boundary
definitions may not strictly follow one of the herein systems, but
will nonetheless overlap with the Kabat CDRs, although they may be
shortened or lengthened in light of prediction or experimental
findings that particular residues or groups of residues or even
entire CDRs do not significantly impact antigen binding. The
methods used herein may utilize CDRs defined according to any of
these systems, although certain embodiments use Kabat or Chothia
defined CDRs.
[0549] The terms "Kabat numbering," "Kabat definitions", and "Kabat
labeling" are used interchangeably herein. These terms, which are
recognized in the art, refer to a system of numbering amino acid
residues which are more variable (i.e., hypervariable) than other
amino acid residues in the heavy and light chain variable regions
of an antibody, or an antigen binding portion thereof (Kabat et
al., Ann. NY Acad. Sci., 190: 382-391 (1971) and Kabat et al.,
Sequences of Proteins of Immunological Interest, Fifth Edition,
U.S. Department of Health and Human Services, NIH Publication No.
91-3242 (1991)). For the heavy chain variable region (VH), the
hypervariable region ranges from amino acid positions 31 to 35 for
CDR1, amino acid positions 50 to 65 for CDR2, and amino acid
positions 95 to 102 for CDR3. For the light chain variable region
(VL), the hypervariable region ranges from amino acid positions 24
to 34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino
acid positions 89 to 97 for CDR3.
[0550] The growth and analysis of extensive public databases of
amino acid sequences of variable heavy and light regions over the
past twenty years have led to the understanding of the typical
boundaries between framework regions (FR) and CDR sequences within
variable region sequences and enabled persons skilled in this art
to accurately determine the CDRs according to Kabat numbering,
Chothia numbering, or other systems. See, e.g., Martin, "Protein
Sequence and Structure Analysis of Antibody Variable Domains," In
Kontermann and Dubel, eds., Antibody Engineering (Springer-Verlag,
Berlin, 2001), chapter 31, pages 432-433. A useful method of
determining the amino acid sequences of Kabat CDRs, and thereby
sequences of Kabat FRs as well, within the amino acid sequences of
variable heavy (VH) and variable light (VL) regions is provided
below:
[0551] To identify a CDR-L1 amino acid sequence: [0552] Starts
approximately 24 amino acid residues from the amino terminus of the
VL region; [0553] Residue before the CDR-L1 sequence is always
cysteine (C); [0554] Residue after the CDR-L1 sequence is always
tryptophan (W), typically Trp-Tyr-Gln (W-Y-Q), but also Trp-Leu-Gln
(W-L-Q), Trp-Phe-Gln (W-F-Q), and Trp-Tyr-Leu (W-Y-L); [0555]
Length is typically 10 to 17 amino acid residues.
[0556] To identify a CDR-L2 amino acid sequence: [0557] Starts
always 16 residues after the end of CDR-L1; [0558] Residues before
the CDR-L2 sequence are generally Ile-Tyr (I-Y), but also Val-Tyr
(V-Y), Ile-Lys (I-K), and Ile-Phe (I-F); [0559] Length is always 7
amino acid residues.
[0560] To identify a CDR-L3 amino acid sequence: [0561] Starts
always 33 amino acids after the end of CDR-L2; [0562] Residue
before the CDR-L3 amino acid sequence is always a cysteine (C);
[0563] Residues after are always Phe-Gly-X-Gly (F-G-X-G) (SEQ ID
NO:7), [0564] where X is any amino acid; [0565] Length is typically
7 to 11 amino acid residues.
[0566] To identify a CDR-H1 amino acid sequence: [0567] Starts
approximately 31 amino acid residues from amino terminus of VH
region and always 9 residues after a cysteine (C); [0568] Residues
before are always Cys-X-X-X-X-X-X-X-X (SEQ ID NO:8), [0569] where X
is any amino acid; [0570] Residue after is always a Trp (W),
typically Trp-Val (W-V), but also Trp-Ile (W-I), and Trp-Ala (W-A);
[0571] Length is typically 5 to 7 amino acid residues.
[0572] To identify a CDR-H2 amino acid sequence: [0573] Starts
always 15 amino acid residues after the end of CDR-H1; [0574]
Residues before are typically Leu-Glu-Trp-Ile-Gly (L-E-W-I-G) (SEQ
ID NO:9), but other variations also; [0575] Residues after are
Lys/Arg-Leu/Ile/Val/Phe/Thr/Ala-Thr/Ser/Ile/Ala
(K/R-L/I/V/F/T/A-T/S/I/A); [0576] Length is typically 16 to 19
amino acid residues.
[0577] To identify a CDR-H3 amino acid sequence: [0578] Starts
always 33 amino acid residues after the end of CDR-H2 and always 3
after a cysteine (C) [0579] Residues before are always Cys-X-X
(C-X-X), where X is any amino acid, typically Cys-Ala-Arg (C-A-R);
[0580] Residues after are always Trp-Gly-X-Gly (W-G-X-G) (SEQ ID
NO:10), where X is any amino acid; [0581] Length is typically 3 to
25 amino acid residues.
[0582] The term "CDR-grafted antibody" refers to antibodies which
comprise heavy and light chain variable region sequences from one
species but in which the sequences of one or more of the CDR
regions of VH and/or VL are replaced with CDR sequences of another
species, such as antibodies having murine heavy and light chain
variable regions in which one or more of the murine CDRs (e.g.,
CDR3) has been replaced with human CDR sequences.
[0583] The term "humanized antibody" refers to antibodies which
comprise heavy and light chain variable region sequences from a
non-human species (e.g., a mouse) but in which at least a portion
of the VH and/or VL sequence has been altered to be more
"human-like," i.e., more similar to human germline variable
sequences. A "humanized antibody" is an antibody or a variant,
derivative, analog, or fragment thereof, which immunospecifically
binds to an antigen of interest and which comprises a framework
(FR) region having substantially the amino acid sequence of a human
antibody and a complementary determining region (CDR) having
substantially the amino acid sequence of a non-human antibody. As
used herein, the term "substantially" in the context of a CDR
refers to a CDR having an amino acid sequence at least 80%, at
least 85%, at least 90%, at least 95%, at least 98% or at least 99%
identical to the amino acid sequence of a non-human antibody CDR. A
humanized antibody comprises substantially all of at least one, and
typically two, variable domains (Fab, Fab', F(ab') 2, FabC, Fv) in
which all or substantially all of the CDR regions correspond to
those of a non-human immunoglobulin (i.e., donor antibody) and all
or substantially all of the framework regions are those of a human
immunoglobulin consensus sequence. In an embodiment, a humanized
antibody also comprises at least a portion of an immunoglobulin
constant region (Fc), typically that of a human immunoglobulin. In
some embodiments, a humanized antibody contains both the light
chain as well as at least the variable domain of a heavy chain. The
antibody also may include the CH1, hinge, CH2, CH3, and CH4 regions
of the heavy chain. In some embodiments, a humanized antibody only
contains a humanized light chain. In some embodiments, a humanized
antibody only contains a humanized heavy chain. In specific
embodiments, a humanized antibody only contains a humanized
variable domain of a light chain and/or humanized heavy chain.
[0584] A humanized antibody can be selected from any class of
immunoglobulins, including IgM, IgG, IgD, IgA, and IgE, and any
isotype, including without limitation IgG1, IgG2, IgG3, and IgG4. A
humanized antibody may comprise sequences from more than one class
or isotype, and particular constant domains may be selected to
optimize desired effector functions using techniques well known in
the art.
[0585] The framework regions and CDRs of a humanized antibody need
not correspond precisely to the parental sequences, e.g., the donor
antibody CDR or the consensus framework may be mutagenized by
substitution, insertion, and/or deletion of at least one amino acid
residue so that the CDR or framework residue at that site does not
correspond to either the donor antibody or the consensus framework.
In a preferred embodiment, such mutations, however, will not be
extensive. Usually, at least 80%, preferably at least 85%, more
preferably at least 90%, and most preferably at least 95% of the
humanized antibody residues will correspond to those of the
parental FR and CDR sequences. As used herein, the term "consensus
framework" refers to the framework region in the consensus
immunoglobulin sequence. As used herein, the term "consensus
immunoglobulin sequence" refers to the sequence formed from the
most frequently occurring amino acids (or nucleotides) in a family
of related immunoglobulin sequences (See, e.g., Winnaker, From
Genes to Clones (Verlagsgesellschaft, Weinheim, 1987)). A
"consensus immunoglobulin sequence" may thus comprise a "consensus
framework region(s)" and/or a "consensus CDR(s)". In a family of
immunoglobulins, each position in the consensus sequence is
occupied by the amino acid occurring most frequently at that
position in the family. If two amino acids occur equally
frequently, either can be included in the consensus sequence.
[0586] An "affinity matured" antibody is an antibody with one or
more alterations in one or more CDRs thereof which result in an
improvement in the affinity of the antibody for a target antigen,
compared to a parent antibody which does not possess the
alteration(s). Exemplary affinity matured antibodies will have
nanomolar or even picomolar affinities for the target antigen. A
variety of procedures for producing affinity matured antibodies are
known in the art. For example, Marks et al., BioTechnology, 10:
779-783 (1992) describes affinity maturation by VH and VL domain
shuffling. Random mutagenesis of CDR and/or framework residues is
described by Barbas et al., Proc. Nat. Acad. Sci. USA, 91:
3809-3813 (1994); Schier et al., Gene, 169: 147-155 (1995); Yelton
et al., J. Immunol., 155: 1994-2004 (1995); Jackson et al., J.
Immunol., 154(7): 3310-3319 (1995); Hawkins et al, J. Mol. Biol.,
226: 889-896 (1992). Selective mutation at selective mutagenesis
positions and at contact or hypermutation positions with an
activity enhancing amino acid residue is described in U.S. Pat. No.
6,914,128 B1.
[0587] The term "multivalent binding protein" denotes a binding
protein comprising two or more antigen binding sites (also referred
to herein as "antigen binding domains"). A multivalent binding
protein is preferably engineered to have three or more antigen
binding sites, and is generally not a naturally occurring antibody.
The term "multispecific binding protein" refers to a binding
protein capable of binding two or more related or unrelated
targets, including a binding protein capable of binding two or more
different epitopes of the same target molecule.
[0588] The term "bispecific antibody", as used herein, refers to
full-length antibodies that are generated by quadroma technology
(see Milstein et al., Nature, 305(5934): 537-540 (1983)), by
chemical conjugation of two different monoclonal antibodies (see,
Staerz et al., Nature, 314(6012): 628-631 (1985)), or by
knob-into-hole or similar approaches which introduces mutations in
the Fc region (see Holliger et al., Proc. Natl. Acad. Sci. USA,
90(14): 6444-6448 (1993)), resulting in multiple different
immunoglobulin species of which only one is the functional
bispecific antibody. By molecular function, a bispecific antibody
binds one antigen (or epitope) on one of its two binding arms (one
pair of HC/LC), and binds a different antigen (or epitope) on its
second arm (a different pair of HC/LC). By this definition, a
bispecific antibody has two distinct antigen binding arms (in both
specificity and CDR sequences), and is monovalent for each antigen
it binds to.
[0589] The term "dual-specific antibody", as used herein, refers to
full-length antibodies that can bind two different antigens (or
epitopes) in each of its two binding arms (a pair of HC/LC) (see
PCT publication WO 02/02773). Accordingly a dual-specific binding
protein has two identical antigen binding arms, with identical
specificity and identical CDR sequences, and is bivalent for each
antigen to which it binds.
[0590] "Dual variable domain" ("DVD") binding proteins of the
invention comprise two or more antigen binding sites and may be
divalent (two antigen binding sites), tetravalent (four antigen
binding sites), or multivalent binding proteins. DVDs may be
monospecific, i.e., capable of binding one antigen (or one specific
epitope), or multispecific, i.e., capable of binding two or more
antigens (i.e., two or more epitopes of the same target antigen
molecule or two or more epitopes of different target antigens). A
preferred DVD binding protein comprises two heavy chain DVD
polypeptides and two light chain DVD polypeptides is referred to as
a "DVD immunoglobulin" or "DVD-Ig". Such a DVD-Ig binding protein
is thus tetrameric and reminiscent of an IgG molecule, but provides
more antigen binding site than an IgG molecule. Thus, each half of
a tetrameric DVD-Ig molecule is reminiscent of one half of an IgG
molecule and comprises a heavy chain DVD polypeptide and a light
chain DVD polypeptide, but unlike a pair of heavy and light chains
of an IgG molecule that provide a single antigen binding domain, a
pair of heavy and light chains of a DVD-Ig provide two or more
antigen binding sites.
[0591] Each antigen binding site of a DVD-Ig binding protein is
derived from a donor ("parental") monoclonal antibody and thus
comprises a heavy chain variable domain (VH) and a light chain
variable domain (VL) with a total of six CDRs involved in antigen
binding per antigen binding site. Accordingly, a DVD-Ig binding
protein that binds two different epitopes (i.e., two different
epitopes of two different antigen molecules or two different
epitopes of the same antigen molecule) comprises an antigen binding
site derived from a first parental monoclonal antibody and an
antigen binding site of a second parental monoclonal antibody.
[0592] A description of the design, expression, and
characterization of DVD-Ig binding molecules is provided in PCT
Publication No. WO 2007/024715, U.S. Pat. No. 7,612,181, and Wu et
al., Nature Biotech., 25: 1290-1297 (2007). A preferred example of
such DVD-Ig molecules comprises a heavy chain that comprises the
structural formula VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first
heavy chain variable domain, VD2 is a second heavy chain variable
domain, C is a heavy chain constant domain, X1 is a linker with the
proviso that it is not CH1, X2 is an Fc region, and n is 0 or 1,
but preferably 1; and a light chain that comprises the structural
formula VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first light chain
variable domain, VD2 is a second light chain variable domain, C is
a light chain constant domain, X1 is a linker with the proviso that
it is not CH1, and X2 does not comprise an Fc region; and n is 0 or
1, but preferably 1. Such a DVD-Ig may comprise two such heavy
chains and two such light chains, wherein each chain comprises
variable domains linked in tandem without an intervening constant
region between variable regions, wherein a heavy chain and a light
chain associate to form tandem functional antigen binding sites,
and a pair of heavy and light chains may associate with another
pair of heavy and light chains to form a tetrameric binding protein
with four functional antigen binding sites. In another example, a
DVD-Ig molecule may comprise heavy and light chains that each
comprise three variable domains (VD1, VD2, VD3) linked in tandem
without an intervening constant region between variable domains,
wherein a pair of heavy and light chains may associate to form
three antigen binding sites, and wherein a pair of heavy and light
chains may associate with another pair of heavy and light chains to
form a tetrameric binding protein with six antigen binding
sites.
[0593] In a preferred embodiment, a DVD-Ig binding protein
according to the invention not only binds the same target molecules
bound by its parental monoclonal antibodies, but also possesses one
or more desirable properties of one or more of its parental
monoclonal antibodies. Preferably, such an additional property is
an antibody parameter of one or more of the parental monoclonal
antibodies. Antibody parameters that may be contributed to a DVD-Ig
binding protein from one or more of its parental monoclonal
antibodies include, but are not limited to, antigen specificity,
antigen affinity, potency, biological function, epitope
recognition, protein stability, protein solubility, production
efficiency, immunogenicity, pharmacokinetics, bioavailability,
tissue cross reactivity, and orthologous antigen binding.
[0594] A DVD-Ig binding protein according to the invention binds at
least one epitope of a human DLL4 protein. Non-limiting examples of
a DVD-Ig binding protein according to the invention include a
DVD-Ig binding protein that binds one or more epitopes of human
DLL4, a DVD-Ig binding protein that binds an epitope of a human
DLL4 and an epitope of a DLL4 of another species (for example,
mouse), and a DVD-Ig binding protein that binds an epitope of a
human DLL4 and an epitope of another target molecule (for example,
VEGFR2 or VEGFR1).
[0595] A "functional antigen binding site" of a binding protein is
one that is capable of binding a target antigen. The antigen
binding affinity of the antigen binding site is not necessarily as
strong as the parent antibody from which the antigen binding site
is derived, but the ability to bind antigen must be measurable
using any one of a variety of methods known for evaluating antibody
binding to an antigen. Moreover, the antigen binding affinity of
each of the antigen binding sites of a multivalent antibody herein
need not be quantitatively the same.
[0596] As used herein, the terms "acceptor" and "acceptor antibody"
refer to an antibody or nucleic acid sequence providing or encoding
at least 80%, at least 85%, at least 90%, at least 95%, at least
98%, or 100% of the amino acid sequences of one or more of the
framework regions (FRs). In some embodiments, the term "acceptor"
refers to the antibody amino acid or nucleic acid sequence
providing or encoding the constant region(s). In yet another
embodiment, the term "acceptor" refers to the antibody amino acid
or nucleic acid sequence providing or encoding one or more of the
framework regions and the constant region(s). In a specific
embodiment, the term "acceptor" refers to a human antibody amino
acid or nucleic acid sequence that provides or encodes at least
80%, preferably, at least 85%, at least 90%, at least 95%, at least
98%, or 100% of the amino acid sequences of one or more of the
framework regions. In accordance with this embodiment, an acceptor
may contain at least 1, at least 2, at least 3, least 4, at least
5, or at least 10 amino acid residues that does (do) not occur at
one or more specific positions of a human antibody. An acceptor
framework region and/or acceptor constant region(s) may be, e.g.,
derived or obtained from a germline antibody gene, a mature
antibody gene, a functional antibody (e.g., antibodies well-known
in the art, antibodies in development, or antibodies commercially
available).
[0597] As used herein, the term "canonical" residue refers to a
residue in a CDR or framework that defines a particular canonical
CDR structure as defined by Chothia et al. (J. Mol. Biol., 196:
901-917 (1987); Chothia et al., J. Mol. Biol., 227: 799-817 (1992),
both are incorporated herein by reference). According to Chothia et
al., critical portions of the CDRs of many antibodies have nearly
identical peptide backbone confirmations despite great diversity at
the level of amino acid sequence. Each canonical structure
specifies primarily a set of peptide backbone torsion angles for a
contiguous segment of amino acid residues forming a loop.
[0598] As used herein, the terms "donor" and "donor antibody" refer
to an antibody providing one or more CDRs. In a preferred
embodiment, the donor antibody is an antibody from a species
different from the antibody from which the framework regions are
obtained or derived. In the context of a humanized antibody, the
term "donor antibody" refers to a non-human antibody providing one
or more CDRs.
[0599] As used herein, the term "framework" or "framework sequence"
refers to the remaining sequences of a variable region minus the
CDRs. Because the exact definition of a CDR sequence can be
determined by different systems (for example, see above), the
meaning of a framework sequence is subject to correspondingly
different interpretations. The six CDRs (CDR-L1, -L2, and -L3 of
light chain and CDR-H1, -H2, and -H3 of heavy chain) also divide
the framework regions on the light chain and the heavy chain into
four sub-regions (FR1, FR2, FR3, and FR4) on each chain, in which
CDR1 is positioned between FR1 and FR2, CDR2 between FR2 and FR3,
and CDR3 between FR3 and FR4. Without specifying the particular
sub-regions as FR1, FR2, FR3, or FR4, a framework region, as
referred by others, represents the combined FRs within the variable
region of a single, naturally occurring immunoglobulin chain. As
used herein, a FR represents one of the four sub-regions, and FRs
represents two or more of the four sub-regions constituting a
framework region.
[0600] Human heavy chain and light chain framework (FR) sequences
are known in the art that can be used as heavy chain and light
chain "acceptor" framework sequences (or simply, "acceptor"
sequences) to humanize a non-human antibody using techniques known
in the art. In an embodiment of the invention, human heavy chain
and light chain acceptor sequences are selected from the framework
sequences listed in publicly available databases such as V-base
(hypertext transfer protocol://vbase.mrc-cpe.cam.ac.uk/) or in the
international ImMunoGeneTics.RTM. (IMGT.RTM.) information system
(hypertext transfer
protocol://imgt.cines.fr/texts/IMGTrepertoire/LocusGenes/). Table
3, below, provides a non-limiting list of examples of human heavy
chain acceptor sequences known in the art. Table 4, below, provides
a non-limiting list of examples of human light chain acceptor
sequences known in the art. In an embodiment of the invention,
human heavy chain and light chain acceptor sequences are selected
from the amino acid sequences described in Table 3 and Table 4,
below, however, other human heavy chain and light acceptors
sequences not listed in Tables 3 and 4 may also be used to humanize
an antibody according to the invention.
TABLE-US-00008 TABLE 3 Heavy Chain Acceptor Sequences. Protein
region/ SEQ Closest ID Germline Amino Acid Sequence NO: Family
12345678901234567890123456789012 11 VH3-7 FR1
EVQLVESGGGLVQPGGSLRLSCAASGF TFS 12 VH3-7 FR2 WVRQAPGKGLEWVA 13
VH3-7 FR3 RFTISRDNAKNSLYLQMNSLRAEDTAV YYCAR 14 JH4 FR4 WGQGTLVTVSS
15 VH3 CONSENUSUS EVQLVESGGGLVQPGGSLRLSCAASGF FR1 TFS 16 VH3
CONSENUSUS WVRQAPGKGLEWVS FR2 17 VH3 CONSENUSUS
RFTISRDNSKNTLYLQMNSLRAEDTAV FR3 YYCAR 18 JH4 FR4 WGQGTLVTVSS 19
VH1-46 FR1 QVQLVQSGAEVKKPGASVKVSCKASG YTFT 20 VH1-46 FR2
WVRQAPGQGLEWMG 21 VH1-46 FR3 RVTMTRDTSTSTVYMELSSLRSEDTAV YYCAR 22
JH4 FR4 WGQGTLVTVSS 23 VH3-30 FR1 QVQLVESGGGVVQPGRSLRLSCAASGF TFS
24 VH3-30 FR2 WVRQAPGKGLEWVA 25 VH3-30 FR3
RFTISRDNSKNTLYLQMNSLRAEDTAV YYCAR 26 JH3 FR4 WGQGTMVTVSS 27 VH3
CONSENUSUS EVQLVESGGGLVQPGGSLRLSCAASGF FR1 TFS 28 VH3 CONSENUSUS
WVRQAPGKGLEWVS FR2 29 VH3 CONSENUSUS RFTISRDNSKNTLYLQMNSLRAEDTAV
FR3 YYCAR 30 JH3 FR4 WGQGTMVTVSS 31 VH2-70/JH6 FR1
EVTLRESGPALVKPTQTLTLTCTFSGFS LS 32 VH2-70/JH6 FR2 WIRQPPGKALEWLA 33
VH2-70/JH6 FR3 RLTISKDTSKNQVVLTMTNMDPVDTAT YYCAR 34 VH2-70/JH6 FR4
WGQGTTVTVSS 35 VH2-26/JH6 FR1 EVTLKESGPVLVKPTETLTLTCTVSGFS LS 36
VH2-26/JH6 FR2 WIRQPPGKALEWLA 37 VH2-26/JH6 FR3
RLTISKDTSKSQVVLTMTNMDPVDTAT YYCAR 38 VH2-26/JH6 FR4 WGQGTTVTVSS 39
VH3-72/JH6 FR1 EVQLVESGGGLVQPGGSLRLSCAASGF TFS 40 VH3-72/JH6 FR2
WVRQAPGKGLEWVG 41 VH3-72/JH6 FR3 RFTISRDDSKNSLYLQMNSLKTEDTAV YYCAR
42 VH3-72/JH6 FR4 WGQGTTVTVSS 43 VH3-21/JH6 FR1
EVQLVESGGGLVKPGGSLRLSCAASGF TFS 44 VH3-21/JH6 FR2 WVRQAPGKGLEWVS 45
VH3-21/JH6 FR3 RFTISRDNAKNSLYLQMNSLRAEDTAV YYCAR 46 VH3-21/JH6 FR4
WGQGTTVTVSS 47 VH1-69/JH6 FR1 EVQLVQSGAEVKKPGSSVKVSCKASG GTFS 48
VH1-69/JH6 FR2 WVRQAPGQGLEWMG 49 VH1-69/JH6 FR3
RVTITADKSTSTAYMELSSLRSEDTAVY YCAR 50 VH1-69/JH6 FR4 WGQGTTVTVSS 51
VH1-18/JH6 FR1 EVQLVQSGAEVKKPGASVKVSCKASG YTFT 52 VH1-18/JH6 FR2
WVRQAPGQGLEWMG 53 VH1-18/JH6 FR3 RVTMTTDTSTSTAYMELRSLRSDDTAV YYCAR
54 VH1-18/JH6 FR4 WGQGTTVTVSS 55 IGHV4-59 FR1
EVQLQESGPGLVKPSETLSLTCTVSGGS IS 56 IGHV4-59 FR2 WIRQPPGKGLEWIG 57
IGHV4-59 FR3 RVTISVDTSKNQFSLKLSSVTAADTAV YYCAR 58 IGHV4-59/
WGQGTLVTVSS JH FR4 59 IGHV3-66 FR1 EVQLVESGGGLVQPGGSLRLSCAVSGG SIS
60 IGHV3-66 FR2 WIRQAPGKGLEWIG 61 IGHV3-66 FR3
RVTISVDTSKNSFYLQMNSLRAEDTAV YYCAR 62 IGHV3-66/ WGQGTLVTVSS JH FR4
63 IGHV4-59 FR1 EVQLQESGPGLVKPGETLSLTCTVSGGS IS 64 IGHV4-59 FR2
WIRQAPGKGLEWIG 65 IGHV4-59 FR3 RVTISVDTSKNQFYLKLSSVRAEDTAV YYCAR 66
IGHV4-59/ WGQGTLVTVSS JH FR4 67 IGHV5-51 FR1
EVQLVQSGTEVKKPGESLKISCKVSGG SIS 68 IGHV5-51 FR2 WIRQMPGKGLEWIG 69
IGHV5-51 FR3 QVTISVDTSFNTFFLQWSSLKASDTAM YYCAR 70 IGHV5-51/JH
WGQGTMVTVSS FR4 71 IGHV2-70 FR1 EVTLRESGPALVKPTQTLTLTCTVSGGS IS 72
IGHV2-70 FR2 WIRQPPGKGLEWIG 73 IGHV2-70 FR3
RVTISVDTSKNQFVLTMTNMDPVDTAT YYCAR 74 IGHV2-70/ WGQGTTVTVSS JH FR4
75 IGHV3-15 FR1 EVQLLESGGGLVKSGGSLRLSCAASGF TFR 76 IGHV3-15 FR2
WVRQAPGKGLEWVA 77 IGHV3-15 FR3 RFTISRDNSKNTLYLQLNSLRAEDTAV YYCAK 78
IGHV3-15/ WGQGTMVTVSS JH FR4 79 IGHV3-43 FR1
EVQLVESGGGVVQPGGSLRLSCAASGF TFG 80 IGHV3-43 FR2 WVRQAPGKGLEWVA 81
IGHV3-43 FR3 RFTISRDNSKNTLYLQLNSLRAEDTAV YYCAK 82 IGHV3-43/
WGQGTMVTVSS JH FR4
TABLE-US-00009 TABLE 4 Light Chain Acceptor Sequences Protein
region/ SEQ Closest ID Germline Sequence NO.: Family
12345678901234567890123456789012 83 O2 FR1 DIQMTQSPSSLSASVGDRVTITC
84 O2 FR2 WYQQKPGKAPKLLIY 85 O2 FR3
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC 86 JK2 FR4 FGQGTKLEIK 87 L2 FR1
EIVMTQSPATLSVSPGERATLSC 88 L2 FR2 WYQQKPGQAPRLLIY 89 L2 FR3
GIPARFSGSGSGTEFTLTISSLQSEDFAVYYC 90 JK2 FR4 FGQGTKLEIK 91 B3/JK4
FR1 DIVMTQSPDSLAVSLGERATINC 92 B3/JK4 FR2 WYQQKPGQPPKLLIY 93 B3/JK4
FR3 GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC 94 B3/JK4 FR4 FGGGTKVEIKR 95
L2/JK4 FR1 EIVMTQSPATLSVSPGERATLSC 96 L2/JK4 FR2 WYQQKPGQAPRLLIY 97
L2/JK4 FR3 GIPARFSGSGSGTEFTLTISSLQSEDFAVYYC 98 L2/JK4 FR4
FGGGTKVEIKR 99 L15/JK4 FR1 DIQMTQSPSSLSASVGDRVTITC 100 L15/JK4 FR2
WYQQKPEKAPKSLIY 101 L15/JK4 FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC
102 L15/JK4 FR4 FGGGTKVEIKR 103 L5/JK4 FR1 DIQMTQSPSSVSASVGDRVTITC
104 L5/JK4 FR2 WYQQKPGKAPKLLIY 105 L5/JK4 FR3
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC 106 L5/JK4 FR4 FGGGTKVEIKR 107
IGLV3-1 FR1 SYELTQPPSVSVSPGQTASITC 108 IGLV3-1 FR2 WYQQKPGQSPVLVIY
109 IGLV3-1 FR3 GIPERFSGSNSGDTATLTISGTQPMDEADYYC 110 IGLV3-1/
FGYGTKVTVL JL FR4 111 IGLV3-1 FR1 SYELTQPPSVSVSPGQTASITC 112
IGLV3-1 FR2 WYQQKPGQSPVLVIY 113 IGLV3-1 FR3
GIPERFSGSNSGDTATLTISGTQPMDEADYYC 114 IGLV3-1/ GGGTKLTVLG JL FR4 115
IGLV3-1 FR1 YELTQPPSVSVSPGQTASITC 116 IGLV3-1 FR2 WYQQKPGQSPVLVIY
117 IGLV3-1 FR3 GIPERFSGSNSGDTATLTISGTQPMDEADYYC 118 IGLV3-1/
GGGTKLTVLG JL FR4 119 IGLV3-1 FR1 LYVLTQPPSVSVSPGQTASITC 120
IGLV3-1 FR2 WYQQKPGQSPVLVIY 121 IGLV3-1 FR3
GIPERFSGSNSGDTATLTISGTQTMDEADYLC 122 IGLV3-1/ FGGGTKVTVLG JL FR4
123 IGKV6D-21 FR1 EYVLTQSPDFQSVTPKEKVTITC 124 IGKV6D-21 FR2
WYQQKPDQSPKLVIY 125 IGKV6D-21 FR3 GVPSRFSGSNSGDDATLTINSLEAEDAATYYC
126 IGKV6D-21/JK FGQGTKVEIKR FR4 127 IGKV3D-15 FR1
EYVLTQSPATLSVSPGERATLSC 128 IGKV3D-15 FR2 WYQQKPGQSPRLVIY 129
IGKV3D-15 FR3 DIPARFSGSNSGDEATLTISSLQSEDFAVYYC 130 IGKV3D-15/JK
FGQGTRLEIKR FR4 131 IGKV4-1 FR1 DYVLTQSPDSLAVSLGERATINC 132 IGKV4-1
FR2 WYQQKPGQSPKLVIY 133 IGKV4-1 FR3
GIPDRFSGSNSGDDATLTISSLQAEDVAVYYC 134 IGKV4-1/ FGGGTKVEIKR JK FR4
135 IGLV3-1 FR1 LPVLTQPPSVSVSPGQTASITC 136 IGLV3-1 FR2
WYQQKPGQSPVLVIY 137 IGLV3-1 FR3 GIPERFSGSNSGNTATLTISGTQTMDEADYLC
138 IGLV3-1/ FGGGTKVTVL JL FR4 139 IGLV3-1 FR1
SYELTQPPSVSVSPGQTASITC 140 IGLV3-1 FR2 WYQQKPGQSPVLVIY 141 IGLV3-1
FR3 GIPERFSGSNSGNTATLTISGTQTMDEADYLC 142 IGLV3-1/ FGGGTKLTVL JL
FR4
[0601] In an embodiment, heavy chain human acceptor framework
sequences from Table 3 for use in generating humanized antibodies
that bind DLL4 according to the invention include a set consisting
of the VH3-7 FR1, the VH3-7 FR2, the VH3-7 FR3, and the JH4 FR4
acceptor sequences; a set consisting of the VH3 consensus FR1, the
VH3 consensus FR2, the VH3 consensus FR3, and the JH4 FR4 acceptor
sequences; a set consisting of the VH1-46 FR1, the VH1-46 FR2, the
VH1-46 FR3, and the JH4 FR4 acceptor sequences; a set consisting of
the VH3-30 FR1, the VH3-30 FR2, the VH3-30 FR3, and the JH3 FR4
acceptor sequences; and a set consisting of the VH3 consensus FR1,
the VH3 consensus FR2, the VH3 consensus FR3, and the JH3 FR4
acceptor sequences.
[0602] In an embodiment, light chain human acceptor framework
sequences from Table 4 for use in generating humanized antibodies
that bind DLL4 according to the invention include a set consisting
of the O2 FR1, O2 FR2, O2 FR3, and JK2 FR4 acceptors sequences and
a set a consisting of the L2 FR1, L2 FR2, L2 FR3, and JK2 FR4
acceptor sequences.
[0603] In an embodiment, a set of human acceptor framework
sequences for use in generating a humanized antibody that binds
DLL4 according to the invention comprises one or more (e.g., any
one, two, three, four, five, six, seven, or eight per binding
domain) of the acceptor framework sequences selected from the group
consisting of: [0604] heavy chain framework-1 (H-FR1):
E-V-Q-L-V-E-S-G-G-G-L-V-Q-P-G-G-S-L-R-L-S-C-A-A-S-G-F-T-F-X.sub.30
(SEQ ID NO:143), wherein X.sub.30 is S, R, or G; [0605] heavy chain
framework-2 (H-FR2): W-V-R-Q-A-P-G-K-G-L-E-W-V-A (SEQ ID NO:144);
[0606] heavy chain framework-3 (H-FR3):
R-F-T-I-S-R-D-N-A-K-X.sub.11-S-L-Y-L-Q-M-N-S-L-R-A-E-D-T-A-V-Y-Y-C-X.sub.-
31-R (SEQ ID NO:145), wherein; [0607] X.sub.11 is N or S; and
[0608] X.sub.31 is A or S; [0609] heavy chain framework-4 (H-FR4):
W-G-Q-G-T-L-V-T-V-S-S(SEQ ID NO:146); [0610] light chain
framework-1 (L-FR1):
D-I-Q-M-T-Q-S-P-S-S-L-S-A-S-V-G-D-R-V-T-I-T-C(SEQ ID NO:147);
[0611] light chain framework-2 (L-FR2):
W-Y-Q-Q-K-P-G-K-X.sub.9-P-K-L-L-I-X.sub.15 (SEQ ID NO:148),
wherein; [0612] X.sub.9 is A or S; and [0613] X.sub.15 is F or Y;
[0614] light chain framework-3 (L-FR3):
G-V-P-S-R-F-S-G-S-G-S-G-T-D-X.sub.15-T-L-T-I-S-S-L-Q-P-E-D-F-A-T-Y-Y-C(SE-
Q ID NO:149), wherein; [0615] X.sub.15 is F or S; and [0616] light
chain framework-4 (L-FR4): F-G-Q-G-T-K-L-E-I-K (SEQ ID NO:150).
[0617] In a preferred embodiment, an antibody that binds DLL4
according to the invention is humanized using a set of human
acceptor sequences consisting of an H-FR1, H-FR2, H-FR3, H-FR-4,
L-FR1, L-FR2, L-FR3, and L-FR4 acceptor sequence described
above.
[0618] As used herein, the term "germline antibody gene" or "gene
fragment" refers to an immunoglobulin sequence encoded by
non-lymphoid cells that have not undergone the maturation process
that leads to genetic rearrangement and mutation for expression of
a particular immunoglobulin. (See, e.g., Shapiro et al., Crit. Rev.
Immunol., 22(3): 183-200 (2002); Marchalonis et al., Adv. Exp. Med.
Biol., 484:13-30 (2001)). One of the advantages provided by various
embodiments of the present invention stems from the recognition
that germline antibody genes are more likely than mature antibody
genes to conserve essential amino acid sequence structures
characteristic of individuals in the species, hence less likely to
be recognized as from a foreign source when used therapeutically in
that species.
[0619] As used herein, the term "key residue" refers to certain
residues within the variable region that have more impact on the
binding specificity and/or affinity of an antibody, in particular a
humanized antibody. A key residue includes, but is not limited to,
one or more of the following: a residue that is adjacent to a CDR,
a potential glycosylation site (can be either N- or O-glycosylation
site), a rare residue, a residue capable of interacting with the
antigen, a residue capable of interacting with a CDR, a canonical
residue, a contact residue between heavy chain variable region and
light chain variable region, a residue within the Vernier zone, and
a residue in the region that overlaps between the Chothia
definition of a variable heavy chain CDR1 and the Kabat definition
of the first heavy chain framework.
[0620] As used herein, "Vernier" zone refers to a subset of
framework residues that may adjust CDR structure and fine-tune the
fit to antigen as described by Foote and Winter (J. Mol. Biol.,
224: 487-499 (1992)). Vernier zone residues form a layer underlying
the CDRs and may impact on the structure of CDRs and the affinity
of the antibody.
[0621] As used herein, the term "neutralizing" refers to
counteracting the biological activity of an antigen when a binding
protein specifically binds the antigen. In an embodiment, the
neutralizing binding protein binds an antigen and reduces its
biological activity by at least about 20%, 40%, 60%, 80%, 85%, 90%,
95%, or more.
[0622] The term "activity" includes activities such as the binding
specificity/affinity of an antibody for an antigen, for example, an
anti-hDLL4 antibody that binds to an DLL4 antigen and/or the
neutralizing potency of an antibody, or an anti-hDLL4 antibody
whose binding to hDLL4 inhibits the biological activity of hDLL4,
e.g. inhibition of PHA blast proliferation or inhibition of
receptor binding in a human Notch receptor binding assay, or PHA
blast interferon-gamma induction assay.
[0623] The term "epitope" includes any polypeptide determinant that
specifically binds to an immunoglobulin or T-cell receptor. In
certain embodiments, epitope determinants include chemically active
surface groupings of molecules such as amino acids, sugar side
chains, phosphoryl, or sulfonyl, and, in certain embodiments, may
have specific three dimensional structural characteristics, and/or
specific charge characteristics. An epitope is a region of an
antigen that is bound by an antibody. An epitope thus consists of
the amino acid residues of a region of an antigen (or fragment
thereof) known to bind to the complementary site on the specific
binding partner. An antigen or antigenic fragment can contain more
than one epitope. Thus, it is understood by persons skilled in this
art that every "antigen binding site" of an antibody molecule binds
an epitope of an antigen molecule and every antigen molecule may
have one, two, several, or many epitopes. Moreover, it is
understood by persons skilled in this art that two independently
isolated antibodies to an antigen molecule may bind at the same
epitope or at two different epitopes on the antigen molecule.
[0624] In certain embodiments, an antibody is said to specifically
bind an antigen when it recognizes its target antigen in a complex
mixture of proteins and/or macromolecules. Antibodies are said to
"bind to the same epitope" if the antibodies cross-compete (one
prevents the binding or modulating effect of the other). In
addition, structural definitions of epitopes (overlapping, similar,
identical) are informative, but functional definitions are often
more relevant as they encompass structural (binding) and functional
(modulation, competition) parameters.
[0625] The term "surface plasmon resonance," as used herein, refers
to an optical phenomenon that allows for the analysis of real-time
biospecific interactions by detection of alterations in protein
concentrations within a biosensor matrix, for example using the
BIAcore.RTM. system (BIAcore International AB, a GE Healthcare
company, Uppsala, Sweden and Piscataway, N.J., US). For further
descriptions, see Jonsson et al., Ann. Biol. Clin., 51: 19-26
(1993); Jonsson et al. BioTechniques, 11: 620-627 (1991); Johnsson
et al., J. Mol. Recognit., 8: 125-131 (1995); and Johnsson et al.,
Anal. Biochem., 198: 268-277 (1991).
[0626] The term "K.sub.on", as used herein, is intended to refer to
the on rate constant for association of a binding protein (e.g., an
antibody) to a cognate partner (e.g., an antigen) to form a binding
partner/cognate partner (e.g., antibody/antigen) complex as is
known in the art. The "K.sub.on" also is known by the terms
"association rate constant," or "k.sub.a," as used interchangeably
herein. This value indicating the binding rate of an antibody to
its target antigen or the rate of complex formation between an
antibody and antigen also is shown by the equation:
Antibody("Ab")+Antigen("Ag").fwdarw.Ab-Ag.
[0627] The term "K.sub.off," as used herein, is intended to refer
to the off rate constant for dissociation of a binding protein
(e.g., an antibody) from the, e.g., antibody/antigen complex as is
known in the art. The "K.sub.off" also is known by the terms
"dissociation rate constant" or "k.sub.d" as used interchangeably
herein. This value indicates the dissociation rate of an antibody
from its target antigen or separation of Ab-Ag complex over time
into free antibody and antigen as shown by the equation below:
Ab+Ag.rarw.Ab-Ag.
[0628] The terms "equilibrium dissociation constant" or "K.sub.D",
as used interchangeably herein, refer to the value obtained in a
titration measurement at equilibrium, or by dividing the
dissociation rate constant (K.sub.off) by the association rate
constant (K.sub.on). The association rate constant, the
dissociation rate constant, and the equilibrium dissociation
constant are used to represent the binding affinity of an antibody
to an antigen. Methods for determining association and dissociation
rate constants are well known in the art. Using fluorescence-based
techniques offers high sensitivity and the ability to examine
samples in physiological buffers at equilibrium. Other experimental
approaches and instruments such as a BIAcore.RTM. surface plasmon
resonance (biomolecular interaction analysis) assay can be used
(e.g., instrument available from BIAcore International AB, a GE
Healthcare company, Uppsala, Sweden). Additionally, a KinExA.RTM.
(Kinetic Exclusion Assay) assay, available from Sapidyne
Instruments (Boise, Id.) can also be used.
[0629] "Label" and "detectable label" mean a moiety attached to a
specific binding partner, such as an antibody or an analyte bound
by the antibody, e.g., to render the reaction between members of a
specific binding pair, such as an antibody and an analyte,
detectable. The specific binding partner, e.g., antibody or
analyte, so labeled is referred to as "detectably labeled". Thus,
the term "labeled binding protein" as used herein, refers to a
protein with a label incorporated that provides for the
identification of the binding protein. In an embodiment, the label
is a detectable marker that can produce a signal that is detectable
by visual or instrumental means, e.g., incorporation of a
radiolabeled amino acid or attachment to a polypeptide of biotinyl
moieties that can be detected by a marked avidin (e.g., an avidin
or a streptavidin containing a fluorescent marker or enzymatic
activity that can be detected by optical or colorimetric methods).
Examples of labels for polypeptides include, but are not limited
to, the following: radioisotopes or radionuclides (e.g., .sup.3H,
.sup.14C, .sup.35S, .sup.90Y, .sup.99Tc, .sup.111In, .sup.125I,
.sup.131I, .sup.177Lu, .sup.166Ho, and .sup.153Sm); chromogens,
fluorescent labels (e.g., FITC, rhodamine, and lanthanide
phosphors), enzymatic labels (e.g., horseradish peroxidase,
luciferase, alkaline phosphatase); chemiluminescent markers;
biotinyl groups; predetermined polypeptide epitopes recognized by a
secondary reporter (e.g., leucine zipper pair sequences, binding
sites for secondary antibodies, metal binding domains, and epitope
tags); and magnetic agents, such as gadolinium chelates.
Representative examples of labels commonly employed for
immunoassays include moieties that produce light, e.g., acridinium
compounds, and moieties that produce fluorescence, e.g.,
fluorescein. Other labels are known in the art or described herein.
In this regard, the moiety itself may not be detectably labeled but
may become detectable upon reaction with yet another moiety. Use of
"detectably labeled" is intended to encompass the latter type of
detectable labeling.
[0630] The term "antibody conjugate" refers to a binding protein,
such as an antibody, chemically linked to a second chemical moiety,
such as a therapeutic or cytotoxic agent. The term "agent" is used
herein to denote a chemical compound, a mixture of chemical
compounds, a biological macromolecule, or an extract made from
biological materials. Preferably, the therapeutic or cytotoxic
agents include, but are not limited to, pertussis toxin, taxol,
cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,
etoposide, tenoposide, vincristine, vinblastine, colchicine,
doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,
mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,
procaine, tetracaine, lidocaine, propranolol, and puromycin and
analogs or homologs thereof.
[0631] The terms "crystal" and "crystallized" as used herein, refer
to a binding protein (e.g., an antibody), or antigen binding
portion thereof, that exists in the form of a crystal. Crystals are
one form of the solid state of matter, which is distinct from other
forms such as the amorphous solid state or the liquid crystalline
state. Crystals are composed of regular, repeating,
three-dimensional arrays of atoms, ions, molecules (e.g., proteins
such as antibodies), or molecular assemblies (e.g.,
antigen/antibody complexes, including Fab/antigen complexes). These
three-dimensional arrays are arranged according to specific
mathematical relationships that are well-understood in the field.
The fundamental unit, or building block, that is repeated in a
crystal is called the asymmetric unit. Repetition of the asymmetric
unit in an arrangement that conforms to a given, well-defined
crystallographic symmetry provides the "unit cell" of the crystal.
Repetition of the unit cell by regular translations in all three
dimensions provides the crystal. See, Giege et al., In
Crystallization of Nucleic Acids and Proteins, a Practical
Approach, 2nd ed., (Ducruix and Giege, eds.) (Oxford University
Press, New York, 1999), chapter 1, pages 1-16.
[0632] The term "polynucleotide" means a polymeric form of two or
more nucleotides, either ribonucleotides or deoxyribonucleotides or
a modified form of either type of nucleotide. The term includes
single and double stranded forms of DNA.
[0633] The term "isolated polynucleotide" shall mean a
polynucleotide (e.g., of genomic, cDNA, or synthetic origin, or
some combination thereof) that, by virtue of its origin, the
"isolated polynucleotide" is not associated with all or a portion
of a polynucleotide with which the "isolated polynucleotide" is
found in nature; is operably linked to a polynucleotide that it is
not linked to in nature; or does not occur in nature as part of a
larger sequence.
[0634] The term "vector," is intended to refer to a nucleic acid
molecule capable of transporting another nucleic acid to which it
has been linked. One type of vector is a "plasmid", which refers to
a circular double stranded DNA loop into which additional DNA
segments may be ligated. Another type of vector is a viral vector,
wherein additional DNA segments may be ligated into the viral
genome. Certain vectors are capable of autonomous replication in a
host cell into which they are introduced (e.g., bacterial vectors
having a bacterial origin of replication and episomal mammalian
vectors). Other vectors (e.g., non-episomal mammalian vectors) can
be integrated into the genome of a host cell upon introduction into
the host cell, and thereby are replicated along with the host
genome. Moreover, certain vectors are capable of directing the
expression of genes to which they are operatively linked. Such
vectors are referred to herein as "recombinant expression vectors"
(or simply, "expression vectors"). In general, expression vectors
of utility in recombinant DNA techniques are often in the form of
plasmids. In the present specification, "plasmid" and "vector" may
be used interchangeably as the plasmid is the most commonly used
form of vector. However, the invention is intended to include such
other forms of expression vectors, such as viral vectors (e.g.,
replication defective retroviruses, adenoviruses and
adeno-associated viruses), which serve equivalent functions. RNA
versions of vectors (including RNA viral vectors) may also find use
in the invention.
[0635] The term "operably linked" refers to a juxtaposition wherein
the components described are in a relationship permitting them to
function in their intended manner. A control sequence "operably
linked" to a coding sequence is ligated in such a way that
expression of the coding sequence is achieved under conditions
compatible with the control sequences. "Operably linked" sequences
include expression control sequences that are contiguous with a
gene of interest, expression control sequences that act in trans,
i.e., located on a different nucleic acid molecule than a gene of
interest, as well as expression control sequences that are located
on the same nucleic acid molecule as, but at a distance from, a
gene of interest. The term "expression control sequence" as used
herein refers to polynucleotide sequences which are necessary to
effect the expression and processing of coding sequences to which
they are ligated. Expression control sequences include appropriate
transcription initiation, termination, promoter and enhancer
sequences; efficient RNA processing signals such as splicing and
polyadenylation signals; sequences that stabilize cytoplasmic mRNA;
sequences that enhance translation efficiency (i.e., Kozak
consensus sequence); sequences that enhance protein stability; and
when desired, sequences that enhance protein secretion. The nature
of such control sequences differs depending upon the host organism;
in prokaryotes, such control sequences generally include a
promoter, a ribosomal binding site, and a transcription termination
sequence; in eukaryotes, generally, such control sequences include
a promoter and a transcription termination sequence. The term
"control sequences" is intended to include components whose
presence is essential for expression and processing, and can also
include additional components whose presence is advantageous, for
example, leader sequences and fusion partner sequences.
[0636] "Transformation," refers to any process by which exogenous
nucleic acid (e.g., a DNA molecule) enters a host cell.
Transformation may occur under natural or artificial conditions
using various methods well known in the art. Transformation may
rely on any known method for the insertion of foreign nucleic acid
sequences into a prokaryotic or eukaryotic host cell. The method is
selected based on the host cell being transformed and may include,
but is not limited to, plasmid uptake across a cellular membrane,
viral infection, electroporation, lipofection, and particle
bombardment. Such "transformed" cells include stably transformed
cells in which the inserted DNA is capable of replication either as
an autonomously replicating plasmid or as part of the host
chromosome. They also include cells which transiently express the
inserted DNA or RNA for limited periods of time.
[0637] The term "recombinant host cell" (or simply "host cell"), is
intended to refer to a cell into which exogenous DNA has been
introduced. In an embodiment, the host cell comprises two or more
(e.g., multiple) nucleic acids encoding antibodies, such as, by way
of non-limiting example, the host cells described in U.S. Pat. No.
7,262,028. Such terms are intended to refer not only to the
particular subject cell, but, also to the progeny of such a cell.
Because certain modifications may occur in succeeding generations
due to either mutation or environmental influences, such progeny
may not, in fact, be identical to the parent cell, but are still
included within the scope of the term "host cell" as used herein.
In an embodiment, host cells include prokaryotic and eukaryotic
cells selected from any of the Kingdoms of life. In another
embodiment, eukaryotic cells include protist, fungal, plant and
animal cells. In another embodiment, host cells include but are not
limited to prokaryotic species, such Escherichia coli; mammalian
cell lines, such as CHO, HEK 293, COS, NS0, SP2, and PER.C6; the
insect cell line Sf9; and fungal cell species, such as
Saccharomyces cerevisiae.
[0638] Standard techniques may be used for recombinant DNA,
oligonucleotide synthesis, and tissue culture and transformation
(e.g., electroporation, lipofection). Enzymatic reactions and
purification techniques may be performed according to
manufacturer's specifications or as commonly accomplished in the
art or as described herein. The foregoing techniques and procedures
may be generally performed according to conventional methods well
known in the art and as described in various general and more
specific references that are cited and discussed throughout the
present specification. See, e.g., Sambrook et al., Molecular
Cloning: A Laboratory Manual, second ed. (Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, 1989).
[0639] "Transgenic organism," as known in the art, refers to an
organism having cells that contain a transgene, wherein the
transgene introduced into the organism (or an ancestor of the
organism) expresses a polypeptide not naturally expressed in the
organism. A "transgene" is a DNA construct, which is stably and
operably integrated into the genome of a cell from which a
transgenic organism develops, directing the expression of an
encoded gene product in one or more cell types or tissues of the
transgenic organism.
[0640] The term "regulate" and "modulate" are used interchangeably,
and, as used herein, refers to a change or an alteration in the
activity of a molecule of interest (e.g., the biological activity
of hDLL4). Modulation may be an increase or a decrease in the
magnitude of a certain activity or function of the molecule of
interest. Exemplary activities and functions of a molecule include,
but are not limited to, binding characteristics, enzymatic
activity, cell receptor activation, and signal transduction.
[0641] Correspondingly, the term "modulator," as used herein, is a
compound capable of changing or altering an activity or function of
a molecule of interest (e.g., the biological activity of hDLL4).
For example, a modulator may cause an increase or decrease in the
magnitude of a certain activity or function of a molecule compared
to the magnitude of the activity or function observed in the
absence of the modulator. In certain embodiments, a modulator is an
inhibitor, which decreases the magnitude of at least one activity
or function of a molecule. Exemplary inhibitors include, but are
not limited to, proteins, peptides, antibodies, peptibodies,
carbohydrates or small organic molecules. Peptibodies have been
described. See, e.g., PCT Publication No. WO01/83525.
[0642] The term "agonist", as used herein, refers to a modulator
that, when contacted with a molecule of interest, causes an
increase in the magnitude of a certain activity or function of the
molecule compared to the magnitude of the activity or function
observed in the absence of the agonist. Particular agonists of
interest may include, but are not limited to, members of the
Notch-signaling pathway, DLL4 polypeptides and nucleic acids,
carbohydrates, or any other molecules that bind to DLL4.
[0643] The term "antagonist" or "inhibitor", as used herein, refers
to a modulator that, when contacted with a molecule of interest
causes a decrease in the magnitude of a certain activity or
function of the molecule compared to the magnitude of the activity
or function observed in the absence of the antagonist. Particular
antagonists of interest include those that block or modulate the
biological or immunological activity of DLL4, especially human DLL4
(hDLL4). Antagonists and inhibitors of hDLL4 may include, but are
not limited to, proteins, nucleic acids, carbohydrates, or any
other molecule, which binds to hDLL4 and/or rodent DLL4.
[0644] As used herein, the term "effective amount" refers to the
amount of a therapy that is sufficient to reduce or ameliorate the
severity and/or duration of a disorder or one or more symptoms
thereof; inhibit or prevent the advancement of a disorder; cause
regression of a disorder; inhibit or prevent the recurrence,
development, onset, or progression of one or more symptoms
associated with a disorder; detect a disorder; or enhance or
improve the prophylactic or therapeutic effect(s) of another
therapy (e.g., prophylactic or therapeutic agent).
[0645] "Patient" and "subject" may be used interchangeably herein
to refer to an animal, such as a mammal, including a primate (for
example, a human, a monkey, and a chimpanzee), a non-primate (for
example, a cow, a pig, a camel, a llama, a horse, a goat, a rabbit,
a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, and
a whale), a bird (e.g., a duck or a goose), and a shark.
Preferably, a patient or subject is a human, such as a human being
treated or assessed for a disease, disorder, or condition; a human
at risk for a disease, disorder, or condition; a human having a
disease, disorder, or condition; and/or human being treated for a
disease, disorder, or condition. More preferably, a patient or
subject is being treated or assessed for cancer or other disease in
which the existing aberrant DLL4 expression supports the cancer or
other disease and inhibition or disruption of DLL4 activity is
desirable to treat the cancer or other disease.
[0646] The term "sample," as used herein, is used in its broadest
sense. A "biological sample," as used herein, includes, but is not
limited to, any quantity of a substance from a living thing or
formerly living thing. Such living things include, but are not
limited to, humans, mice, rats, monkeys, dogs, rabbits and other
animals. Such substances include, but are not limited to, blood,
(e.g., whole blood), plasma, serum, urine, amniotic fluid, synovial
fluid, endothelial cells, leukocytes, monocytes, other cells,
organs, tissues, bone marrow, lymph nodes and spleen.
[0647] "Component," "components," and "at least one component,"
refer generally to a capture antibody, a detection or conjugate
antibody, a control, a calibrator, a series of calibrators, a
sensitivity panel, a container, a buffer, a diluent, a salt, an
enzyme, a co-factor for an enzyme, a detection reagent, a
pretreatment reagent/solution, a substrate (e.g., as a solution), a
stop solution, and the like that can be included in a kit for assay
of a test sample, such as a patient urine, serum or plasma sample,
in accordance with the methods described herein and other methods
known in the art. Thus, in the context of the present disclosure,
"at least one component," "component," and "components" can include
a polypeptide or other analyte as above, such as a composition
comprising an analyte such as a polypeptide, which is optionally
immobilized on a solid support, such as by binding to an
anti-analyte (e.g., anti-polypeptide) antibody. Some components can
be in solution or lyophilized for reconstitution for use in an
assay.
[0648] "Risk" refers to the possibility or probability of a
particular event occurring either presently or at some point in the
future. "Risk stratification" refers to an array of known clinical
risk factors that allows physicians to classify patients into a
low, moderate, high or highest risk of developing a particular
disease, disorder or condition.
[0649] "Specific" and "specificity" in the context of an
interaction between members of a specific binding pair (e.g., an
antigen or fragment thereof and an antibody or antigen binding
fragment thereof) refer to the selective reactivity of the
interaction. The phrase "specifically binds to" and analogous
phrases refer to the ability of binding proteins, such as
antibodies (or antigen binding fragments thereof), to bind
specifically to a molecule of interest (or a fragment thereof) and
not bind specifically to other entities.
[0650] "Specific binding partner" is a member of a specific binding
pair. A specific binding pair comprises two different molecules,
which specifically bind to each other through chemical or physical
means. Therefore, in addition to antigen and antibody specific
binding pairs, other specific binding pairs can include biotin and
avidin (or streptavidin), carbohydrates and lectins, complementary
nucleotide sequences, effector and receptor molecules, cofactors
and enzymes, enzyme inhibitors and enzymes, and the like.
Furthermore, specific binding pairs can include members that are
analogs of the original specific binding members, for example, an
analyte-analog. Immunoreactive specific binding members include
antigens, antigen fragments, and antibodies, including monoclonal
and polyclonal antibodies as well as complexes, fragments, and
variants (including fragments of variants) thereof, whether
isolated or recombinantly produced.
[0651] "Variant" as used herein means a polypeptide that differs
from a given polypeptide (e.g., DLL4 polypeptide or anti-DLL4
antibody) in amino acid sequence by the addition (e.g., insertion),
deletion, or conservative substitution of amino acids, but that
retains the biological activity of the given polypeptide (e.g., a
variant DLL4 may compete with a wildtype DLL4 for binding with an
anti-DLL4 antibody if the variant DLL4 retains the original
antibody binding site (epitope) of the wildtype DLL4). A
conservative substitution of an amino acid, i.e., replacing an
amino acid with a different amino acid of similar properties (e.g.,
hydrophilicity and degree and distribution of charged regions) is
recognized in the art as typically involving a minor change. These
minor changes can be identified, in part, by considering the
hydropathic index of amino acids, as understood in the art (see,
e.g., Kyte et al., J. Mol. Biol., 157: 105-132 (1982)). The
hydropathic index of an amino acid is based on a consideration of
its hydrophobicity and charge. It is known in the art that amino
acids of similar hydropathic indexes can be substituted and still
retain protein function. In one aspect, amino acids having
hydropathic indexes of .+-.2 are substituted. The hydrophilicity of
amino acids also can be used to reveal substitutions that would
result in proteins retaining biological function. A consideration
of the hydrophilicity of amino acids in the context of a peptide
permits calculation of the greatest local average hydrophilicity of
that peptide, a useful measure that has been reported to correlate
well with antigenicity and immunogenicity (see, e.g., U.S. Pat. No.
4,554,101). Substitution of amino acids having similar
hydrophilicity values can result in peptides retaining biological
activity, for example immunogenicity, as is understood in the art.
In one aspect, substitutions are performed with amino acids having
hydrophilicity values within .+-.2 of each other. Both the
hydrophobicity index and the hydrophilicity value of amino acids
are influenced by the particular side chain of that amino acid.
Consistent with that observation, amino acid substitutions that are
compatible with biological function are understood to depend on the
relative similarity of the amino acids, and particularly the side
chains of those amino acids, as revealed by the hydrophobicity,
hydrophilicity, charge, size, and other properties. "Variant" also
can be used to describe a polypeptide or fragment thereof that has
been differentially processed, such as by proteolysis,
phosphorylation, or other post-translational modification, yet
retains its biological activity or antigen reactivity, e.g., the
ability to bind to DLL4. Use of "variant" herein is intended to
encompass fragments of a variant unless otherwise contradicted by
context.
[0652] The term "sample", as used herein, is used in its broadest
sense. A "biological sample", as used herein, includes, but is not
limited to, any quantity of a substance from a living thing or
formerly living thing. Such living things include, but are not
limited to, humans, mice, rats, monkeys, dogs, rabbits and other
animals. Such substances include, but are not limited to, blood,
serum, urine, synovial fluid, cells, organs, tissues, bone marrow,
lymph nodes, and spleen.
I. Antibodies that Bind Human DLL4.
[0653] One aspect of the present invention provides isolated rat
monoclonal antibodies, or antigen-binding portions thereof, that
bind to DLL4 with high affinity, a slow off rate, and/or high
neutralizing capacity. Another aspect of the invention provides
chimeric antibodies that bind DLL4. In another aspect, the
invention provides CDR grafted antibodies, or antigen-binding
portions thereof, that DLL4. Another aspect of the invention
provides humanized antibodies, or antigen-binding portions thereof,
that bind DLL4. In an embodiment, the antibodies, or portions
thereof, are isolated antibodies or isolated portions thereof. In
another embodiment, the antibodies, or antigen-binding portions
thereof, of the invention are neutralizing anti-DLL antibodies.
Advantageously, such antibodies or antigen-binding portions thereof
that bind DLL4 find use as therapeutic agents that can be
administered to an individual (human or other mammal). Preferably,
the antibodies or antigen-binding portions thereof of the invention
are neutralizing anti-DLL4 and/or anti-VEGFR2 antibodies.
A. Method of Making Anti-DLL4 Antibodies.
[0654] Antibodies of the present invention may be made by any of a
number of techniques known in the art. Aspects of various
techniques that may be employed to obtain DLL4 monoclonal
antibodies according to the invention are described below.
1. Anti-DLL4 Monoclonal Antibodies Using Hybridoma Technology.
[0655] Monoclonal antibodies can be prepared using a wide variety
of techniques known in the art including the use of hybridoma,
recombinant, and phage display technologies, or a combination
thereof. For example, monoclonal antibodies can be produced using
hybridoma techniques including those known in the art and taught,
for example, in Harlow et al., Antibodies: A Laboratory Manual,
second edition, (Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, 1988); Hammerling, et al., In Monoclonal Antibodies and
T-Cell Hybridomas, (Elsevier, New York, 1981). It is also noted
that the term "monoclonal antibody" as used herein is not limited
to antibodies produced through hybridoma technology. The term
"monoclonal antibody" refers to an antibody that is derived from a
single clone, including any eukaryotic, prokaryotic, or phage
clone, and not the method by which it is produced.
[0656] In an embodiment, the present invention provides methods of
generating monoclonal antibodies as well as antibodies produced by
the method comprising culturing a hybridoma cell secreting an
antibody of the invention wherein, preferably, the hybridoma is
generated by fusing splenocytes isolated from an animal, e.g., a
rat or a mouse, immunized with DLL4 with myeloma cells and then
screening the hybridomas resulting from the fusion for hybridoma
clones that secrete an antibody able to bind a polypeptide of the
invention. Briefly, rats can be immunized with a DLL4 antigen (see,
Examples, below). In a preferred embodiment, the DLL4 antigen is
administered with an adjuvant to stimulate the immune response.
Such adjuvants include complete or incomplete Freund's adjuvant,
RIBI (muramyl dipeptides) or ISCOM (immunostimulating complexes).
Such adjuvants may protect the polypeptide from rapid dispersal by
sequestering it in a local deposit, or they may contain substances
that stimulate the host to secrete factors that are chemotactic for
macrophages and other components of the immune system. Preferably,
if a polypeptide is being administered, the immunization schedule
will involve two or more administrations of the polypeptide, spread
out over several weeks; however, a single administration of the
polypeptide may also be used.
[0657] After immunization of an animal with a DLL4 antigen,
antibodies and/or antibody-producing cells may be obtained from the
animal. An anti-DLL4 antibody-containing serum is obtained from the
animal by bleeding or sacrificing the animal. The serum may be used
as it is obtained from the animal, an immunoglobulin fraction may
be obtained from the serum, or the anti-DLL4 antibodies may be
purified from the serum. Serum or immunoglobulins obtained in this
manner are polyclonal, thus having a heterogeneous array of
properties.
[0658] Once an immune response is detected, e.g., antibodies
specific for the antigen DLL4 are detected in the rat serum, the
rat spleen is harvested and splenocytes isolated. The splenocytes
are then fused by well-known techniques to any suitable myeloma
cells, for example cells from cell line SP20 available from the
American Type Culture Collection (ATCC, Manassas, Va., US).
Hybridomas are selected and cloned by limited dilution. The
hybridoma clones are then assayed by methods known in the art for
cells that secrete antibodies capable of binding DLL4. Ascites
fluid, which generally contains high levels of antibodies, can be
generated by immunizing rats with positive hybridoma clones.
[0659] In another embodiment, antibody-producing immortalized
hybridomas may be prepared from the immunized animal. After
immunization, the animal is sacrificed and the splenic B cells are
fused to immortalized myeloma cells as is well known in the art.
See, e.g., Harlow and Lane, supra. In a preferred embodiment, the
myeloma cells do not secrete immunoglobulin polypeptides (a
non-secretory cell line). After fusion and antibiotic selection,
the hybridomas are screened using DLL4, or a portion thereof, or a
cell expressing DLL4. In a preferred embodiment, the initial
screening is performed using an enzyme-linked immunosorbent assay
(ELISA) or a radioimmunoassay (RIA), preferably an ELISA. An
example of ELISA screening is provided in PCT Publication No. WO
00/37504.
[0660] Anti-DLL4 antibody-producing hybridomas are selected,
cloned, and further screened for desirable characteristics,
including robust hybridoma growth, high antibody production, and
desirable antibody characteristics, as discussed further below.
Hybridomas may be cultured and expanded in vivo in syngeneic
animals, in animals that lack an immune system, e.g., nude mice, or
in cell culture in vitro. Methods of selecting, cloning and
expanding hybridomas are well known to those of ordinary skill in
the art.
[0661] In a preferred embodiment, hybridomas are rat hybridomas, as
described herein. In another embodiment, hybridomas are produced in
a non-human, non-rat species such as mice, sheep, pigs, goats,
cattle, or horses. In yet another preferred embodiment, the
hybridomas are human hybridomas, in which a human non-secretory
myeloma is fused with a human cell expressing an anti-DLL4
antibody.
[0662] Antibody fragments that recognize specific epitopes may be
generated by known techniques. For example, Fab and F(ab').sub.2
fragments of the invention may be produced by proteolytic cleavage
of immunoglobulin molecules, using enzymes such as papain (to
produce two identical Fab fragments) or pepsin (to produce a
F(ab').sub.2 fragment). A F(ab').sub.2 fragment of an IgG molecule
retains the two antigen-binding sites of the larger ("parent") IgG
molecule, including both light chains (containing the variable
light chain and constant light chain regions), the CH1 domains of
the heavy chains, and a disulfide-forming hinge region of the
parent IgG molecule. Accordingly, a F(ab').sub.2 fragment is still
capable of crosslinking antigen molecules like the parent IgG
molecule.
2. Anti-DLL4 Monoclonal Antibodies Using SLAM.
[0663] In another aspect of the invention, recombinant antibodies
are generated from single, isolated lymphocytes using a procedure
referred to in the art as the selected lymphocyte antibody method
(SLAM), as described in U.S. Pat. No. 5,627,052; PCT Publication
No. WO 92/02551; and Babcook et al., Proc. Natl. Acad. Sci. USA,
93: 7843-7848 (1996). In this method, single cells secreting
antibodies of interest, e.g., lymphocytes derived from any one of
the immunized animals described in Section I.A.1 (above), are
screened using an antigen-specific hemolytic plaque assay, wherein
the antigen DLL4, a subunit of DLL4, or a fragment thereof, is
coupled to sheep red blood cells using a linker, such as biotin,
and used to identify single cells that secrete antibodies with
specificity for DLL4. Following identification of
antibody-secreting cells of interest, heavy- and light-chain
variable region cDNAs are rescued from the cells by reverse
transcriptase-PCR (RT-PCR) and these variable regions can then be
expressed, in the context of appropriate immunoglobulin constant
regions (e.g., human constant regions), in mammalian host cells,
such as COS or CHO cells. The host cells transfected with the
amplified immunoglobulin sequences, derived from in vivo selected
lymphocytes, can then undergo further analysis and selection in
vitro, for example, by panning the transfected cells to isolate
cells expressing antibodies to DLL4. The amplified immunoglobulin
sequences further can be manipulated in vitro, such as by in vitro
affinity maturation method. See, for example, PCT Publication No.
WO 97/29131 and PCT Publication No. WO 00/56772.
3. Anti-DLL4 Monoclonal Antibodies Using Transgenic Animals.
[0664] In another embodiment of the instant invention, antibodies
are produced by immunizing a non-human animal comprising some, or
all, of the human immunoglobulin locus with a DLL4 antigen. In an
embodiment, the non-human animal is a XENOMOUSE.RTM. transgenic
mouse, an engineered mouse strain that comprises large fragments of
the human immunoglobulin loci and is deficient in mouse antibody
production. See, e.g., Green et al., Nature Genetics, 7: 13-21
(1994) and U.S. Pat. Nos. 5,916,771; 5,939,598; 5,985,615;
5,998,209; 6,075,181; 6,091,001; 6,114,598; and 6,130,364. See also
PCT Publication Nos. WO 91/10741; WO 94/02602; WO 96/34096; WO
96/33735; WO 98/16654; WO 98/24893; WO 98/50433; WO 99/45031; WO
99/53049; WO 00/09560; and WO 00/37504. The XENOMOUSE.RTM.
transgenic mouse produces an adult-like human repertoire of fully
human antibodies, and generates antigen-specific human monoclonal
antibodies. The XENOMOUSE.RTM. transgenic mouse contains
approximately 80% of the human antibody repertoire through
introduction of megabase sized, germline configuration YAC
fragments of the human heavy chain loci and x light chain loci. See
Mendez et al., Nature Genetics, 15: 146-156 (1997), Green and
Jakobovits, J. Exp. Med., 188: 483-495 (1998), the disclosures of
which are hereby incorporated by reference.
4. Anti-DLL4 Monoclonal Antibodies Using Recombinant Antibody
Libraries.
[0665] In vitro methods also can be used to make the antibodies of
the invention, wherein an antibody library is screened to identify
an antibody having the desired DLL4-binding specificity. Methods
for such screening of recombinant antibody libraries are well known
in the art and include methods described in, for example, U.S. Pat.
No. 5,223,409 (Ladner et al.); PCT Publication No. WO 92/18619
(Kang et al.); PCT Publication No. WO 91/17271 (Dower et al.); PCT
Publication No. WO 92/20791 (Winter et al.); PCT Publication No. WO
92/15679 (Markland et al.); PCT Publication No. WO 93/01288
(Breitling et al.); PCT Publication No. WO 92/01047 (McCafferty et
al.); PCT Publication No. WO 92/09690 (Garrard et al.); Fuchs et
al., Bio/Technology, 9: 1369-1372 (1991); Hay et al., Hum. Antibod.
Hybridomas, 3: 81-85 (1992); Huse et al., Science, 246: 1275-1281
(1989); McCafferty et al., Nature, 348: 552-554 (1990); Griffiths
et al., EMBO J., 12: 725-734 (1993); Hawkins et al., J. Mol. Biol.,
226: 889-896 (1992); Clackson et al., Nature, 352: 624-628 (1991);
Gram et al., Proc. Natl. Acad. Sci. USA, 89: 3576-3580 (1992);
Garrard et al., Bio/Technology, 9: 1373-1377 (1991); Hoogenboom et
al., Nucl. Acids Res., 19: 4133-4137 (1991); Barbas et al., Proc.
Natl. Acad. Sci. USA, 88: 7978-7982 (1991); US Patent Application
Publication No. 2003/0186374; and PCT Publication No. WO 97/29131,
the contents of each of which are incorporated herein by
reference.
[0666] The recombinant antibody library may be from a subject
immunized with DLL4, or a portion of DLL4. Alternatively, the
recombinant antibody library may be from a naive subject, i.e., one
who has not been immunized with DLL4, such as a human antibody
library from a human subject who has not been immunized with human
DLL4. Antibodies of the invention are selected by screening the
recombinant antibody library with the peptide comprising human DLL4
to thereby select those antibodies that recognize DLL4. Methods for
conducting such screening and selection are well known in the art,
such as described in the references in the preceding paragraph. To
select antibodies of the invention having particular binding
affinities for DLL4, such as those that dissociate from human DLL4
with a particular K.sub.off rate constant, the art-known method of
surface plasmon resonance can be used to select antibodies having
the desired K.sub.off rate constant. To select antibodies of the
invention having a particular neutralizing activity for hDLL4, such
as those with a particular IC.sub.50, standard methods known in the
art for assessing the inhibition of DLL4 activity may be used.
[0667] In one aspect, the invention pertains to an isolated
antibody, or an antigen-binding portion thereof, that binds human
DLL4. Preferably, the antibody is a neutralizing antibody. In
various embodiments, the antibody is a recombinant antibody or a
monoclonal antibody.
[0668] For example, antibodies of the present invention can also be
generated using various phage display methods known in the art. In
phage display methods, functional antibody domains are displayed on
the surface of phage particles which carry the polynucleotide
sequences encoding them. Such phage can be utilized to display
antigen-binding domains expressed from a repertoire or
combinatorial antibody library (e.g., human or murine). Phage
expressing an antigen binding domain that binds the antigen of
interest can be selected or identified with antigen, e.g., using
labeled antigen or antigen bound or captured to a solid surface or
bead. Phage used in these methods are typically filamentous phage
including fd and M13 binding domains expressed from phage with Fab,
Fv, or disulfide stabilized Fv antibody domains recombinantly fused
to either the phage gene III or gene VIII protein. Examples of
phage display methods that can be used to make the antibodies of
the present invention include those disclosed in Brinkmann et al.,
J. Immunol. Methods, 182: 41-50 (1995); Ames et al., J. Immunol.
Methods, 184:177-186 (1995); Kettleborough et al., Eur. J.
Immunol., 24: 952-958 (1994); Persic et al., Gene, 187: 9-18
(1997); Burton et al., Advances in Immunology, 57: 191-280 (1994);
PCT Publication No. WO 92/01047; PCT Publication Nos. WO 90/02809;
WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO
95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484;
5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908;
5,516,637; 5,780,225; 5,658,727; 5,733,743; and 5,969,108.
[0669] As described in the above references, after phage selection,
the antibody coding regions from the phage can be isolated and used
to generate whole antibodies including human antibodies or any
other desired antigen binding fragment, and expressed in any
desired host, including mammalian cells, insect cells, plant cells,
yeast, and bacteria, e.g., as described in detail below. For
example, techniques to recombinantly produce Fab, Fab', and
F(ab').sub.2 fragments can also be employed using methods known in
the art such as those disclosed in PCT publication No. WO 92/22324;
Mullinax et al., BioTechniques, 12(6): 864-869 (1992); Sawai et
al., Am. J. Reprod. Immunol., 34: 26-34 (1995); and Better et al.,
Science, 240: 1041-1043 (1988). Examples of techniques which can be
used to produce single-chain Fvs and antibodies include those
described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al.,
Methods in Enzymology, 203: 46-88 (1991); Shu et al., Proc. Natl.
Acad. Sci. USA, 90: 7995-7999 (1993); and Skerra et al., Science,
240: 1038-1041 (1988).
[0670] Alternative to screening of recombinant antibody libraries
by phage display, other methodologies known in the art for
screening large combinatorial libraries can be applied to the
identification of antibodies of the invention. One type of
alternative expression system is one in which the recombinant
antibody library is expressed as RNA-protein fusions, as described
in PCT Publication No. WO 98/31700 (Szostak and Roberts), and in
Roberts and Szostak, Proc. Natl. Acad. Sci. USA, 94: 12297-12302
(1997). In this system, a covalent fusion is created between an
mRNA and the peptide or protein that it encodes by in vitro
translation of synthetic mRNAs that carry puromycin, a peptidyl
acceptor antibiotic, at their 3' end. Thus, a specific mRNA can be
enriched from a complex mixture of mRNAs (e.g., a combinatorial
library) based on the properties of the encoded peptide or protein,
e.g., antibody, or portion thereof, such as binding of the
antibody, or portion thereof, to the dual specificity antigen.
Nucleic acid sequences encoding antibodies, or portions thereof,
recovered from screening of such libraries can be expressed by
recombinant means as described above (e.g., in mammalian host
cells) and, moreover, can be subjected to further affinity
maturation by either additional rounds of screening of mRNA-peptide
fusions in which mutations have been introduced into the originally
selected sequence(s), or by other methods for affinity maturation
in vitro of recombinant antibodies, as described above. A preferred
example of this methodology, is the PROfusion display technology
employed in the Examples (infra).
[0671] In another approach the antibodies of the present invention
can also be generated using yeast display methods known in the art.
In yeast display methods, genetic methods are used to tether
antibody domains to the yeast cell wall and display them on the
surface of yeast. In particular, such yeast can be utilized to
display antigen-binding domains expressed from a repertoire or
combinatorial antibody library (e.g., human or murine). Examples of
yeast display methods that can be used to make the antibodies of
the present invention include those disclosed in U.S. Pat. No.
6,699,658 (Wittrup et al.) incorporated herein by reference.
B. Production of Recombinant DLL4 Antibodies
[0672] Antibodies of the present invention may be produced by any
of a number of techniques known in the art. For example, expression
from host cells, wherein expression vector(s) encoding the heavy
and light chains is (are) transfected into a host cell by standard
techniques. The various forms of the term "transfection" are
intended to encompass a wide variety of techniques commonly used
for the introduction of exogenous DNA into a prokaryotic or
eukaryotic host cell, e.g., electroporation, calcium-phosphate
precipitation, DEAE-dextran transfection and the like. Although it
is possible to express the antibodies of the invention in either
prokaryotic or eukaryotic host cells, expression of antibodies in
eukaryotic cells is preferable, and most preferable in mammalian
host cells, because such eukaryotic cells (and in particular
mammalian cells) are more likely than prokaryotic cells to assemble
and secrete a properly folded and immunologically active
antibody.
[0673] Exemplary mammalian host cells for expressing the
recombinant antibodies of the invention include Chinese Hamster
Ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub
and Chasin, Proc. Natl. Acad. Sci. USA, 77: 4216-4220 (1980), used
with a DHFR selectable marker, e.g., as described in Kaufman and
Sharp, J. Mol. Biol., 159: 601-621 (1982), NS0 myeloma cells, COS
cells, and SP2 cells. When recombinant expression vectors encoding
antibody genes are introduced into mammalian host cells, the
antibodies are produced by culturing the host cells for a period of
time sufficient to allow for expression of the antibody in the host
cells or, more preferably, secretion of the antibody into the
culture medium in which the host cells are grown. Antibodies can be
recovered from the culture medium using standard protein
purification methods.
[0674] Host cells can also be used to produce functional antibody
fragments, such as Fab fragments or scFv molecules. It will be
understood that variations on the above procedure are within the
scope of the present invention. For example, it may be desirable to
transfect a host cell with DNA encoding functional fragments of
either the light chain and/or the heavy chain of an antibody of
this invention. Recombinant DNA technology may also be used to
remove some, or all, of the DNA encoding either or both of the
light and heavy chains that is not necessary for binding to the
antigens of interest. The molecules expressed from such truncated
DNA molecules are also encompassed by the antibodies of the
invention. In addition, bifunctional antibodies may be produced in
which one heavy and one light chain are an antibody of the
invention (i.e., binds human DLL4) and the other heavy and light
chain are specific for an antigen other than human DLL4 by
crosslinking an antibody of the invention to a second antibody by
standard chemical crosslinking methods.
[0675] In a preferred system for recombinant expression of an
antibody, or antigen-binding portion thereof, of the invention, a
recombinant expression vector encoding both the antibody heavy
chain and the antibody light chain is introduced into dhfr-CHO
cells by calcium phosphate-mediated transfection. Within the
recombinant expression vector, the antibody heavy and light chain
genes are each operatively linked to CMV enhancer/AdMLP promoter
regulatory elements to drive high levels of transcription of the
genes. The recombinant expression vector also carries a DHFR gene,
which allows for selection of CHO cells that have been transfected
with the vector using methotrexate selection/amplification. The
selected transformant host cells are cultured to allow for
expression of the antibody heavy and light chains and intact
antibody is recovered from the culture medium. Standard molecular
biology techniques are used to prepare the recombinant expression
vector, transfect the host cells, select for transformants, culture
the host cells and recover the antibody from the culture medium.
Still further the invention provides a method of synthesizing a
recombinant antibody of the invention by culturing a host cell of
the invention in a suitable culture medium until a recombinant
antibody of the invention is synthesized. The method can further
comprise isolating the recombinant antibody from the culture
medium.
1. Anti-DLL4 Antibodies.
[0676] Amino acid sequences of VH and VL regions of isolated rat
monoclonal antibodies that bind human DLL4 are shown for clones
38H12, 1A11, 37D10, 32C7, 14G1, 14A11, and 15D6 in Table 9 (See,
Example 4, below). The isolated anti-DLL4 antibody CDR sequences
described herein establish a family of DLL4 binding proteins,
isolated in accordance with this invention, and comprising
polypeptides that include the CDR sequences derived therefrom and
affinity matured clones thereof. Sequences of variable regions and
CDRs of the monoclonal antibodies and affinity matured derivatives
thereof are listed in Tables 9, 11, 16, 20, and 21. To generate and
to select CDRs for binding proteins according to the invention
having preferred DLL4 binding and/or neutralizing activity with
respect to human DLL4, standard methods known in the art for
generating binding proteins of the present invention and assessing
the DLL4 binding and/or neutralizing characteristics of those
binding protein may be used, including but not limited to those
specifically described herein.
[0677] Based on an alignment of the amino acid sequences of the
CDRs of the heavy chain variable regions (VH) and the light chain
variable regions (VL) of the anti-DLL4 antibody clones described
herein, the invention provides a DLL4 binding protein comprising an
antigen binding domain capable of binding human DLL4, said antigen
binding domain comprising at least one or more of the six CDRs,
i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDRL-3, defined
below:
[0678] CDR-H1 is selected from the group consisting of: [0679]
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5 (SEQ ID NO:151), wherein;
[0680] X.sub.1 is N, H, or Y; [0681] X.sub.2 is F; [0682] X.sub.3
is P; [0683] X.sub.4 is M; and [0684] X.sub.5 is A or S; [0685]
residues 31-35 of SEQ ID NO:157 (CDR-H1 38H12); [0686] residues
31-35 of SEQ ID NO:161 (CDR-H1 37D10); [0687] residues 31-35 of SEQ
ID NO:163 (CDR-H1 32C7); [0688] residues 31-35 of SEQ ID NO:165
(CDR-H1 14G1); [0689] residues 31-35 of SEQ ID NO:167 (CDR-H1
14A11); [0690] residues 31-35 of SEQ ID NO:169 (CDR-H1 15D6);
[0691] residues 31-35 of SEQ ID NO:171 (CDR-H1 VH.1 1A11); [0692]
residues 31-35 of SEQ ID NO:172 (CDR-H1 VH.1a 1A11); [0693]
residues 31-35 of SEQ ID NO:173 (CDR-H1 VH.1b 1A11); [0694]
residues 31-35 of SEQ ID NO:174 (CDR-H1 VH.2a 1A11); [0695]
residues 31-35 of SEQ ID NO:179 (CDR-H1 VH.1 38H12); [0696]
residues 31-35 of SEQ ID NO:180 (CDR-H1 VH.1A 38H12); [0697]
residues 31-35 of SEQ ID NO:181 (CDR-H1 VH.1b 38H12); [0698]
residues 31-35 of SEQ ID NO:182 (CDR-H1 VH.2a 38H12); [0699]
residues 31-35 of SEQ ID NO:187 (CDR-H1 h1A11VH.1); [0700] residues
31-35 of SEQ ID NO:188 (CDR-H1 h1A11.A6); [0701] residues 31-35 of
SEQ ID NO:189 (CDR-H1 h1A11.A8); [0702] residues 31-35 of SEQ ID
NO:190 (CDR-H1 h1A11.C6); [0703] residues 31-35 of SEQ ID NO:191
(CDR-H1 h1A11.A11); [0704] residues 31-35 of SEQ ID NO:192 (CDR-H1
h1A11.B5); [0705] residues 31-35 of SEQ ID NO:193 (CDR-H1
h1A11.E12); [0706] residues 31-35 of SEQ ID NO:194 (CDR-H1
h1A11.G3); [0707] residues 31-35 of SEQ ID NO:195 (CDR-H1
h1A11.F5); and [0708] residues 31-35 of SEQ ID NO:196 (CDR-H1
h1A11.H2);
[0709] CDR-H2 is selected from the group consisting of: [0710]
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X-
.sub.10-X.sub.11-X.sub.12-X.sub.13-X.sub.14-X.sub.15-X.sub.16-X.sub.17
(SEQ ID NO:152), wherein; [0711] X.sub.1 is T or S; [0712] X.sub.2
is I; [0713] X.sub.3 is S; [0714] X.sub.4 is S or G; [0715] X.sub.5
is S; [0716] X.sub.6 is D; [0717] X.sub.7 is G, A, D, S, or E;
[0718] X.sub.8 is T or W; [0719] X.sub.9 is T, P, or A; [0720]
X.sub.10 is Y, S, T, or N; [0721] X.sub.11 is Y or I; [0722]
X.sub.12 is R or G; [0723] X.sub.13 is D; [0724] X.sub.14 is 5;
[0725] X.sub.15 is V; [0726] X.sub.16 is K; and [0727] X.sub.17 is
G; [0728] residues 50-66 of SEQ ID NO:157 (CDR-H2 38H12); [0729]
residues 50-68 of SEQ ID NO:161 (CDR-H2 37D10); [0730] residues
50-66 of SEQ ID NO:163 (CDR-H2 32C7); [0731] residues 50-66 of SEQ
ID NO:165 (CDR-H2 14G1); [0732] residues 50-66 of SEQ ID NO:167
(CDR-H2 14A11); [0733] residues 50-66 of SEQ ID NO:169 (CDR-H2
15D6); [0734] residues 50-66 of SEQ ID NO:171 (CDR-H2 VH.1 1A11);
[0735] residues 50-66 of SEQ ID NO:172 (CDR-H2 VH.1a 1A11); [0736]
residues 50-66 of SEQ ID NO:173 (CDR-H2 VH.1b 1A11); [0737]
residues 50-66 of SEQ ID NO:174 (CDR-H2 VH.2a 1A11); [0738]
residues 50-66 of SEQ ID NO:179 (CDR-H2 VH.1 38H12); [0739]
residues 50-66 of SEQ ID NO:180 (CDR-H2 VH.1A 38H12); [0740]
residues 50-66 of SEQ ID NO:181 (CDR-H2 VH.1b 38H12); [0741]
residues 31-35 of SEQ ID NO:182 (CDR-H1 VH.2a 38H12); [0742]
residues 50-66 of SEQ ID NO:187 (CDR-H2 h1A11VH.1); [0743] residues
50-66 of SEQ ID NO:188 (CDR-H2 h1A11.A6); [0744] residues 50-66 of
SEQ ID NO:189 (CDR-H2 h1A11.A8); [0745] residues 50-66 of SEQ ID
NO:190 (CDR-H2 h1A11.C6); [0746] residues 50-66 of SEQ ID NO:191
(CDR-H2 h1A11.A11); [0747] residues 50-66 of SEQ ID NO:192 (CDR-H2
h1A11.B5); [0748] residues 50-66 of SEQ ID NO:193 (CDR-H2
h1A11.E12); [0749] residues 50-66 of SEQ ID NO:194 (CDR-H2
h1A11.G3); [0750] residues 50-66 of SEQ ID NO:195 (CDR-H2
h1A11.F5); and [0751] residues 50-66 of SEQ ID NO:196 (CDR-H2
h1A11.H2);
[0752] CDR-H3 is selected from the group consisting of: [0753]
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9
(SEQ ID NO:153), wherein; [0754] X.sub.1 is G; [0755] X.sub.2 is Y;
[0756] X.sub.3 is Y; [0757] X.sub.4 is N; [0758] X.sub.5 is S;
[0759] X.sub.6 is P; [0760] X.sub.7 is F; [0761] X.sub.8 is A; and
[0762] X.sub.9 is Y, F, or S; [0763] residues 99-107 of SEQ ID
NO:157 (CDR-H3 38H12); [0764] residues 101-111 of SEQ ID NO:161
(CDR-H3 37D10); [0765] residues 99-105 of SEQ ID NO:163 (CDR-H3
32C7); [0766] residues 99-105 of SEQ ID NO:165 (CDR-H3 14G1);
[0767] residues 99-110 of SEQ ID NO:167 (CDR-H3 14A11); [0768]
residues 99-110 of SEQ ID NO:169 (CDR-H3 15D6); [0769] residues
99-107 of SEQ ID NO:171 (CDR-H3 VH.1 1A11); [0770] residues 99-107
of SEQ ID NO:172 (CDR-H3 VH.1a 1A11); [0771] residues 99-107 of SEQ
ID NO:173 (CDR-H3 VH.1b 1A11); [0772] residues 99-107 of SEQ ID
NO:174 (CDR-H3 VH.2a 1A11); [0773] residues 99-107 of SEQ ID NO:179
(CDR-H3 VH.1 38H12); [0774] residues 99-107 of SEQ ID NO:180
(CDR-H3 VH.1A 38H12); [0775] residues 99-107 of SEQ ID NO:181
(CDR-H2 VH.1b 38H12); [0776] residues 99-107 of SEQ ID NO:182
(CDR-H1 VH.2a 38H12); [0777] residues 99-107 of SEQ ID NO:187
(CDR-H3 h1A11VH.1); [0778] residues 99-107 of SEQ ID NO:188 (CDR-H3
h1A11.A6); [0779] residues 99-107 of SEQ ID NO:189 (CDR-H3
h1A11.A8); [0780] residues 99-107 of SEQ ID NO:190 (CDR-H3
h1A11.C6); [0781] residues 99-107 of SEQ ID NO:191 (CDR-H3
h1A11.A11); [0782] residues 99-107 of SEQ ID NO:192 (CDR-H3
h1A11.B5); [0783] residues 99-107 of SEQ ID NO:193 (CDR-H3
h1A11.E12); [0784] residues 99-107 of SEQ ID NO:194 (CDR-H3
h1A11.G3); [0785] residues 99-107 of SEQ ID NO:195 (CDR-H3
h1A11.F5); and [0786] residues 99-107 of SEQ ID NO:196 (CDR-H3
h1A11.H2);
[0787] CDR-L1 is selected from the group consisting of: [0788]
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X-
.sub.10-X.sub.11 (SEQ ID NO:154), wherein; [0789] X.sub.1 is R;
[0790] X.sub.2 is A; [0791] X.sub.3 is S; [0792] X.sub.4 is E or Q;
[0793] X.sub.5 is D or E; [0794] X.sub.6 is I; [0795] X.sub.7 is Y
or W; [0796] X.sub.8 is S, I, Y, N, or R; [0797] X.sub.9 is N;
[0798] X.sub.10 is L; and [0799] X.sub.11 is A; [0800] residues
24-34 of SEQ ID NO:158 (CDR-L1 38H12); [0801] residues 24-34 of SEQ
ID NO:162 (CDR-L1 37D10); [0802] residues 24-34 of SEQ ID NO:164
(CDR-L1 32C7); [0803] residues 24-34 of SEQ ID NO:166 (CDR-L1
14G1); [0804] residues 23-37 of SEQ ID NO:168 (CDR-L1 14A11);
[0805] residues 23-37 of SEQ ID NO:170 (CDR-L1 15D6); [0806]
residues 24-34 of SEQ ID NO:175 (CDR-L1 VL.1 1A11); [0807] residues
24-34 of SEQ ID NO:176 (CDR-L1 VL.1a 1A11); [0808] residues 24-34
of SEQ ID NO:177 (CDR-L1 VL.1b 1A11); [0809] residues 24-34 of SEQ
ID NO:178 (CDR-L1 VL.2a 1A11); [0810] residues 24-34 of SEQ ID
NO:183 (CDR-L1 VL.1 38H12); [0811] residues 24-34 of SEQ ID NO:184
(CDR-L1 VL.1a 38H12); [0812] residues 24-34 of SEQ ID NO:185
(CDR-L1 VL.1b 38H12); [0813] residues 24-34 of SEQ ID NO:186
(CDR-L1 VL.2a 38H12); [0814] residues 24-34 of SEQ ID NO:197
(CDR-L1 h1A11VL.1); [0815] residues 24-34 of SEQ ID NO:198 (CDR-L1
h1A11.A2); [0816] residues 24-34 of SEQ ID NO:199 (CDR-L1
h1A11.A12); [0817] residues 24-34 of SEQ ID NO:200 (CDR-L1
h1A11.A7); [0818] residues 24-34 of SEQ ID NO:201 (CDR-L1
h1A11.B4); [0819] residues 24-34 of SEQ ID NO:202 (CDR-L1
h1A11.B5); and [0820] residues 24-34 of SEQ ID NO:203 (CDR-L1
h1A11.E12);
[0821] CDR-L2 is selected from group consisting of: [0822]
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7 (SEQ ID
NO:155), wherein; [0823] X.sub.1 is D; [0824] X.sub.2 is T; [0825]
X.sub.3 is N or S; [0826] X.sub.4 is N, D, S, I, Y, or V; [0827]
X.sub.5 is L; [0828] X.sub.6 is A; and [0829] X.sub.7 is D; [0830]
residues 50-56 of SEQ ID NO:158 (CDR-L2 38H12); [0831] residues
50-56 of SEQ ID NO:162 (CDR-L2 37D10); [0832] residues 50-56 of SEQ
ID NO:164 (CDR-L2 32C7); [0833] residues 50-56 of SEQ ID NO:166
(CDR-L2 14G1); [0834] residues 53-59 of SEQ ID NO:168 (CDR-L2
14A11); [0835] residues 53-59 of SEQ ID NO:170 (CDR-L2 15D6);
[0836] residues 50-56 of SEQ ID NO:175 (CDR-L2 VL.1 1A11); [0837]
residues 50-56 of SEQ ID NO:176 (CDR-L2 VL.1a 1A11); [0838]
residues 50-56 of SEQ ID NO:177 (CDR-L2 VL.1b 1A11); [0839]
residues 50-56 of SEQ ID NO:178 (CDR-L2 VL.2a 1A11); [0840]
residues 50-56 of SEQ ID NO:183 (CDR-L2 VL.1 38H12); [0841]
residues 50-56 of SEQ ID NO:184 (CDR-L2 VL.1a 38H12); [0842]
residues 50-56 of SEQ ID NO:185 (CDR-L2 VL.1b 38H12); [0843]
residues 50-56 of SEQ ID NO:186 (CDR-L2 VL.2a 38H12); [0844]
residues 50-56 of SEQ ID NO:197 (CDR-L2 h1A11VL.1); [0845] residues
50-56 of SEQ ID NO:198 (CDR-L2 h1A11.A2); [0846] residues 50-56 of
SEQ ID NO:199 (CDR-L2 h1A11.A12); [0847] residues 50-56 of SEQ ID
NO:200 (CDR-L2 h1A11.A7); [0848] residues 50-56 of SEQ ID NO:201
(CDR-L2 h1A11.B4); [0849] residues 50-56 of SEQ ID NO:202 (CDR-L2
h1A11.B5); and [0850] residues 50-56 of SEQ ID NO:203 (CDR-L2
h1A11.E12); and
[0851] CDR-L3 is selected from the group consisting of: [0852]
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9
(SEQ ID NO:156), wherein; [0853] X.sub.1 is Q; [0854] X.sub.2 is Q;
[0855] X.sub.3 is Y; [0856] X.sub.4 is N, D, or T; [0857] X.sub.5
is N, Y, or W; [0858] X.sub.6 is Y or V; [0859] X.sub.7 is P;
[0860] X.sub.8 is P; and [0861] X.sub.9 is T; [0862] residues 89-97
of SEQ ID NO:158 (CDR-L3 38H12); [0863] residues 89-97 of SEQ ID
NO:162 (CDR-L3 37D10); [0864] residues 89-97 of SEQ ID NO:164
(CDR-L3 32C7); [0865] residues 89-98 of SEQ ID NO:166 (CDR-L3
14G1); [0866] residues 92-100 of SEQ ID NO:168 (CDR-L3 14A11);
[0867] residues 92-100 of SEQ ID NO:170 (CDR-L3 15D6); [0868]
residues 89-97 of SEQ ID NO:175 (CDR-L3 VL.1 1A11); [0869] residues
89-97 of SEQ ID NO:176 (CDR-L3 VL.1a 1A11); [0870] residues 89-97
of SEQ ID NO:177 (CDR-L3 VL.1b 1A11); [0871] residues 89-97 of SEQ
ID NO:178 (CDR-L3 VL.2a 1A11); [0872] residues 89-97 of SEQ ID
NO:183 (CDR-L3 VL.1 38H12); [0873] residues 89-97 of SEQ ID NO:184
(CDR-L3 VL.1a 38H12); [0874] residues 89-97 of SEQ ID NO:185
(CDR-L3 VL.1b 38H12); [0875] residues 89-97 of SEQ ID NO:186
(CDR-L3 VL.2a 38H12); [0876] residues 89-97 of SEQ ID NO:197
(CDR-L3 h1A11VL.1); [0877] residues 89-97 of SEQ ID NO:198 (CDR-L3
h1A11.A2); [0878] residues 89-97 of SEQ ID NO:199 (CDR-L3
h1A11.A12); [0879] residues 89-97 of SEQ ID NO:200 (CDR-L3
h1A11.A7); [0880] residues 89-97 of SEQ ID NO:201 (CDR-L3
h1A11.B4); [0881] residues 89-97 of SEQ ID NO:202 (CDR-L3
h1A11.B5); and [0882] residues 89-97 of SEQ ID NO:203 (CDR-L3
h1A11.E12).
[0883] Preferably, a DLL4 binding protein comprises at least one
CDR described above, more preferably any two CDRs described above,
more preferably any three CDRs described above, even more
preferably any four CDRs described above, still more preferably any
five CDRs described above, and most preferably any six CDRs
described above (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and
CDR-L3 as described above). A particularly preferred DLL4 binding
protein comprising three CDRs comprises CDR-H1, CDR-H2, and CDR-H3
as described above.
[0884] Preferably, a DLL4 binding protein comprising one or more
CDRs described above binds human ("hu", "h") DLL4 and also one or
more DLL4 proteins selected from the group consisting of: mouse
("murine", "mu") DLL4, cynomolgus monkey ("cynomolgus", "cyno")
DLL4, and rat DLL4.
[0885] Preferably, a DLL4 binding protein comprising one or more
CDRs described above binds human ("hu") DLL4 and also cynomolgus
monkey ("cynomolgus", "cyno") DLL4.
2. Anti-DLL4 Chimeric Antibodies.
[0886] A chimeric antibody is a molecule in which different
portions of the antibody are derived from different animal species,
such as antibodies having a variable region derived from a murine
monoclonal antibody and a human immunoglobulin constant region. See
e.g., Morrison, Science, 229: 1202-1207 (1985); Oi et al.,
BioTechniques, 4: 214 (1986); Gillies et al., J. Immunol. Methods,
125: 191-202 (1989); U.S. Pat. Nos. 5,807,715; 4,816,567; and
4,816,397. In addition, techniques developed for the production of
"chimeric antibodies" by splicing genes from a mouse antibody
molecule of appropriate antigen specificity together with genes
from a human antibody molecule of appropriate biological activity
can be used. See, for example, Morrison et al., Proc. Natl. Acad.
Sci. USA, 81: 6851-6855 (1984); Neuberger et al., Nature, 312:
604-608 (1984); Takeda et al., Nature, 314: 452-454 (1985), which
are incorporated herein by reference in their entireties.
3. Anti-DLL4 CDR Grafted Antibodies.
[0887] The isolated anti-DLL4 antibody CDR sequences of the
invention may be used to make CDR-grafted antibodies to modulate
the properties of the original antibody. Such properties include
but are not limited to binding kinetics, affinity, biological
activities, species cross-reactivity, molecule cross-reactivity,
epitope, physicochemical properties, pharmacokinetic properties,
pharmacodynamic properties, or pharmacological properties.
CDR-grafted antibodies comprise heavy and light chain variable
region sequences from a human antibody or a non-human primate
antibody wherein one or more of the CDR regions of VH and/or VL are
replaced with CDR sequences of the original anti-DLL4 antibody. A
framework sequence from any human or non-human primate antibody may
serve as the template for CDR grafting. However, straight chain
replacement onto such a framework often leads to some loss of
binding affinity to the antigen. The more homologous a human, or
other species, antibody is to the original human antibody, the less
likely the possibility that combining the CDRs with the new human
framework or non-human primate framework will introduce distortions
in the CDRs that could reduce affinity or other properties.
Therefore, it is preferable that the variable framework that is
chosen to replace the human variable region framework apart from
the CDRs has at least a 30% sequence identity with the human
antibody variable region framework. It is more preferable that the
variable region framework that is chosen to replace the human
variable region framework apart from the CDRs has at least a 40%
sequence identity with the human antibody variable region
framework. It is more preferable that the variable region framework
that is chosen to replace the human variable framework apart from
the CDRs has at least a 50% sequence identity with the human
antibody variable region framework. It is more preferable that the
variable region framework that is chosen to replace the human
variable framework apart from the CDRs has at least a 60% sequence
identity with the human antibody variable region framework. It is
more preferable that the new human or non-human primate and the
original human variable region framework apart from the CDRs has at
least 70% sequence identity. It is even more preferable that the
new human or non-human primate and the original human variable
region framework apart from the CDRs has at least 75% sequence
identity. It is most preferable that the new human or non-human
primate and the original human variable region framework apart from
the CDRs has at least 80% sequence identity. Even using a highly
homologous human or non-human primate framework to graft CDRs of
the original human anti-DLL4 antibody, the resulting grafted
antibody may still lose binding affinity to antigen to some degree.
In this case, to regain the affinity it is necessary to include at
least one or more key framework residue(s) substitution of the
original antibody to the corresponding position of the newly
grafted antibody. Such a key residue may be selected from the group
consisting of: [0888] a residue adjacent to a CDR; [0889] a
glycosylation site residue; [0890] a rare residue; [0891] a residue
capable of interacting with human DLL4 [0892] a canonical residue;
[0893] a contact residue between heavy chain variable region and
light chain variable region; [0894] a residue within a Vernier
zone; and [0895] a residue in a region that overlaps between a
Chothia-defined variable heavy chain CDR1 and a Kabat-defined first
heavy chain framework.
4. Anti-DLL4 Humanized Antibodies.
[0896] While the compositions of the present invention eliminate
the requirement to make humanized antibodies, humanized DLL4
antibodies may be prepared using compositions of the invention.
Humanized antibodies are antibody molecules from non-human species
antibody that binds the desired antigen having one or more
complementarity determining regions (CDRs) from the non-human
species and framework regions from a human immunoglobulin molecule.
Known human Ig sequences are disclosed at web sites available via
the world wide web (www.), e.g.,
ncbi.nlm.nih.gov/entrez/query.fcgi; atcc.org/phage/hdb.html;
sciquest.com/; abcam.com/; antibodyresource.com/onlinecomp.html;
public.iastate.edu/.about.pedro-/research_tools.html;
mgen.uniheidelberg.de/SD/IT/IT.html;
whfreeman.com/immunology-/CH05/kuby05.htm;
library.thinkquest.org/12429/Immune/Antibody.html;
hhmi.org/grants/lectures/1996/vlab/;
path.-cam.ac.uk/.about.mrc7/mikeimages.html; antibodyresource.com/;
mcb.harvard.edu/BioLinks-/Immunology.html; immunologylink.com/;
pathbox.wustl.edu/.about.hcenter/index.html;
bio-tech.ufl.edu/.about.hcl/; pebio.com/pa/340913-/340913.html;
nal.usda.gov/awic/pubs/antibody/;
m.ehimeu.acjp/.about.yasuhito-/Elisa.html; biodesign.com/table.asp;
icnet.uk/axp/facs/davies/lin-ks.html;
biotech.ufl.edu-/.about.fccl/protocol.html;
isac-net.org/sites_geo.html;
aximtl.imt.uni-marburg.de/.about.rek/AEP-Start.html;
baserv.uci.kun.nl/.about.jraats/links1.html;
recab.uni-hd.de/immuno.bme.nwu.edu/;
mrc-cpe.cam.ac.uk/imt-doc/public/INTRO.html;
ibt.unam.mx/-vir/V_mice.html; imgt.cnusc.fr:8104/;
biochem.ucl.ac.uk/.about.martin/abs/index.html;
anti-body.bath.ac.uk/; abgen.cvm.tamu.edu/lab/wwwabgen.html;
unizh.ch/.about.honegger/AHO-seminar/Slide01.html;
cryst.bbk.ac.uk/.about.ubcg07s/;
nimr.mrc.ac.uk/CC/ccaewg/ccaewg.htm;
path.cam.ac.uk/.about.mrc7/humanisation/TAHHP.html;
ibt.unam.mx/vir/structure/stat_aim.-html;
biosci.missouri.edu/smithgp/index.html;
cryst.bioc.cam.ac.uk/.about.fmolina/Webpages-/Pept/spottech.html;
jerini.de/frroducts.htm; patents.ibm.com/ibm.html. Kabat et al.,
Sequences of Proteins of Immunological Interest, U.S. Dept. Health
(1983), each entirely incorporated herein by reference. Such
imported sequences can be used to reduce immunogenicity or reduce,
enhance or modify binding, affinity, on-rate, off-rate, avidity,
specificity, half-life, or any other suitable characteristic, as
known in the art.
[0897] Framework residues in the human framework regions may be
substituted with the corresponding residue from the CDR donor
antibody to alter, preferably improve, antigen binding. These
framework substitutions are identified by methods well known in the
art, e.g., by modeling of the interactions of the CDR and framework
residues to identify framework residues important for antigen
binding and sequence comparison to identify unusual framework
residues at particular positions. (See, e.g., U.S. Pat. No.
5,585,089 (Queen et al.); Riechmann et al., Nature, 332: 323-327
(1988), which are incorporated herein by reference in their
entireties.) Three-dimensional immunoglobulin models are commonly
available and are familiar to those skilled in the art. Computer
programs are available which illustrate and display probable
three-dimensional conformational structures of selected candidate
immunoglobulin sequences. Inspection of these displays permits
analysis of the likely role of the residues in the functioning of
the candidate immunoglobulin sequence, i.e., the analysis of
residues that influence the ability of the candidate immunoglobulin
to bind its antigen. In this way, FR residues can be selected and
combined from the consensus and import sequences so that the
desired antibody characteristic, such as increased affinity for the
target antigen(s), is achieved. In general, the CDR residues are
directly and most substantially involved in influencing antigen
binding. Antibodies can be humanized using a variety of techniques
known in the art, such as but not limited to those described in
Jones et al., Nature, 321: 522-525 (1986); Verhoeyen et al.,
Science, 239: 1534-1536 (1988), Sims et al., J. Immunol., 151:
2296-2308 (1993); Chothia and Lesk, J. Mol. Biol., 196: 901-917
(1987), Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285-4289
(1992); Presta et al., J. Immunol., 151: 2623-2632 (1993), Padlan,
E. A., Molecular Immunology, 28(4/5): 489-498 (1991); Studnicka et
al., Protein Engineering, 7(6): 805-814 (1994); Roguska. et al.,
Proc. Natl. Acad. Sci. USA, 91:969-973 (1994); PCT Publication Nos.
WO 91/09967, WO 99/06834 (PCT/US98/16280), WO 97/20032
(PCT/US96/18978), WO 92/11272 (PCT/US91/09630), WO 92/03461
(PCT/US91/05939), WO 94/18219 (PCT/US94/01234), WO 92/01047
(PCT/GB91/01134), WO 93/06213 (PCT/GB92/01755), WO90/14443,
WO90/14424, and WO90/14430; European Publication Nos. EP 0 592 106,
EP 0 519 596, and EP 0 239 400; U.S. Pat. Nos. 5,565,332;
5,723,323; 5,976,862; 5,824,514; 5,817,483; 5,814,476; 5,763,192;
5,723,323; 5,766,886; 5,714,352; 6,204,023; 6,180,370; 5,693,762;
5,530,101; 5,585,089; 5,225,539; and 4,816,567, each entirely
incorporated herein by reference, included references cited
therein.
C. Production of Antibodies and Antibody-Producing Cell Lines.
[0898] Preferably, anti-DLL4 antibodies of the present invention
exhibit a high capacity to reduce or to neutralize tumor
angiogenesis activity, e.g., as assessed by any one of several in
vitro and in vivo assays known in the art. For example, these
antibodies neutralize DLL4 interaction in the Notch-signaling
pathway with IC.sub.50 values in DLL4 in the range of at least
about 10.sup.-7 M, or about 10.sup.-8 M. Preferably, anti-DLL4
antibodies of the present invention also exhibit a high capacity to
reduce or to neutralize DLL4 activity.
[0899] In preferred embodiments, an isolated antibody, or
antigen-binding portion thereof, binds human DLL4, wherein the
antibody, or antigen-binding portion thereof, dissociates from
human DLL4 with a K.sub.off rate constant of about 0.1 s.sup.-1 or
less, as determined by surface plasmon resonance, or which inhibits
DLL4 and/or human DLL4 activity with an IC.sub.50 of about
1.times.10.sup.-6 M or less. Alternatively, the antibody, or an
antigen-binding portion thereof, may dissociate from human DLL4
with a K.sub.off rate constant of about 1.times.10.sup.-2s.sup.-1
or less, as determined by surface plasmon resonance, or may inhibit
human DLL4 and/or human DLL4 activity with an IC.sub.50 of about
1.times.10.sup.-7 M or less. Alternatively, the antibody, or an
antigen-binding portion thereof, may dissociate from human DLL4
with a K.sub.off rate constant of about 1.times.10.sup.-3 s.sup.-1
or less, as determined by surface plasmon resonance, or may inhibit
human DLL4 with an IC.sub.50 of about 1.times.10.sup.-8M or less.
Alternatively, the antibody, or an antigen-binding portion thereof,
may dissociate from human DLL4 with a K.sub.off rate constant of
about 1.times.10.sup.-4 s.sup.-1 or less, as determined by surface
plasmon resonance, or may inhibit DLL4 activity with an IC.sub.50
of about 1.times.10.sup.-9M or less. Alternatively, the antibody,
or an antigen-binding portion thereof, may dissociate from human
DLL4 with a K.sub.off rate constant of about
1.times.10.sup.-5s.sup.-1 or less, as determined by surface plasmon
resonance, or may inhibit DLL4 and/or human DLL4 activity with an
IC.sub.50 of about 1.times.10.sup.-10 M or less. Alternatively, the
antibody, or an antigen-binding portion thereof, may dissociate
from human DLL4 with a K.sub.off rate constant of about
1.times.10.sup.-5 s.sup.-1 or less, as determined by surface
plasmon resonance, or may inhibit DLL4 and/or human DLL4 activity
with an IC.sub.50 of about 1.times.10.sup.-11M or less.
[0900] In certain embodiments, the antibody comprises a heavy chain
constant region, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM,
or IgD constant region. Preferably, the heavy chain constant region
is an IgG1 heavy chain constant region or an IgG4 heavy chain
constant region. Furthermore, the antibody can comprise a light
chain constant region, either a kappa light chain constant region
or a lambda light chain constant region. Preferably, the antibody
comprises a kappa light chain constant region. Alternatively, the
antibody portion can be, for example, a Fab fragment or a single
chain Fv fragment.
[0901] Replacements of amino acid residues in the Fc portion to
alter antibody effector function are known in the art (see, U.S.
Pat. Nos. 5,648,260 and 5,624,821 (Winter et al.)). The Fc portion
of an antibody mediates several important effector functions e.g.
cytokine induction, ADCC, phagocytosis, complement dependent
cytotoxicity (CDC), and half-life/clearance rate of antibody and
antigen-antibody complexes. In some cases these effector functions
are desirable for therapeutic antibody but in other cases might be
unnecessary or even deleterious, depending on the therapeutic
objectives. Certain human IgG isotypes, particularly IgG1 and IgG3,
mediate ADCC and CDC via binding to Fc.gamma.Rs and complement C1q,
respectively. Neonatal Fc receptors (FcRn) are the critical
components determining the circulating half-life of antibodies. In
still another embodiment at least one amino acid residue is
replaced in the constant region of the antibody, for example the Fc
region of the antibody, such that effector functions of the
antibody are altered.
[0902] One embodiment provides a labeled binding protein wherein an
antibody or antibody portion of the invention is derivatized or
linked to another functional molecule (e.g., another peptide or
protein). For example, a labeled binding protein of the invention
can be derived by functionally linking an antibody or antibody
portion of the invention (by chemical coupling, genetic fusion,
noncovalent association or otherwise) to one or more other
molecular entities, such as another antibody (e.g., a bispecific
antibody or a diabody), a detectable agent, a cytotoxic agent, a
pharmaceutical agent, and/or a protein or peptide that can mediate
associate of the antibody or antibody portion with another molecule
(such as a streptavidin core region or a polyhistidine tag).
[0903] Useful detectable agents with which an antibody or antibody
portion of the invention may be derivatized include fluorescent
compounds. Exemplary fluorescent detectable agents include
fluorescein, fluorescein isothiocyanate, rhodamine,
5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin and
the like. An antibody may also be derivatized with detectable
enzymes, such as alkaline phosphatase, horseradish peroxidase,
glucose oxidase and the like. When an antibody is derivatized with
a detectable enzyme, it is detected by adding additional reagents
that the enzyme uses to produce a detectable reaction product. For
example, when the detectable agent horseradish peroxidase is
present, the addition of hydrogen peroxide and diaminobenzidine
leads to a colored reaction product, which is detectable. An
antibody may also be derivatized with biotin, and detected through
indirect measurement of avidin or streptavidin binding.
[0904] Another embodiment of the invention provides a crystallized
DLL4 binding protein. Preferably, the invention relates to crystals
of DLL4 binding proteins described herein, including whole
anti-DLL4 antibodies, fragments thereof, as well as antibody
constructs and binding protein conjugates (including antibody
conjugates) as disclosed herein, and formulations and compositions
comprising such crystals. In one embodiment, the crystallized
binding protein has a greater half-life in vivo than the soluble
counterpart of the binding protein. In another embodiment the
binding protein retains biological activity after crystallization.
Crystallized binding proteins of the invention may be produced
according methods known in the art and as disclosed in PCT
Publication No. WO 02/72636, incorporated herein by reference.
[0905] Another embodiment of the invention provides a glycosylated
binding protein wherein the antibody or antigen-binding portion
thereof comprises one or more carbohydrate residues. Nascent in
vivo protein production may undergo further processing, known as
post-translational modification. In particular, sugar (glycosyl)
residues may be added enzymatically, a process known as
glycosylation. The resulting proteins bearing covalently linked
oligosaccharide side chains are known as glycosylated proteins or
glycoproteins. Protein glycosylation depends on the amino acid
sequence of the protein of interest, as well as the host cell in
which the protein is expressed. Different organisms may produce
different glycosylation enzymes (e.g., glycosyltransferases and
glycosidases), and have different substrates (nucleotide sugars)
available. Due to such factors, protein glycosylation pattern, and
composition of glycosyl residues, may differ depending on the host
system in which the particular protein is expressed. Glycosyl
residues useful in the invention may include, but are not limited
to, glucose, galactose, mannose, fucose, n-acetylglucosamine and
sialic acid. Preferably the glycosylated binding protein comprises
glycosyl residues such that the glycosylation pattern is human.
[0906] It is known to those skilled in the art that differing
protein glycosylation may result in differing protein
characteristics. For instance, the efficacy of a therapeutic
protein produced in a microorganism host, such as yeast, and
glycosylated utilizing the yeast endogenous pathway may be reduced
compared to that of the same protein expressed in a mammalian cell,
such as a CHO cell line. Such glycoproteins may also be immunogenic
in humans and show reduced half-life in vivo after administration.
Specific receptors in humans and other animals may recognize
specific glycosyl residues and promote the rapid clearance of the
protein from the bloodstream. Other adverse effects may include
changes in protein folding, solubility, susceptibility to
proteases, trafficking, transport, compartmentalization, secretion,
recognition by other proteins or factors, antigenicity, or
allergenicity. Accordingly, a practitioner may prefer a therapeutic
protein with a specific composition and pattern of glycosylation,
for example glycosylation composition and pattern identical, or at
least similar, to that produced in human cells or in the
species-specific cells of the intended subject animal.
[0907] Expressing glycosylated proteins different from that of a
host cell may be achieved by genetically modifying the host cell to
express heterologous glycosylation enzymes. Using techniques known
in the art a practitioner may generate antibodies or
antigen-binding portions thereof exhibiting human protein
glycosylation. For example, yeast strains have been genetically
modified to express non-naturally occurring glycosylation enzymes
such that glycosylated proteins (glycoproteins) produced in these
yeast strains exhibit protein glycosylation identical to that of
animal cells, especially human cells (US Patent Application
Publication Nos. 2004/0018590 and 2002/0137134).
[0908] Further, it will be appreciated by one skilled in the art
that a protein of interest may be expressed using a library of host
cells genetically engineered to express various glycosylation
enzymes, such that member host cells of the library produce the
protein of interest with variant glycosylation patterns. A
practitioner may then select and isolate the protein of interest
with particular novel glycosylation patterns. Preferably, the
protein having a particularly selected novel glycosylation pattern
exhibits improved or altered biological properties.
D. Uses of DLL4 Binding Proteins.
[0909] Given their ability to bind to human DLL4 and murine DLL4,
the DLL4 binding proteins described herein, including antibodies
and portions thereof, can be used to detect or measure DLL4 in a
sample (e.g., in a mixture, solution, or biological sample, such as
blood, serum, or plasma), using any of the conventional
immunoassays known in the art, such as an enzyme linked
immunosorbent assays (ELISA), a radioimmunoassay (RIA), or a tissue
immunohistochemistry. The invention provides a method for detecting
human DLL4 and/or murine DLL4 in a sample comprising contacting a
sample with a DLL4 binding protein and detecting either the DLL4
binding protein bound to human DLL4 and/or murine DLL4 or the
unbound binding protein to thereby detect human DLL4 and/or murine
DLL4 in the sample. A DLL4 binding protein described herein can be
directly or indirectly labeled with a detectable substance to
facilitate detection of the bound or unbound DLL4 binding protein.
Suitable detectable substances include various enzymes, prosthetic
groups, fluorescent materials, luminescent materials and
radioactive materials. Examples of suitable enzymes include
horseradish peroxidase, alkaline phosphatase, .beta.-galactosidase,
or acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; and examples of suitable radioactive material include
.sup.3H, .sup.14C, .sup.35S, .sup.90Y, .sup.99Tc, .sup.111In,
.sup.125I, .sup.131I, .sup.177Lu, .sup.166Ho, or .sup.153Sm.
[0910] Biological samples that can be assayed for DLL4 include
urine, feces, blood, serum, plasma, perspiration, saliva, oral swab
(cheek, tongue, throat), vaginal swab, rectal swab, dermal swab,
dermal scrape, tissue biopsy, as well as any other tissue sample
that can be obtained by methods available in the art.
[0911] Alternative to labeling the binding protein, human DLL4 can
be assayed in biological fluids by a competition immunoassay
utilizing recombinant human (rh) DLL4 standards labeled with a
detectable substance and an unlabeled DLL4 binding protein
described herein. In this assay, the biological sample, the labeled
rhDLL4 standards, and the DLL4 binding protein are combined and the
amount of labeled rhDLL4 standard bound to the unlabeled binding
protein is determined. The amount of human DLL4 in the biological
sample is inversely proportional to the amount of labeled rhDLL4
standard bound to the DLL4 binding protein. Similarly, human DLL4
can also be assayed in biological fluids by a competition
immunoassay utilizing rhDLL4 standards labeled with a detectable
substance and an unlabeled DLL4 binding protein described
herein.
[0912] The DLL4 binding proteins of the invention preferably are
capable of neutralizing DLL4 activity, in particular hDLL4
activity, both in vitro and in vivo. Accordingly, such binding
proteins of the invention can be used to inhibit DLL4 activity,
e.g., in a cell culture containing DLL4, in human subjects, or in
other mammalian subjects expressing a DLL4 with which a binding
protein of the invention cross-reacts. In one embodiment, the
invention provides a method for inhibiting DLL4 activity comprising
contacting a DLL4 with a DLL4 antibody or antibody portion of the
invention such that DLL4 activity is inhibited. For example, in a
cell culture containing or suspected of containing DLL4, an
antibody or antibody portion of the invention can be added to the
culture medium to inhibit DLL4 activity in the culture.
[0913] In another embodiment, the invention provides a method for
reducing DLL4 activity in a subject, advantageously from a subject
suffering from a disease or disorder in which DLL4 or DLL4 activity
is detrimental. The invention provides methods for reducing DLL4 or
DLL4 activity in a subject suffering from such a disease or
disorder, which method comprises administering to the subject a
DLL4 binding protein of the invention such that DLL4 or DLL4
activity in the subject is reduced. Preferably, the DLL4 is human
DLL4, and the subject is a human subject. Alternatively, the
subject can be a mammal expressing a DLL4 to which a DLL4 binding
protein of the invention is capable of binding. Still further, the
subject can be a mammal into which DLL4 has been introduced (e.g.,
by administration of DLL4 or by expression of a DLL4 transgene). An
antibody or other DLL4 binding protein of the invention can be
administered to a human subject for therapeutic purposes. Moreover,
a DLL4 binding protein of the invention can be administered to a
non-human mammal expressing a DLL4 with which the binding protein
is capable of binding for veterinary purposes or as an animal model
of human disease. Regarding the latter, such animal models may be
useful for evaluating the therapeutic efficacy of antibodies and
other DLL4 binding proteins of the invention (e.g., testing of
dosages and time courses of administration).
[0914] As used herein, the term "a disorder in which DLL4 and/or
Notch signaling activity is detrimental" is intended to include
diseases, such as cancer, and other disorders in which the presence
of DLL4 and/or Notch signaling activity in a subject suffering from
the disorder has been shown to be or is suspected of being either
responsible for the pathophysiology of the disorder or a factor
that contributes to a worsening of the disorder. Accordingly, a
disorder in which DLL4 and/or Notch signaling activity is
detrimental is a disorder in which reduction of DLL4 and/or Notch
signaling activity is expected to alleviate the symptoms and/or
progression of the disorder (e.g., tumor growth). Such disorders
may be evidenced, for example, by an increase in angiogenesis in a
subject suffering from the disorder (e.g., an increase in the
concentration of various proteins known in the art to increase in
serum, plasma, synovial fluid, etc., of the subject during tumor
growth and formation), which can be detected, for example, using an
anti-DLL4 antibody as described above. Non-limiting examples of
disorders that can be treated with the antibodies of the invention
include those disorders discussed in the section below pertaining
to pharmaceutical compositions of the antibodies of the
invention.
II. Pharmaceutical Compositions and Therapeutic Uses.
[0915] The invention also provides pharmaceutical compositions
comprising a DLL4 binding protein of the invention and a
pharmaceutically acceptable carrier. The pharmaceutical
compositions comprising DLL4 binding proteins of the invention are
for use in, but not limited to, diagnosing, detecting, or
monitoring a disorder; in preventing, treating, managing, or
ameliorating a disorder or one or more symptoms thereof and/or in
research. In a specific embodiment, a composition comprises one or
more DLL4 binding proteins of the invention.
[0916] In another embodiment, the pharmaceutical composition
comprises one or more binding proteins of the invention and one or
more prophylactic or therapeutic agents other than binding proteins
of the invention for treating a disorder in which DLL4 and/or DLL4
activity is detrimental. Preferably, the prophylactic or
therapeutic agents known to be useful for or having been or
currently being used in the prevention, treatment, management, or
amelioration of a disorder, such as cancer or a tumor, or one or
more symptoms thereof. In accordance with these embodiments, the
composition may further comprise of a carrier, diluent, or
excipient.
[0917] The binding proteins of the invention can be incorporated
into pharmaceutical compositions suitable for administration to a
subject. Typically, the pharmaceutical composition comprises a DLL4
binding protein (or DLL4 binding portion thereof) of the invention
and a pharmaceutically acceptable carrier. As used herein,
"pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents, and the like that
are physiologically compatible. Examples of pharmaceutically
acceptable carriers include one or more of water, saline, phosphate
buffered saline, dextrose, glycerol, ethanol and the like, as well
as combinations thereof. In many cases, it will be preferable to
include isotonic agents, for example, sugars, polyalcohols such as
mannitol, sorbitol, or sodium chloride in the composition.
Pharmaceutically acceptable carriers may further comprise minor
amounts of auxiliary substances such as wetting or emulsifying
agents, preservatives or buffers, which enhance the shelf life or
effectiveness of the antibody or antibody portion.
[0918] Various delivery systems are known and can be used to
administer one or more DLL4 binding proteins of the invention or
the combination of one or more binding proteins of the invention
and a prophylactic agent or therapeutic agent useful for
preventing, managing, treating, or ameliorating a disorder or one
or more symptoms thereof, e.g., reducing tumor angiogenesis,
encapsulation in liposomes, microparticles, microcapsules,
recombinant cells capable of expressing the DLL4 binding protein,
receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol.
Chem., 262: 4429-4432 (1987)), construction of a nucleic acid as
part of a retroviral or other vector, etc. Methods of administering
a prophylactic or therapeutic agent of the invention include, but
are not limited to, parenteral administration (e.g., intradermal,
intramuscular, intraperitoneal, intravenous and subcutaneous),
epidural administration, intratumoral administration, and mucosal
administration (e.g., intranasal and oral routes). In addition,
pulmonary administration can be employed, e.g., by use of an
inhaler or nebulizer, and formulation with an aerosolizing agent.
See, e.g., U.S. Pat. Nos. 6,019,968; 5,985,320; 5,985,309; 5,934,
272; 5,874,064; 5,855,913; 5,290,540; and 4,880,078; and PCT
Publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO
98/31346, and WO 99/66903, each of which is incorporated herein by
reference their entireties. In one embodiment, a DLL4 binding
protein of the invention, combination therapy, or a composition of
the invention is administered using Alkermes AIR.RTM. pulmonary
drug delivery technology (Alkermes, Inc., Cambridge, Mass., US). In
a specific embodiment, prophylactic or therapeutic agents of the
invention are administered intramuscularly, intravenously,
intratumorally, orally, intranasally, pulmonary, or subcutaneously.
The prophylactic or therapeutic agents may be administered by any
convenient route, for example by infusion or bolus injection, by
absorption through epithelial or mucocutaneous linings (e.g., oral
mucosa, rectal and intestinal mucosa, etc.) and may be administered
together with other biologically active agents. Administration can
be systemic or local.
[0919] In a specific embodiment, it may be desirable to administer
the prophylactic or therapeutic agents of the invention locally to
the area in need of treatment; this may be achieved by, for
example, and not by way of limitation, local infusion, by
injection, or by means of an implant, said implant being of a
porous or non-porous material, including membranes and matrices,
such as sialastic membranes, polymers, fibrous matrices (e.g.,
Tissuel.RTM.), or collagen matrices. In one embodiment, an
effective amount of one or more DLL4 binding proteins of the
invention antagonists is administered locally to the affected area
to a subject to prevent, treat, manage, and/or ameliorate a
disorder or a symptom thereof. In another embodiment, an effective
amount of one or more DLL4 binding proteins of the invention is
administered locally to the affected area in combination with an
effective amount of one or more therapies (e.g., one or more
prophylactic or therapeutic agents) other than a binding protein of
the invention of a subject to prevent, treat, manage, and/or
ameliorate a disorder or one or more symptoms thereof.
[0920] In another embodiment, the prophylactic or therapeutic agent
can be delivered in a controlled release or sustained release
system. In one embodiment, a pump may be used to achieve controlled
or sustained release (see, Langer (Science, 249: 1527-1533 (1990));
Sefton, CRC Crit. Ref. Biomed. Eng., 14: 201-240 (1987); Buchwald
et al., Surgery, 88: 507-516 (1980); Saudek et al., N. Engl. J.
Med., 321: 574-579 (1989)). In another embodiment, polymeric
materials can be used to achieve controlled or sustained release of
the therapies of the invention. See, e.g., Goodson, J. M, In
Medical Applications of Controlled Release, Vol. II, Applications
and Evaluations, (Langer and Wise, eds.), (CRC Press Inc., Boca
Raton, 1984), chapter 6, pages 115-138; Controlled Drug
Bioavailability, Drug Product Design and Performance, Smolen and
Ball (eds.) (Wiley, New York, 1984); Langer and Peppas, J.
Macromol. Sci. Rev. Macromol. Chem. Phys., C23: 61-126 (1983); see
also, Levy et al., Science, 228: 190-192 (1985); During et al.,
Ann. Neurol., 25: 351-356 (1989); Howard et al., J. Neurosurg., 71:
105-112 (1989); U.S. Pat. Nos. 5,679,377; 5,916,597; 5,912,015;
5,989,463; and 5,128,326; and PCT Publication Nos. WO 99/15154 and
WO 99/20253. Examples of polymers used in sustained release
formulations include, but are not limited to, poly(2-hydroxy ethyl
methacrylate), poly(methyl methacrylate), poly(acrylic acid),
poly(ethylene-co-vinyl acetate), poly(methacrylic acid),
polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone),
poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol),
polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and
polyorthoesters. In a preferred embodiment, the polymer used in a
sustained release formulation is inert, free of leachable
impurities, stable on storage, sterile, and biodegradable. In yet
another embodiment, a controlled or sustained release system can be
placed in proximity of the prophylactic or therapeutic target, thus
requiring only a fraction of the systemic dose (see, e.g., Goodson,
In Medical Applications of Controlled Release, (1984), pages
115-138).
[0921] Controlled release systems are discussed in the review by
Langer (Science, 249: 1527-1533 (1990)). Any technique known to one
of skill in the art can be used to produce sustained release
formulations comprising one or more therapeutic agents of the
invention. See, e.g., U.S. Pat. No. 4,526,938; PCT Publication Nos.
WO 91/05548 and WO 96/20698; Ning et al., "Intratumoral
Radioimmunotherapy of a Human Colon Cancer Xenograft Using a
Sustained-Release Gel," Radiother. Oncol., 39: 179-189 (1996); Song
et al., "Antibody Mediated Lung Targeting of Long-Circulating
Emulsions," PDA J. Pharm. Sci. Tech., 50: 372-377 (1996); Cleek et
al., "Biodegradable Polymeric Carriers for a bFGF Antibody for
Cardiovascular Application," Proceed. Intl. Symp. Control. Rel.
Bioact. Mater., 24: 853-854 (1997), and Lam et al.,
"Microencapsulation of Recombinant Humanized Monoclonal Antibody
for Local Delivery," Proceed. Intl. Symp. Control Rel. Bioact.
Mater.: 24: 759-760 (1997), each of which is incorporated herein by
reference in their entireties.
[0922] In a specific embodiment, where the composition of the
invention is a nucleic acid encoding a prophylactic or therapeutic
agent, the nucleic acid can be administered in vivo to promote
expression of its encoded prophylactic or therapeutic agent, by
constructing it as part of an appropriate nucleic acid expression
vector and administering it so that it becomes intracellular, e.g.,
by use of a retroviral vector (see U.S. Pat. No. 4,980,286), or by
direct injection, or by use of microparticle bombardment (e.g., a
gene gun; Biolistic, DuPont), or coating with lipids or
cell-surface receptors or transfecting agents, or by administering
it in linkage to a homeobox-like peptide which is known to enter
the nucleus (see, e.g., Joliot et al., Proc. Natl. Acad. Sci. USA,
88: 1864-1868 (1991)). Alternatively, a nucleic acid can be
introduced intracellularly and incorporated within host cell DNA
for expression by homologous recombination.
[0923] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include, but are not limited
to, parenteral (e.g., intravenous), intradermal, subcutaneous,
oral, intranasal (e.g., inhalation), transdermal (e.g., topical),
transmucosal, and rectal administration. In a specific embodiment,
the composition is formulated in accordance with routine procedures
as a pharmaceutical composition adapted for intravenous,
subcutaneous, intramuscular, oral, intranasal, or topical
administration to human beings. Typically, compositions for
intravenous administration are solutions in sterile isotonic
aqueous buffer. Where necessary, the composition may also include a
solubilizing agent and a local anesthetic such as lignocaine to
ease pain at the site of the injection.
[0924] If compositions of the invention are to be administered
topically, the compositions can be formulated in the form of an
ointment, cream, transdermal patch, lotion, gel, shampoo, spray,
aerosol, solution, emulsion, or other form well-known to one of
skill in the art. See, e.g., Remington's Pharmaceutical Sciences
and Introduction to Pharmaceutical Dosage Forms, 19th ed., (Mack
Publishing Co., Easton, Pa., 1995). For non-sprayable topical
dosage forms, viscous to semi-solid or solid forms comprising a
carrier or one or more excipients compatible with topical
application and having a dynamic viscosity preferably greater than
water are typically employed. Suitable formulations include,
without limitation, solutions, suspensions, emulsions, creams,
ointments, powders, liniments, salves, and the like, which are, if
desired, sterilized or mixed with auxiliary agents (e.g.,
preservatives, stabilizers, wetting agents, buffers, or salts) for
influencing various properties, such as, for example, osmotic
pressure. Other suitable topical dosage forms include sprayable
aerosol preparations wherein the active ingredient, preferably in
combination with a solid or liquid inert carrier, is packaged in a
mixture with a pressurized volatile (e.g., a gaseous propellant,
such as FREON.RTM.) or in a squeeze bottle. Moisturizers or
humectants can also be added to pharmaceutical compositions and
dosage forms if desired. Examples of such additional ingredients
are well known in the art.
[0925] If a method of the invention comprises intranasal
administration of a composition, the composition can be formulated
in an aerosol form, spray, mist or in the form of drops. In
particular, prophylactic or therapeutic agents for use according to
the present invention can be conveniently delivered in the form of
an aerosol spray presentation from pressurized packs or a
nebulizer, with the use of a suitable propellant (e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas).
In the case of a pressurized aerosol the dosage unit may be
determined by providing a valve to deliver a metered amount.
Capsules and cartridges (composed of, e.g., gelatin) for use in an
inhaler or insufflator may be formulated containing a powder mix of
the compound and a suitable powder base such as lactose or
starch.
[0926] If a method of the invention comprises oral administration,
compositions can be formulated orally in the form of tablets,
capsules, cachets, gelcaps, solutions, suspensions, and the like.
Tablets or capsules can be prepared by conventional means with
pharmaceutically acceptable excipients such as binding agents
(e.g., pregelatinised maize starch, polyvinylpyrrolidone, or
hydroxypropyl methylcellulose); fillers (e.g., lactose,
microcrystalline cellulose, or calcium hydrogen phosphate);
lubricants (e.g., magnesium stearate, talc, or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulphate). The tablets may be
coated by methods well-known in the art. Liquid preparations for
oral administration may take the form of, but not limited to,
solutions, syrups or suspensions, or they may be presented as a dry
product for constitution with water or other suitable vehicle
before use. Such liquid preparations may be prepared by
conventional means with pharmaceutically acceptable additives such
as suspending agents (e.g., sorbitol syrup, cellulose derivatives,
or hydrogenated edible fats); emulsifying agents (e.g., lecithin or
acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl
alcohol, or fractionated vegetable oils); and preservatives (e.g.,
methyl or propyl-p-hydroxybenzoates or sorbic acid). The
preparations may also contain buffer salts, flavoring, coloring,
and sweetening agents as appropriate. Preparations for oral
administration may be suitably formulated for slow release,
controlled release, or sustained release of a prophylactic or
therapeutic agent(s).
[0927] A method of the invention may comprise pulmonary
administration, e.g., by use of an inhaler or nebulizer, of a
composition formulated with an aerosolizing agent. See, e.g., U.S.
Pat. Nos. 6,019,968; 5,985,320; 5,985,309; 5,934,272; 5,874,064;
5,855,913; 5,290,540; and 4,880,078; and PCT Publication Nos. WO
92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO 99/66903,
each of which is incorporated herein by reference their entireties.
In a specific embodiment, an antibody of the invention, combination
therapy, and/or composition of the invention is administered using
Alkermes AIR.RTM. pulmonary drug delivery technology (Alkermes,
Inc., Cambridge, Mass., US).
[0928] A method of the invention may comprise administration of a
composition formulated for parenteral administration by injection
(e.g., by bolus injection or continuous infusion). Formulations for
injection may be presented in unit dosage form (e.g., in ampoules
or in multi-dose containers) with an added preservative. The
compositions may take such forms as suspensions, solutions, or
emulsions in oily or aqueous vehicles, and may contain formulatory
agents such as suspending, stabilizing and/or dispersing agents.
Alternatively, the active ingredient may be in powder form for
constitution with a suitable vehicle (e.g., sterile pyrogen-free
water) before use.
[0929] A method of the invention may additionally comprise
administration of compositions formulated as depot preparations.
Such long acting formulations may be administered by implantation
(e.g., subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compositions may be formulated
with suitable polymeric or hydrophobic materials (e.g., as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives (e.g., as a sparingly soluble
salt).
[0930] Methods of the invention encompass administration of
compositions formulated as neutral or salt forms. Pharmaceutically
acceptable salts include those formed with anions such as those
derived from hydrochloric, phosphoric, acetic, oxalic, tartaric
acids, etc., and those formed with cations such as those derived
from sodium, potassium, ammonium, calcium, ferric hydroxides,
isopropylamine, triethylamine, 2-ethylamino ethanol, histidine,
procaine, etc.
[0931] Generally, the ingredients of compositions are supplied
either separately or mixed together in unit dosage form, for
example, as a dry lyophilized powder or water free concentrate in a
hermetically sealed container such as an ampoule or sachette
indicating the quantity of active agent. Where the mode of
administration is infusion, a composition can be dispensed with an
infusion bottle containing sterile pharmaceutical grade water or
saline. Where the mode of administration is by injection, an
ampoule of sterile water for injection or saline can be provided so
that the ingredients may be mixed prior to administration.
[0932] In particular, the invention also provides that one or more
of the prophylactic or therapeutic agents or pharmaceutical
compositions of the invention is packaged in a hermetically sealed
container such as an ampoule or sachette indicating the quantity of
the agent. In one embodiment, one or more of the prophylactic or
therapeutic agents, or pharmaceutical compositions of the invention
is supplied as a dry sterilized lyophilized powder or water free
concentrate in a hermetically sealed container and can be
reconstituted (e.g., with water or saline) to the appropriate
concentration for administration to a subject. Preferably, one or
more of the prophylactic or therapeutic agents or pharmaceutical
compositions of the invention is supplied as a dry sterile
lyophilized powder in a hermetically sealed container at a unit
dosage of at least 5 mg, more preferably at least 10 mg, at least
15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50
mg, at least 75 mg, or at least 100 mg. The lyophilized
prophylactic or therapeutic agents or pharmaceutical compositions
of the invention should be stored at between 2.degree. C. and
8.degree. C. in its original container and the prophylactic or
therapeutic agents, or pharmaceutical compositions of the invention
should be administered within 1 week, preferably within 5 days,
within 72 hours, within 48 hours, within 24 hours, within 12 hours,
within 6 hours, within 5 hours, within 3 hours, or within 1 hour
after being reconstituted. In an alternative embodiment, one or
more of the prophylactic or therapeutic agents or pharmaceutical
compositions of the invention is supplied in liquid form in a
hermetically sealed container indicating the quantity and
concentration of the agent. Preferably, the liquid form of the
administered composition is supplied in a hermetically sealed
container at least 0.25 mg/ml, more preferably at least 0.5 mg/ml,
at least 1 mg/ml, at least 2.5 mg/ml, at least 5 mg/ml, at least 8
mg/ml, at least 10 mg/ml, at least 15 mg/kg, at least 25 mg/ml, at
least 50 mg/ml, at least 75 mg/ml, or at least 100 mg/ml. The
liquid form should be stored at between 2.degree. C. and 8.degree.
C. in its original container.
[0933] The binding proteins of the invention can be incorporated
into a pharmaceutical composition suitable for parenteral
administration. Preferably, the binding protein will be prepared as
an injectable solution containing 0.1-250 mg/ml antibody. The
injectable solution can be composed of either a liquid or
lyophilized dosage form in a flint or amber vial, ampoule or
pre-filled syringe. The buffer can be L-histidine (1-50 mM),
optimally 5-10 mM, at pH 5.0 to 7.0 (optimally pH 6.0). Other
suitable buffers include but are not limited to, sodium succinate,
sodium citrate, sodium phosphate or potassium phosphate. Sodium
chloride can be used to modify the toxicity of the solution at a
concentration of 0-300 mM (optimally 150 mM for a liquid dosage
form). Cryoprotectants can be included for a lyophilized dosage
form, principally 0-10% sucrose (optimally 0.5-1.0%). Other
suitable cryoprotectants include trehalose and lactose. Bulking
agents can be included for a lyophilized dosage form, principally
1-10% mannitol (optimally 2-4%). Stabilizers can be used in both
liquid and lyophilized dosage forms, principally 1-50 mM
L-methionine (optimally 5-10 mM). Other suitable bulking agents
include glycine, arginine, can be included as 0-0.05%
polysorbate-80 (optimally 0.005-0.01%). Additional surfactants
include but are not limited to polysorbate 20 and BRIJ
surfactants.
[0934] The compositions of this invention may be in a variety of
forms. These include, for example, liquid, semi-solid and solid
dosage forms, such as liquid solutions (e.g., injectable and
infusible solutions), dispersions or suspensions, tablets, pills,
powders, liposomes and suppositories. The preferred form depends on
the intended mode of administration and therapeutic application.
Typical preferred compositions are in the form of injectable or
infusible solutions, such as compositions similar to those used for
passive immunization of humans with other antibodies. The preferred
mode of administration is parenteral (e.g., intravenous,
subcutaneous, intraperitoneal, intramuscular). In a preferred
embodiment, a DLL4 binding protein described herein is administered
by intravenous infusion or injection. In another preferred
embodiment, a DLL4 binding protein is administered by intramuscular
or subcutaneous injection.
[0935] Therapeutic compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
composition can be formulated as a solution, microemulsion,
dispersion, liposome, or other ordered structure suitable to high
drug concentration. Sterile injectable solutions can be prepared by
incorporating the active compound (i.e., antibody or antibody
portion) in the required amount in an appropriate solvent with one
or a combination of ingredients enumerated above, as required,
followed by filtered sterilization. Generally, dispersions are
prepared by incorporating the active compound into a sterile
vehicle that contains a basic dispersion medium and the required
other ingredients from those enumerated above. In the case of
sterile, lyophilized powders for the preparation of sterile
injectable solutions, the preferred methods of preparation are
vacuum drying and spray-drying that yields a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof. The proper fluidity of a
solution can be maintained, for example, by the use of a coating
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. Prolonged
absorption of injectable compositions can be brought about by
including, in the composition, an agent that delays absorption, for
example, monostearate salts and gelatin.
[0936] The DLL4 binding proteins of the present invention can be
administered by a variety of methods known in the art, although for
many therapeutic applications, the preferred route/mode of
administration is subcutaneous injection, intravenous injection, or
infusion. As will be appreciated by the skilled artisan, the route
and/or mode of administration will vary depending upon the desired
results. In certain embodiments, the active compound may be
prepared with a carrier that will protect the compound against
rapid release, such as a controlled release formulation, including
implants, transdermal patches, and microencapsulated delivery
systems. Biodegradable, biocompatible polymers can be used, such as
ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, and polylactic acid. Many methods for
the preparation of such formulations are patented or generally
known to those skilled in the art. See, e.g., Sustained and
Controlled Release Drug Delivery Systems, J. R. Robinson, ed.,
(Marcel Dekker, Inc., New York, 1978).
[0937] In certain embodiments, a binding protein of the invention
may be orally administered, for example, with an inert diluent or
an assimilable edible carrier. The compound (and other ingredients,
if desired) may also be enclosed in a hard or soft shell gelatin
capsule, compressed into tablets, or incorporated directly into the
subject's diet. For oral therapeutic administration, the compounds
may be incorporated with excipients and used in the form of
ingestible tablets, buccal tablets, troches, capsules, elixirs,
suspensions, syrups, wafers, and the like. To administer a compound
of the invention by other than parenteral administration, it may be
necessary to coat the compound with, or co-administer the compound
with, a material to prevent its inactivation.
[0938] Supplementary active compounds can also be incorporated into
the compositions. In certain embodiments, a binding protein of the
invention is coformulated with and/or coadministered with one or
more additional therapeutic agents that are useful for treating
disorders in which DLL4 activity is detrimental. For example, an
anti-huDLL4 antibody or antibody portion of the invention may be
coformulated and/or coadministered with one or more additional
antibodies that bind other targets (e.g., antibodies that bind
other cytokines or that bind cell surface molecules). Furthermore,
one or more binding proteins of the invention may be used in
combination with two or more of the foregoing therapeutic agents.
Such combination therapies may advantageously utilize lower dosages
of the administered therapeutic agents, thus avoiding possible
toxicities or complications associated with the various
monotherapies.
[0939] In certain embodiments, a DLL4 binding protein of the
invention is linked to a half-life extending vehicle known in the
art. Such vehicles include, but are not limited to, the Fc domain,
polyethylene glycol, and dextran. Such vehicles are described,
e.g., in U.S. Pat. No. 6,660,843 B1 and published PCT Publication
No. WO 99/25044, which are hereby incorporated by reference.
[0940] In a specific embodiment, nucleic acid sequences comprising
nucleotide sequences encoding a binding protein of the invention or
another prophylactic or therapeutic agent of the invention are
administered to treat, prevent, manage, or ameliorate a disorder or
one or more symptoms thereof by way of gene therapy. Gene therapy
refers to therapy performed by the administration to a subject of
an expressed or expressible nucleic acid. In this embodiment of the
invention, the nucleic acids produce their encoded binding protein
or prophylactic or therapeutic agent of the invention that mediates
a prophylactic or therapeutic effect.
[0941] Any of the methods for gene therapy available in the art can
be used according to the present invention. For general reviews of
the methods of gene therapy, see Goldspiel et al., Clin. Pharmacy,
12: 488-505 (1993); Wu and Wu, Biotherapy, 3: 87-95 (1991);
Tolstoshev, Ann. Rev. Pharmacol. Toxicol., 32: 573-596 (1993);
Mulligan, Science, 260: 926-932 (1993); and Morgan and Anderson,
Ann. Rev. Biochem., 62: 191-217 (1993); Robinson, C., Trends
Biotechnol., 11(5):155 (1993). Methods commonly known in the art of
recombinant DNA technology which can be used are described in
Ausubel et al. (eds.), Current Protocols in Molecular Biology (John
Wiley & Sons, New York, 1993); and Kriegler, Gene Transfer and
Expression, A Laboratory Manual, (Stockton Press, New York, 1990).
Detailed descriptions of various methods of gene therapy are
disclosed in US Patent Application Publication No. 20050042664 A1,
which is incorporated herein by reference.
[0942] In another aspect, this invention provides a method of
treating (e.g. curing, suppressing, ameliorating, delaying, or
preventing the onset of, or preventing recurrence or relapse of) or
preventing a DLL4-associated tumor in a subject. The method
includes administering to a subject a DLL4 binding protein, e.g.,
an anti-DLL4 antibody or fragment thereof as described herein, in
an amount sufficient to treat or prevent the DLL-associated tumor
or cancer. The DLL4 antagonist, i.e., the anti-DLL4 antibody or
fragment thereof, may be administered to a subject alone or in
combination with other therapeutic modalities as described
herein.
[0943] DLL4 plays a critical role in the pathology associated with
a variety of diseases involving immune and inflammatory elements,
in particular cancer and tumor angiogenesis. Examples of
DLL4-associated disorders include, but are not limited to, those
disorders that adversely affect the following biological processes:
neuronal function and development; stabilization of arterial
endothelial fate and angiogenesis; regulation of crucial cell
communication events between endocardium and myocardium during both
the formation of the valve primordial and ventricular development
and differentiation; cardiac valve homeostasis, as well as
implications in other human disorders involving the cardiovascular
system; timely cell lineage specification of both endocrine and
exocrine pancreas; influencing of binary fate decisions of cells
that must choose between the secretory and absorptive lineages in
the gut; expansion of the hematopoietic stem cell compartment
during bone development and participation in commitment to the
osteoblastic lineage such as osteoporosis; regulation of cell-fate
decision in mammary glands at several distinct development stages;
and certain non-nuclear mechanisms, such as control of the actin
cytoskeleton through the tyrosine kinase Abl. More specifically,
DLL4-associated disorders include, but are not limited to, cancers,
T-ALL (T-cell acute lymphoblastic leukemia), CADASIL (cerebral
autosomal dominant arteriopathy with subcortical infarcts and
leukoencephalopathy), MS (multiple sclerosis), tetralogy of Fallot
(TOF), and Alagille syndrome (AS), macular degeneration and
age-related macular degeneration diseases, and other angiogenesis
independent and dependent diseases characterized by aberrant DLL4
expression or activity.
[0944] Preferably, DLL4 binding proteins, such as antibodies and
antigen-binding portions thereof as described herein, are used to
treat cancers and tumors.
[0945] Binding proteins according to the invention can be used
alone or in combination, i.e., more than one DLL4-binding protein
described herein, to treat a cancer, a tumor, or other disorder in
which binding to, inhibition of, and/or neutralization of DLL4 is
considered desirable or otherwise beneficial to the health of an
individual.
[0946] It should be understood that DLL4 binding proteins of the
invention can also be used alone or in combination with an
additional agent, e.g., a therapeutic agent, said additional agent
being selected by the skilled practitioner for its intended
purpose. For example, the additional agent can be a therapeutic
agent that is recognized in the art as being useful to treat a
cancer, tumor, or other disease or condition in which binding to or
inhibition of DLL4 is considered to be desirable or advantageous
for treating the cancer, tumor, or other disease or condition. The
additional agent also can be an agent that imparts a beneficial
attribute to the therapeutic composition, e.g., an agent which
affects the viscosity of the composition.
[0947] It should further be understood that the combinations which
are to be included within this invention are those combinations
useful for their intended purpose. The agents set forth below are
illustrative for purposes and not intended to be limited. The
combinations, which are part of this invention, can be the
antibodies of the present invention and at least one additional
agent selected from the lists below. The combination can also
include more than one additional agent, e.g., two or three
additional agents, if the combination is such that the formed
composition can perform its intended function.
[0948] Preferred combinations of therapeutic agents may interfere
at different points in the pro-tumorigenic or pro-angiogenic
signaling pathways. Preferred examples of therapeutic agents useful
in the methods and compositions of the invention include
antineoplastic agents, radiotherapy, and chemotherapy such as DNA
alkylating agents, cisplatin, carboplatin, anti-tubulin agents,
paclitaxel, docetaxel, taxol, doxorubicin, gemcitabine, gemzar,
anthracyclines, adriamycin, topoisomerase I inhibitors,
topoisomerase II inhibitors, 5-fluorouracil (5-FU), leucovorin,
irinotecan, receptor tyrosine kinase inhibitors (e.g., erlotinib,
gefitinib), COX-2 inhibitors (e.g., celecoxib), and kinase
inhibitors.
[0949] The DLL4 binding proteins of the invention may also be
administered in combination with agents, such as methotrexate,
6-MP, azathioprine sulphasalazine, mesalazine, olsalazine
chloroquinine/hydroxychloroquine, pencillamine, aurothiomalate
(intramuscular and oral), azathioprine, colchicine, corticosteroids
(oral, inhaled and local injection), beta-2 adrenoreceptor agonists
(salbutamol, terbutaline, salmeteral), xanthines (theophylline,
aminophylline), cromoglycate, nedocromil, ketotifen, ipratropium
and oxitropium, cyclosporin, FK506, rapamycin, mycophenolate
mofetil, leflunomide, NSAIDs, for example, ibuprofen,
corticosteroids such as prednisolone, phosphodiesterase inhibitors,
adensosine agonists, antithrombotic agents, complement inhibitors,
adrenergic agents, agents which interfere with signaling by
proinflammatory cytokines such as TNF.alpha. or IL-1 (e.g. IRAK,
NIK, IKK, p38 or MAP kinase inhibitors), IL-1.beta. converting
enzyme inhibitors, TNF.alpha. converting enzyme (TACE) inhibitors,
T-cell signaling inhibitors such as kinase inhibitors,
metalloproteinase inhibitors, sulfasalazine, azathioprine,
6-mercaptopurines, angiotensin converting enzyme inhibitors,
soluble cytokine receptors and derivatives thereof (e.g., soluble
p55 or p75 TNF receptors and the derivatives p75TNFRIgG
(Enbrel.TM.) and p55TNFRIgG (Lenercept), sIL-1RI, sIL-1RII,
sIL-6R), antiinflammatory cytokines (e.g., IL-4, IL-10, IL-11,
IL-13 and TGF.beta.), celecoxib, folic acid, hydroxychloroquine
sulfate, rofecoxib, etanercept, infliximab, naproxen, valdecoxib,
sulfasalazine, methylprednisolone, meloxicam, methylprednisolone
acetate, gold sodium thiomalate, aspirin, triamcinolone acetonide,
propoxyphene napsylate/apap, folate, nabumetone, diclofenac,
piroxicam, etodolac, diclofenac sodium, oxaprozin, oxycodone hcl,
hydrocodone bitartrate/apap, diclofenac sodium/misoprostol,
fentanyl, anakinra, human recombinant, tramadol hcl, salsalate,
sulindac, cyanocobalamin/fa/pyridoxine, acetaminophen, alendronate
sodium, prednisolone, morphine sulfate, lidocaine hydrochloride,
indomethacin, glucosamine sulf/chondroitin, amitriptyline HCl,
sulfadiazine, oxycodone HCl/acetaminophen, olopatadine hcl,
misoprostol, naproxen sodium, omeprazole, cyclophosphamide,
rituximab, IL-1 TRAP, MRA, CTLA4-IG, IL-18 BP, anti-IL-18,
anti-IL15, BIRB-796, SCIO-469, VX-702, AMG-548, VX-740,
Roflumilast, IC-485, CDC-801, and Mesopram.
[0950] Non-limiting examples of therapeutic agents for cancers with
which a DLL4 binding protein of the invention can be
co-administered or used in combination include the following:
budenoside; epidermal growth factor; sulfasalazine;
aminosalicylates; 6-mercaptopurine; azathioprine; metronidazole;
lipoxygenase inhibitors; mesalamine; olsalazine; balsalazide;
antioxidants; thromboxane inhibitors; IL-1 receptor antagonists;
anti-IL-1.beta. monoclonal antibodies; anti-IL-6 monoclonal
antibodies; growth factors; elastase inhibitors;
pyridinyl-imidazole compounds; and antibodies to or antagonists of
other human cytokines or growth factors, for example, TNF, LT,
IL-1, IL-2, IL-6, IL-7, IL-8, IL-15, IL-16, IL-17, IL-18, EMAP-II,
GM-CSF, FGF, and PDGF. Antibodies of the invention, or antigen
binding portions thereof, can be combined with antibodies to cell
surface molecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30,
CD40, CD45, CD69, CD90, or their ligands.
[0951] Other examples of therapeutic agents with which a DLL4
binding protein of the invention can be combined include the
following: TNF antagonists, for example, anti-TNF antibodies, D2E7
(PCT Publication No. WO 97/29131; HUMIRA.RTM.), CA2
(REMICADE.RTM.), CDP 571, TNFR-Ig constructs (p75TNFRIgG
(ENBREL.RTM.) and p55TNFRIgG (LENERCEPT)), and PDE4 inhibitors.
Binding proteins of the invention can be combined with mesalamine,
prednisone, azathioprine, mercaptopurine, infliximab,
methylprednisolone sodium succinate, diphenoxylate/atrop sulfate,
loperamide hydrochloride, methotrexate, omeprazole, folate,
ciprofloxacin/dextrose-water, hydrocodone bitartrate/apap,
tetracycline hydrochloride, fluocinonide, metronidazole,
thimerosal/boric acid, cholestyramine/sucrose, ciprofloxacin
hydrochloride, hyoscyamine sulfate, meperidine hydrochloride,
midazolam hydrochloride, oxycodone hcl/acetaminophen, promethazine
hydrochloride, sodium phosphate, sulfamethoxazole/trimethoprim,
celecoxib, polycarbophil, propoxyphene napsylate, hydrocortisone,
multivitamins, balsalazide disodium, codeine phosphate/apap,
colesevelam hcl, cyanocobalamin, folic acid, levofloxacin,
methylprednisolone, natalizumab, and interferon-gamma.
[0952] Non-limiting examples of therapeutic agents with which a
binding protein of the invention can be combined include the
following: aspirin, nitroglycerin, isosorbide mononitrate,
metoprolol succinate, atenolol, metoprolol tartrate, amlodipine
besylate, diltiazem hydrochloride, isosorbide dinitrate,
clopidogrel bisulfate, nifedipine, atorvastatin calcium, potassium
chloride, furosemide, simvastatin, verapamil hcl, digoxin,
propranolol hydrochloride, carvedilol, lisinopril, spironolactone,
hydrochlorothiazide, enalapril maleate, nadolol, ramipril,
enoxaparin sodium, heparin sodium, valsartan, sotalol
hydrochloride, fenofibrate, ezetimibe, bumetanide, losartan
potassium, lisinopril/hydrochlorothiazide, felodipine, captopril,
and bisoprolol fumarate.
[0953] The pharmaceutical compositions of the invention may include
a "therapeutically effective amount" or a "prophylactically
effective amount" of a binding protein of the invention. A
"therapeutically effective amount" refers to an amount effective,
at dosages and for periods of time necessary, to achieve the
desired therapeutic result. A therapeutically effective amount of
the binding protein may be determined by a person skilled in the
art and may vary according to factors such as the disease state,
age, sex, and weight of the individual, and the ability of the
binding protein to elicit a desired response in the individual. A
therapeutically effective amount is also one in which any toxic or
detrimental effects of the binding protein are outweighed by the
therapeutically beneficial effects. A "prophylactically effective
amount" refers to an amount effective, at dosages and for periods
of time necessary, to achieve the desired prophylactic result.
Typically, since a prophylactic dose is used in subjects prior to
or at an earlier stage of disease, the prophylactically effective
amount will be less than the therapeutically effective amount.
[0954] Dosage regimens may be adjusted to provide the optimum
desired response (e.g., a therapeutic or prophylactic response).
For example, a single bolus may be administered, several divided
doses may be administered over time or the dose may be
proportionally reduced or increased as indicated by the exigencies
of the therapeutic situation. It is especially advantageous to
formulate parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the mammalian subjects to be treated; each unit
containing a predetermined quantity of active compound calculated
to produce the desired therapeutic effect in association with the
required pharmaceutical carrier. The specification for the dosage
unit forms of the invention are dictated by and directly dependent
on (a) the unique characteristics of the active compound and the
particular therapeutic or prophylactic effect to be achieved, and
(b) the limitations inherent in the art of compounding such an
active compound for the treatment of sensitivity in
individuals.
[0955] An exemplary, non-limiting range for a therapeutically or
prophylactically effective amount of a DLL4 binding protein of the
invention is 0.1-20 mg/kg, more preferably 1-10 mg/kg. It is to be
noted that dosage values may vary with the type and severity of the
condition to be alleviated. It is to be further understood that for
any particular subject, specific dosage regimens should be adjusted
over time according to the individual need and the professional
judgment of the person administering or supervising the
administration of the compositions, and that dosage ranges set
forth herein are exemplary only and are not intended to limit the
scope or practice of the claimed composition.
III. Use in Immunotechniques.
[0956] Any of a variety of immunodetection assay formats may be
adapted to employ a DLL4 binding protein of the invention for use
in detecting DLL4 present in a mixture, solution, or biological
sample. Such immunodetection assay formats include but are not
limited to radioimmunoassay (RIA), immunoprecipitation,
enzyme-linked immunosorbent assay (ELISA), immunoblot (e.g.,
Western blot), immunostrips (e.g., immunodipsticks) that comprise a
DLL4 binding protein of the invention adsorbed or immobilized to
substrate, fluorescence activated cell sorting (FACS), and the
like. A DLL4 binding protein described herein can be adsorbed or
immobilized to a substrate, e.g., a resin particle or other
material, for use in an affinity column or any other affinity
format available in the art to purify DLL4 from a sample. Detection
of DLL4 using a DLL4 binding protein of the invention can be
conducted in vitro on a mixture, solution, or in biological sample.
A biological sample that can be contacted with a DLL4 binding
protein of the invention to detect or measure DLL4 in the sample
includes, but is not limited to, urine, saliva, oral swab (buccal,
lingual, or throat swab), dermal swab, dermal scrape, rectal swab,
vaginal swab, whole blood sample, plasma sample, serum sample,
tissue biopsy, and any other sample obtained from an individual by
a procedure known in the art. In another embodiment, a DLL4 binding
protein may be employed to detect DLL4 in vivo such as various
tomography and scanning methods, including but not limited to X-ray
computer assisted tomography (CT), magnetic resonance imaging
(MRI), and positron emission tomography (PET).
[0957] It will be readily apparent to those skilled in the art that
other suitable modifications and adaptations of the methods of the
invention described herein are obvious and may be made using
suitable equivalents without departing from the scope of the
invention or the embodiments disclosed herein. Having now described
the present invention in detail, the same will be more clearly
understood by reference to the following examples, which are
included for purposes of illustration only and are not intended to
be limiting of the invention.
EXAMPLES
Example 1: In Vitro Assays Used to Determine the Functional
Activity of DLL4 Antibodies
Example 1.1: Affinity Determination Using BIACORE.RTM. Surface
Plasmon Resonance Technology
[0958] The BIACORE.RTM. surface plasmon resonance assay (Biacore,
Inc., Piscataway, N.J., US) determines the affinity of antibodies
with kinetic measurements of on-rate and off-rate constants.
Binding of DLL4 antibodies to a purified recombinant DLL4
extracellular domain (ECD) was determined by surface plasmon
resonance-based measurements with a Biacore.RTM. instrument (either
a Biacore 2000, Biacore 3000, or Biacore T100; GE Healthcare,
Piscataway, N.J., US) using running buffer HBS-EPB (10 mM HEPES [pH
7.4], 150 mM NaCl, 3 mM EDTA, 0.1 mg/ml BSA and 0.005% surfactant
P20) at 25.degree. C. For example, approximately 9000 RU of goat
anti-human Fc specific polyclonal antibody (Thermo Fisher
Scientific Inc., Rockford, Ill., US) diluted in 10 mM sodium
acetate (pH 4.5) is directly immobilized across a CMS research
grade biosensor chip using a standard amine coupling kit according
to manufacturer's instructions and procedures at 25 .mu.g/ml.
Unreacted moieties on the biosensor surface were blocked with
ethanolamine. For kinetic analysis, rate equations derived from the
1:1 Langmuir binding model were fitted simultaneously to multiple
antigen injections (using global fit analysis) with the use of
Scrubber 2 (BioLogic Software), Biacore Biaevaluation 4.0.1
software or Biacore T100 Evaluation software. Purified antibodies
were diluted in running buffer for capture across goat anti-human
Fc reaction surfaces. Antibodies to be captured as a ligand (1
.mu.g/ml) were injected over reaction matrices at a flow rate of 10
.mu.l/min. During the assay, all measurements were referenced
against the capture surface alone (i.e., with no captured anti-DLL4
antibody). The association and dissociation rate constants,
K.sub.on (M.sup.-1s.sup.-1) and K.sub.off (s.sup.-1) were
determined under a continuous flow rate of 80 .mu.l/min. Rate
constants were derived by making kinetic binding measurements at
different antigen concentrations ranging from 1.23-900 nM, as a
3-fold dilution series, and included buffer-only injections (to be
used for double referencing). The equilibrium dissociation constant
K.sub.D (M) of the reaction between antibodies and the target
antigen was then calculated from the kinetic rate constants by the
following formula: K.sub.D=K.sub.off/K.sub.on. Binding was recorded
as a function of time and kinetic rate constants were calculated.
In this assay, on-rates as fast as 10.sup.6 M.sup.-1s.sup.-1 and
off-rates as slow as 10.sup.6 s.sup.-1 could be measured.
Example 1.2: Binding of DLL4 Antibodies to Soluble DLL4
Extracellular Domain as Determined by ELISA
Method 1 (Capture ELISA).
[0959] 96-well Nunc-Immuno plates (#439454) were coated with 5
.mu.g/ml antibody against human IgG (Fcg fragment specific, Jackson
ImmunoResearch, #109-005-098, 100 .mu.l/well) in D-PBS (Gibco
#14190) and incubated overnight at 4.degree. C. ELISA plates were
washed 3 times with wash buffer (PBS, 0.05% Tween-20) and then
blocked with 200 ml/well blocking buffer (D-PBS, 1% BSA, 1 mM
CaCl.sub.2, 0.05% Tween-20) for 1 hour at 25.degree. C. Plates were
washed 3 times and incubated with 100 .mu.l/well DLL4 antibodies
(0.0001-100 nM, 10-fold serial dilution in blocking buffer) for 1
hour at 25.degree. C., and then washed again 3 times. Plates
containing captured DLL4 antibody were incubated with
biotin-labeled human DLL4 extracellular domain (10 nM in blocking
buffer, 100 .mu.l/well) for 1 hour at 25.degree. C., washed 3
times, and incubated with streptavidin conjugated with HRP (KPL
#474-3000, 1:10,000 dilution in blocking buffer, 100 .mu.l/well)
for 1 hour at 25.degree. C. After the final wash, plates were
incubated with 100 .mu.l/well ELISA substrate (1-Step Ultra
TMB-ELISA, Pierce #340280). The reaction was stopped after 2
minutes at 25.degree. C. with 100 .mu.l/well 2 N H.sub.2SO.sub.4
and the absorbance was read at 450 nm. Data were analyzed using
Graphpad Prism software and EC.sub.50 values were reported.
Method 2 (Copper Coated Plate).
[0960] 96-well copper-coated plates (Thermo Scientific #15143) were
washed 3 times with wash buffer (PBS, 0.05% Tween-20) before use
and then incubated with 100 .mu.l/well of 6.times.His-tagged
recombinant DLL4 extracellular domain (ECD) ("6.times.His"
disclosed as SEQ ID NO: 206) at 1 .mu.g/ml in PBS, 1 hour at
25.degree. C. with shaking. Plates were then washed 3 times. 100
.mu.l/well of recombinant rat/human chimeric or recombinant human
anti-DLL4 antibodies were then added to the plate (0.00164-27 nM,
4-fold serial dilution in ELISA buffer=PBST, 10% Superblock (Pierce
#37515)) for 1 hour at 25.degree. C. with shaking and then washed
again 3 times. Plates were incubated with goat anti-human HRP
(Pierce #31412) (1:40,000 dilution in ELISA buffer, 100 .mu.l/well)
for 1 hour at 25.degree. C. with shaking, then washed 3 times.
After the final wash, plates were incubated with 100 .mu.l/well
ELISA substrate (Sigma #T8665). The reaction was stopped after 8
minutes at 25.degree. C. with 100 .mu.l/well 1N HCl and the
absorbance was read at 450 nm. Data were analyzed using Graphpad
Prism software, and EC50 values were reported.
Example 1.3: Binding of DLL4 Monoclonal Antibodies to the Surface
of Human Tumor Cell Lines as Assessed by Flow Cytometry (FACS)
[0961] Stable cell lines overexpressing cell-surface DLL4 were
harvested from tissue culture flasks, washed four times and
resuspended in phosphate buffered saline (PBS) containing 1% bovine
serum albumin and 1 mM CaCl.sub.2 (FACS buffer). 1.5.times.10.sup.5
cells were incubated with antibodies at various concentrations in
FACS buffer for 60 minutes on ice. Cells were washed twice and 50
.mu.L of R-phycoerythrin-conjugated anti-rat IgG, F(ab').sub.2
fragment (1:200 dilution in FACS buffer) (Jackson ImmunoResearch,
West Grove, Pa., Cat.#112-116-072) were added. Following an
incubation on ice (4.degree. C., 60 minutes), cells were washed
three times and resuspended in FACS buffer. Fluorescence was
measured using a Becton Dickinson FACSCalibur-HTS (Becton
Dickinson, San Jose, Calif., US). Data were analyzed using Graphpad
Prism software and EC.sub.50 values were reported as the
concentration of antibody to achieve 50% of maximal DLL4 antibodies
binding to DLL4 expressing cells.
Example 1.4: Inhibition of Notch-1 Interaction with Soluble DLL4
Extracellular Domain by DLL4 Antibodies (Competition ELISA)
[0962] 96-well Nunc-Immuno plates (#439454 for huDLL4 ELISA) and
96-well Costar plates (#9018 for muDLL4 ELISA) were coated with 16
nM human Notch-1 (R&D Systems #3647-TK, 100 .mu.l/well in
D-PBS) and incubated overnight at 4.degree. C. Plates were then
washed 3 times with wash buffer (PBS, 0.05% Tween-20) and blocked
with 200 .mu.l/well blocking buffer (D-PBS, 1% BSA, 1 mM
CaCl.sub.2, 0.05% Tween-20) for 1 hour at 25.degree. C. While
blocking, biotin labeled DLL4 extracellular domain (14 nM) was
mixed with antibody (30 pM-66 nM, 3-fold serial dilution in
blocking buffer) for 1 hour at 25.degree. C. with shaking. Assay
plates were washed after blocking, and incubated with DLL4/antibody
mixtures (100 .mu.l/well, 1 hour at 25.degree. C. with shaking).
Plates were washed again and 100 .mu.l/well streptavidin conjugated
with HRP (Fitzgerald #65R-S104PHRPx, diluted 1:5,000 in blocking
buffer) was added for 1 hour at 25.degree. C. with shaking. After a
final wash, plates were developed using 100 .mu.l/well substrate
(TMB Sigma #T8665), and the reaction was stopped using 100
.mu.l/well 1N HCl, and the absorbance was read at 450 nm. Data were
analyzed using Graphpad Prism software and IC.sub.50 values were
reported as the concentration of antibody to achieve 50% reduction
of DLL4 bound to Notch1.
Example 1.5: Blocking of Soluble Notch Binding to
DLL4-Overexpressing 293G Cells by Anti-DLL4 Monoclonal Antibodies
as Assessed by Flow Cytometry (Competition FACS)
[0963] Notch blocking assay: Briefly, stable cell lines
overexpressing cell-surface DLL4 were harvested from tissue culture
flasks and re-suspended in phosphate buffered saline (PBS)
containing 1% bovine serum albumin and 1 mM CaCl.sub.2 (FACS
buffer). HEK293G-DLL4 cells were dispensed into 96-well plate
(v-bottom) at 1.5.times.10.sup.5 cells/well in FACS buffer. After
spinning down cells and discarding the supernatant, 50 .mu.L of
purified IgG with appropriate dilution were added to each well, and
incubated on ice at 4.degree. C. for 60 minutes, followed by
addition of 50 4/well of Notch1-biotin at 0.2 .mu.g/mL for human
DLL4-293G or 2.0 .mu.g/mL for mouse DLL4-293G (1.0 or 0.1 .mu.g/mL
final) for an additional 1 hour incubation ice at 4.degree. C.
After washing the cells two times with FACS buffer, 50 .mu.L of
R-phycoerythrin-conjugated streptavidin (1:150 dilution in FACS
buffer) (Jackson ImmunoResearch, West Grove, Pa., Cat.#016-110-084)
were added. Following an incubation on ice (4.degree. C., 60
minutes), cells were washed three times and resuspended in FACS
buffer. Fluorescence was measured using a Becton Dickinson
FACSCalibur-HTS (Becton Dickinson, San Jose, Calif.). Data were
analyzed using Graphpad Prism software, and IC.sub.50 values were
reported as the concentration of antibody to achieve 50% reduction
of Notch1 bound to DLL4 expressing cells.
Example 1.6: Inhibition of DLL4-Dependent Notch Activation in
EA.Hy926 Cells by DLL4 Antibodies Using Notch Reporter Assay
[0964] 96-well black clear-bottom tissue culture plates were seeded
overnight with 7,000 cells/well engineered EA.hy926 cells
expressing luciferase driven by a Notch-responsive promoter.
Antibodies serially diluted from 200 nM were mixed for 15 minutes
with equal volume of 5,000 HEK293G cells/well expressing
full-length DLL4. The 293G/DLL4 cells were co-cultured with
EA.hy926 Notch reporter cells for 24 hours in the presence of
testing antibodies. Luciferase activity was analyzed by Promega's
substrate (Promega # E2940). Data was analyzed using Graphpad Prism
software, and IC.sub.50 values were reported as the concentration
of antibody to achieve 50% reduction of DLL4-induced Notch
activation.
Example 1.7: Analytical Methods and Techniques for Physicochemical
Property Characterizations
PEG Precipitation Method.
[0965] The use of PEG for inducing phase separation of a solid
protein according to principles of volume exclusion represents a
feasible approach to assess the solubility of a protein. PEG has
several advantages over other precipitants, including minimal
denaturation of proteins at ambient temperatures (does not affect
tertiary structure of proteins) and within the range of 4.degree.
C. to 30.degree. C. temperature control is not required, i.e.,
precipitation studies can be performed at ambient temperature at
the laboratory bench.
[0966] Generally, the precipitation of proteins by PEGs is
explained on the basis of volume exclusion effects. According to
this theory, proteins are sterically excluded from the regions of
solvent that are occupied by PEG linear chains. As a result,
proteins are concentrated and eventually precipitated when their
solubility is exceeded. In thermodynamic terms, the steric
exclusion leads to an increase in the chemical potential of the
protein until it exceeds that of the pure solid state, resulting in
protein precipitation. This happens mainly because of a large
unfavorable free energy of interaction between PEG and proteins,
reducing the preferential hydration of protein due to steric
exclusion effects. In aqueous solutions, preferential hydration
helps to maintain the native structure of proteins. Generally,
volume exclusion has been shown to become more effective with
increasing molecular weight of the PEG, i.e., less PEG is needed to
precipitate proteins with increasing PEG molecular weight.
[0967] A PEG molecular weight of 3000 was chosen for estimating the
solubility of the antibodies covered by this patent. A 50% PEG
solution was made by dissolving PEG in deionized water in the ratio
of one gram of PEG to 1 mL of water. The PEG solution was then
added to a solution of antibody which was initially at a
concentration of less than or equal to 0.5 mg/ml and a volume of
0.5 mL. The PEG solution was continually added and mixed until the
first instance of cloudiness persists. The percentage of PEG 3000
needed to cause this precipitation is calculated as
(50).times.(volume of PEG 3000 solution added)/(initial volume of
antibody solution before PEG addition).
[0968] The percentage of PEG 3000 needed for precipitation was
compared to the percentage needed for precipitation of protein with
known water solubility. For example, the water solubility of
adalimumab exceeds 200 mg/mL. Consequently, if the percentage of
PEG 3000 required to precipitate a protein of interest is similar
to the percentage needed to precipitate adalimumab, then the
predicted solubility of that protein will be similar to the
solubility adalimumab.
Real Solubility Method.
[0969] Real solubility is determined by using Amicon centrifugal
filters to concentrate a protein in solution until the protein is
observed to precipitate out of solution or until the minimum volume
to which the protein can be concentrated within the filter unit is
reached. For the latter, 15 mL Amicon centrifugal filters have a
minimum volume of .about.50 .mu.l while 4 mL Amicon centrifugal
filters have a minimum volume of .about.15 .mu.l.
[0970] First, a protein was dialyzed into a specific
formulation(s). For these studies, the antibody amount was 10 mg or
much less. Then the protein solution was inserted into the Amicon
centrifugal filter retentate chamber. The chamber was lined with a
nitrocellulose membrane with pores that permitted molecules of less
than 10 to 30 kilodaltons to pass when subjected to centrifugal
force. Antibodies, which were typically above 140 kilodaltons, were
retained while water, buffer molecules, small excipients, and salts
passed through. The centrifugal filter was then centrifuged
according to manufacturer specifications until the protein was
observed to precipitate out of solution or until the minimum volume
to which the protein can be concentrated within the filter unit was
reached.
[0971] After centrifugation, the protein solution was removed from
the retentate chamber, and the concentration was measured by
ultraviolet absorbance. The solution was then kept at 25.degree. C.
and 5.degree. C. for 1 to 2 days and was monitored for signs of
precipitation.
Near UV-CD Technique.
[0972] Near UV-CD spectroscopy provides important information about
the tertiary structure of proteins and was one of the most used
techniques in this regards. CD refers to the differential
absorption of the left and right circularly polarized components of
plane polarized radiation. For proteins, the chromophores in the
near UVCD region (250-320 nm) are the aromatic amino acids (i.e.,
tryptophan, tyrosine, and phenylalanine) and the disulfide bonds,
and the CD effect occurs when the chromophores are present in an
asymmetric (buried) environment. Signals in the region from 250-270
nm are attributable to phenylalanine residues, signals from 270-290
nm are attributable to tyrosine, and those from 280-300 nm are
attributable to tryptophan. Disulfide bonds give rise to broad weak
signals throughout the near-UV spectrum. The near-UV CD spectrum
can be sensitive to small changes in tertiary structure such as
those due to protein-protein interactions and/or changes in
formulation conditions.
[0973] There are a number of other factors that can influence the
CD spectra of aromatic amino acids. Among these are (1) the
rigidity of the protein, (2) the nature of hydrogen bonding, and
(3) interactions between various aromatic amino acids.
Additionally, proteins with large number of such amino acids can
have smaller CD bands due to the cancellation of the positive and
negative bands.
[0974] Briefly, a protein dialyzed into the desired formulation(s)
at 1 mg/ml and was scanned from 250-320 nm or 240-320 nm with a
Jasco 800 CD spectrometer. The corresponding formulation without
protein was also scanned, and the readings subtracted from that of
the scan of the protein solution. A near UV-CD spectra was a plot
of molar ellipticities versus wavelength from 250 or 240 to 320
nm.
[0975] For antibodies in general, a near UV-CD spectrum with a
semi-sigmoidal profile indicates good tertiary structure folding
while a flatter and less featured profile indicates a greater
tendency to unfold. Compact folding is associated with good
stability while poor folding exposes the hydrophobic interior which
may lead to hydrophobic interactions among protein molecules
resulting in the formation of undesired aggregates.
DSC Technique.
[0976] The thermal stability of the antibodies was assessed using a
differential scanning calorimetry (DSC) instrument. The DSC
instrument used was an automated VP-DSC equipment with Capillary
Cell (Microcal, GE Healthcare Ltd./Microcal, Buckinghamshire, UK).
Unfolding of molecules was studied applying a 1.degree. C./minute
scan rate over a 25.degree. C.-95.degree. C. temperature range for
samples at 1 mg/mL. Additional measurement parameters applied were
a fitting period of 16 seconds, a pre-scan wait time of 10 minutes,
and measurements were performed in none-feedback mode. Per
individual measurement, 420 .mu.L of sample/blank were filled into
the DSC measurement sample holder, with a plate fill scheme as
provided below. The thermograms obtained were fitted to a non two
state model to obtain the midpoint temperatures and enthalpies of
the different transitions.
[0977] An additional requirement for successful biologics
development candidate is that the protein remains in its native
state and conformation. A protein in aqueous solution is in
equilibrium between the native (folded) conformation and its
denatured (unfolded) conformation. The stability of the native
state is based on the magnitude of the Gibbs free energy (DG) of
the system and the thermodynamic relationship between enthalpy (DH)
and entropy (DS) changes. A positive DG indicates the native state
is more stable than the denatured state--the more positive the DG,
the greater the stability. For a protein to unfold, stabilizing
forces need to be broken. Conformational entropy overcomes
stabilizing forces allowing the protein to unfold at temperatures
where entropy becomes dominant. DSC measures DH of protein
unfolding due to heat denaturation. As a general rule, it can be
stated that the higher the transition midpoint (the Tm), the more
stable the protein at lower temperatures. During the same
experiment, DSC also measures the change in heat capacity (DCp) for
protein denaturation. Heat capacity changes associated with protein
unfolding are primarily due to changes in hydration of side chains
that were buries in the native state, but become solvent exposed in
the denatured state. DSC has been shown to be a valuable predictor
of liquid formulation stability for proteins and other biological
macromolecules (Remmele, R. L. Jr., Gombotz, W. R., BioPharm 13,
36-46, 2000, and; Remmele, R. L. Jr., Nightlinger, N. S.,
Srinivasen, S., Gombotz, W. R., Pharm. Res. 15, 200-208, 1998).
SEC Technique.
[0978] Size exclusion chromatography (SEC) was used to separate
proteins based on size. Proteins are carried in an aqueous mobile
phase and through a porous stationary phase resin packed in a
column. The retention time in the column is a function of the
hydrodynamic size of the protein and the size of the pores in the
packed resin bed. Smaller molecules can penetrate into smaller
pores in the resin and are retained longer than larger molecules.
Upon elution from the column, the proteins are detected by UV
absorbance. The SEC method used a TSK gel guard (TOSOH Biosciences,
Montgomeryville, Pa., cat. no. 08543) and a TSK gel G3000SWxL
(TOSOH Biosciences, Montgomeryville, Pa., cat. no. 08541). The
mobile phase was 100 mM Na.sub.2HPO.sub.4, 200 mM Na.sub.2SO.sub.4,
pH 6.8. The flow rate was 0.25 mL/minute. Injection volume was 20
.mu.L of 1 mg/mL sample. The column temperature was room
temperature. The autosampler temperature was 2-8.degree. C. The
total run time was 55 minutes. The detection was based on UV
absorbance at 214 nm wavelength, with band width set at 8 nm, using
reference wavelength at 360 nm with band width 100 nm.
Freeze-Thaw Method.
[0979] Antibody solutions at 1 mg/ml in the desired formulation(s)
were frozen at -80.degree. C. for at least 4 hours and then thawed
at 30.degree. C. in a water bath. The solutions were then refrozen
at -80.degree. C. This was repeated for 5 cycles. After certain
freeze-thaw cycles, e.g., second and fourth, a portion of the
solution was withdrawn for analysis by SEC before refreezing.
Freeze-thaw stability testing was done at low protein concentration
in order obtain a "worse-case scenario" due to greater exposure of
protein molecules to the denaturing ice-water interfaces. At higher
concentrations, proportionally less protein encounters the
ice-water interface, instead interacting with other protein
molecules.
Accelerated Stability Method.
[0980] Antibody solutions at 1 mg/ml in the desired formulation(s)
were passed through 0.22 .mu.m PVDF filters under sterile
conditions and incubated at 40.degree. C. and/or 50.degree. C. for
at least 21 days. At 7 days and 21 days, aliquots were withdrawn
under sterile conditions and subjected to analysis by SEC.
Solutions were then returned to incubation.
Example 2: Generation of Rat Anti-DLL4 Monoclonal Antibodies by Rat
Hybridoma Technology
[0981] Rats were immunized according to the methods known in the
art (for example, E Harlow, D. Lane, Antibody: A Laboratory Manual,
(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
1998). A human cell line expressing full-length human DLL4 as well
as recombinant mouse DLL4-ECD (DLL4 extracellular domain) proteins
was used as immunogen. Mouse cell lines expressing either human
DLL4 or mouse DLL4 were used for determining anti-sera titer and
for screening hybridomas secreting antigen-specific antibodies.
Immunizing dosages contained 1.times.10.sup.6 cells/rat/injection
for both primary and boost immunizations. To increase immune
response to mouse DLL4, the rats were further boosted with
recombinant mouse DLL4-ECD in emulsion form with an incomplete
Freud's adjuvant (Sigma, St. Louis, Mo., US). Briefly,
adjuvant-antigen mixture was prepared by first gently mixing the
adjuvant in a vial using a vortex. The desired amount of adjuvant
was removed from the vial and put into an autoclaved 1.5 mL
microcentrifuge tube. The antigen was prepared in PBS or saline
with concentration ranging from 0.5-1.0 mg/ml. The calculated
amount of antigen was then added to the microcentrifuge tube with
the adjuvant, and the solution was mixed by gently vortexing for 2
minutes to generate water-in-oil emulsion. The adjuvant-antigen
solution was then drawn into the proper syringe for animal
injection. A total of 50 .mu.g of antigen was injected in a volume
of 50-100 .mu.l. Each animal was immunized, and then boosted for 2
to 3 times depending on the titer. Animals with good titers were
given a final subcutaneous boost with cell line expressing human
DLL4 before fusion.
Hybridoma Fusion and Screening.
[0982] Cells of murine myeloma cell line (SP2/0-Ag14, ATCC
CRL-1581) were cultured to reach the log phase stage right before
fusion. Immunized rat spleen cells were prepared sterilely and
fused with myeloma cells according to the methods known in the art
(for example, E Harlow, D. Lane, Antibody: A Laboratory Manual,
(Cold Spring Harbor
[0983] Laboratory Press, Cold Spring Harbor, N.Y., 1998); Kohler G,
and Milstein C., "Continuous cultures of fused cell secreting
antibody of predefined specificity," Nature, 256: 495-497 (1975)).
Fused "hybrid cells" were subsequently dispensed into 96-well
plates in DMEM/20% FCS/HAT media. Surviving hybridoma colonies were
observed under the microscope seven to ten days post-fusion. After
two weeks, the supernatant from each well was subjected to
cell-based binding screening using mouse cell lines expressing
either recombinant human DLL4 or mouse DLL4. Briefly, the mixture
(1:1 ratio) of mouse cell lines expressing either human or mouse
DLL4 was dispensed into a 96-well (round bottom) plate at
1.times.10.sup.6 cells/well and incubated with hybridoma
supernatant (50 .mu.l) at 4.degree. C. for 1 hour. Cells were then
washed 3 times with FACS buffer (PBS+2% BSA), HRP-goat anti-rat
Ig-PE (phycoerythrin) was used for detection in FACS machine.
Hybridomas were screened using ELISA format according the following
procedure. ELISA plates were coated with 50 .mu.l of either human
DLL4 or mouse DLL4 (2.0 .mu.g/ml in PBS) overnight at 4.degree. C.
Plates were washed 3 times with 250 .mu.l PBS/0.5% Tween.sub.20 and
blocked with 200 .mu.l blocking buffer (2% BSA in PBS with 0.5%
Tween.sub.20). Diluted sera or hybridoma supernatant (100 .mu.l)
was added to each well, and incubated at room temperature for 1
hour. Plates were then washed 3 times with PBS/0.5% Tween.sub.20,
HRP-goat anti-rat-IgG was used for detection, and binding ODs were
observed at 450 nm. Positive hybridoma secreting antibody that
binds to either human DLL4 or mouse DLL4 or both were then selected
and transferred to 24-well plates and subcloned by limiting
dilution to ensure the clonality of the cell line. The isotype of
each monoclonal antibody was determined using the Zymed's Mouse
MonoAb-ID Kit. Hybridoma clones producing antibodies that showed
high specific binding activity were subcloned and purified (Table
5), and affinity (Biacore) and potency (Notch blocking FACS and
reporter assay) of the antibodies were characterized as
follows.
TABLE-US-00010 TABLE 5 A List of Anti-DLL4 Antibodies Generated
Using Rat Hybridoma Technology FACS binding Human Mouse Hybridoma
Isotype DLL4 DLL4 MC10- IgG2a/k + + 37D10.1C2 MC10- IgG2a/k + +
40B10.3C3 MC10- IgG2a/k + + 32C7.5A4 MC10- IgG2a/k + - 38H12.2G8
MC13- IgG2a/K + + 14A11.3A4 MC13-14G1.1B4 IgG2a/K + - MC13-1A11.2E1
IgG1/K + + MC13-13E4.4A3 IgG1/K + + MC13-15D6.1G7 IgG2a/K + + "+ "
indicates antibody bound to cells; "-" indicates antibody did not
bind to cells
Example 3. In Vitro Characterization of Anti-DLL4 Rat Monoclonal
Antibodies
[0984] The antigen binding affinities of these rat monoclonal
antibodies (mAbs) were determined by the BIACORE technology as
described in Example 1.1, and are shown in Tables 6 and 7, below.
Their in vitro activities were further examined using other methods
described in Example 1, and are summarized in Table 8. Further
characterization determined 37D10 and 40B10 were identical rat
mAbs.
TABLE-US-00011 TABLE 6 Biacore Kinetics of Anti-DLL4 Rat Hybridoma
Antibodies Binding to Human and Cynomolgus Monkey DLL4. Kinetics on
Biacore huDLL4 ECD cynoDLL4 ECD k.sub.a k.sub.d K.sub.D k.sub.a
k.sub.d K.sub.D Clone (M.sup.-1s.sup.-1) (s.sup.-1) (nM)
(M.sup.-1s.sup.-1) (s.sup.-1) (nM) 38H12 4.0 .times. 10.sup.+5 1.1
.times. 10.sup.-4 0.3 2.6 .times. 10.sup.+5 9.8 .times. 10.sup.-5
0.4 1A11 1.9 .times. 10.sup.+5 1.1 .times. 10.sup.-3 5.8 1.1
.times. 10.sup.+5 9.3 .times. 10.sup.-4 8.1 37D10 = 1.4 .times.
10.sup.+5 7.0 .times. 10.sup.-2 484 4.0 .times. 10.sup.+4 Too
<617 40B10 fast* 32C7 2.8 .times. 10.sup.+5 7.0 .times.
10.sup.-6 0.03 2.2 .times. 10.sup.+5 6.3 .times. 10.sup.-6 0.03
15D6 1.1 .times. 10.sup.+5 1.0 .times. 10.sup.-3 9.2 5.6 .times.
10.sup.+4 8.3 .times. 10.sup.-4 14.9 14A11 1.2 .times. 10.sup.+5
1.2 .times. 10.sup.-3 10.3 1.1 .times. 10.sup.+5 1.2 .times.
10.sup.-3 11.6 14G1 5.4 .times. 10.sup.+4 3.9 .times. 10.sup.-4 7.2
3.9 .times. 10.sup.+4 4.1 .times. 10.sup.-4 10.5 13E4 2.3 .times.
10.sup.+5 3.9 .times. 10.sup.-4 1.7 1.5 .times. 10.sup.+5 4.2
.times. 10.sup.-4 2.8 hu = human; cyno = cynomolgus monkey; *=
falls out of measurement range
TABLE-US-00012 TABLE 7 Biacore Kinetics of Anti-DLL4 Rat Hybridoma
Antibodies Binding Murine and Rat DLL4. Kinetics on Biacore muDLL4
ECD ratDLL4 ECD k.sub.a k.sub.d K.sub.D k.sub.a k.sub.d K.sub.D
Clone (M.sup.-1s.sup.-1) (s.sup.-1) (nM) (M.sup.-1s.sup.-1)
(s.sup.-1) (nM) 38H12 N/B N/B N/B N/B N/B N/B 1A11 1.3 .times.
10.sup.+5 2.9 .times. 10.sup.-3 23 N/B N/B N/B 37D10 = 1.0 .times.
10.sup.+5 1.35 .times. 10.sup.-2 135 N/D N/D N/D 40B10 32C7 4.8
.times. 10.sup.+4 4.1 .times. 10.sup.-3 88 N/D N/D N/D 15D6 7.4
.times. 10.sup.+4 5.1 .times. 10.sup.-5 0.7 N/B N/B N/B 14A11 7.0
.times. 10.sup.-4 9.0 .times. 10.sup.-5 1.3 N/B N/B N/B 14G1 N/B
N/B N/B N/B N/B N/B 13E4 1.8 .times. 10.sup.+5 1.9 .times.
10.sup.-4 1.1 N/B N/B N/B N/B = no significant binding; N/D = not
determined, mu = mouse
TABLE-US-00013 TABLE 8 In Vitro Characterization of Rat Hybridoma
Derived Anti-DLL4 Antibodies. Functional Blockade Assays Notch
Direct activation Binding Assays Inhibition Binding (IC.sub.50,
ELISA FACS Competition Competition nM) (EC.sub.50, (EC.sub.50,
ELISA FACS huDLL4 nM) nM) (IC.sub.50, nM) (IC.sub.50, nM) cell/
DLL4 DLL4 DLL4 ECD/ huNotch-1/ Notch ECD Cells huNotch-1 DLL4 cells
reporter Rat mAb hu mu hu mu hu mu hu mu cells 1A11 0.06 0.11 34.8
0.8 0.78 0.79 8.4 0.8 2.9 38H12 0.06 -- 1.2 -- 3.63 -- 1.7 -- 0.5
37D10 = 0.12 0.14 2.3 0.7 5.39 3.36 5.5 3.8 -- 40B10 32C7 0.08 0.15
3.1 0.8 -- -- -- -- Agonist 14G1 N/D N/D 8.6 >50.9 N/D N/D 4.2
-- 6.1 14A11 N/D N/D >50.9 2.1 -- 19.8 205 1.6 2.7 15D6 N/D N/D
15 1.6 -- -- 25 1.5 3.2 13E4 N/D N/D 26 1.3 -- -- 5.2 >200 --
"--" = no activity or under detection limit of the assays used;
"N/D" = not determined; hu = human; cyno = cynomolgus monkey; mu =
mouse.
Example 4. Deduction of Variable Region Protein Sequences of
Anti-DLL4 Rat Monoclonal Antibodies by DNA Cloning and
Sequencing
[0985] Total RNA was extracted from hybridoma cell pellets using
RNeasy mini kit (Qiagen, catalog #74104) using the following
protocol. 600 .mu.l of buffer RLT were added to disrupt cells by
pipetting up and down several times. The cell lysate was
homogenized by passing it 10 times through a 20-gauge needle fitted
to an RNase-free syringe. One volume of 70% ethanol was added to
the homogenized lysate and mixed well by pipetting. Up to 700 .mu.l
at a time of the sample were added to an RNeasy spin column and
spun for 15 seconds at 10,000 rpm, discarding flow through. 700
.mu.l of buffer RW1 were added to the column and spun for 15
seconds at 10,000 rpm, discarding flow through. 500 .mu.l of buffer
RPE were added to wash the column membrane and spun for 15 seconds
at 10,000 rpm, discarding flow through. The same step was repeated
one more time, but the column was centrifuged for 2 minutes. Sample
was then centrifuged for 1 minute at 10,000 rpm to eliminate any
carryover of buffer RPE. RNA was eluted with 30 .mu.l of RNase-free
water by centrifuging for 1 minute at 10,000 rpm. Subsequently, 2
.mu.g of total RNA were used to synthesize first-strand cDNA using
SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen,
catalog #11904-018) according to following protocol: 2 .mu.g of
RNA+2 .mu.l dNTP+2 .mu.l Oligo (dT)+DEPC-H.sub.2O (to 20 .mu.l)
were incubated at 65.degree. C. for 5 minutes, then transferred to
ice for at least 1 minute. The sample was then added to the
following mixture: 4 .mu.l of 10.times.RT buffer+8 .mu.l 25 mM
MgCl.sub.2+4 .mu.l 0.1 M DTT+2 .mu.l RNase OUT and incubated at
42.degree. C. for 2 minutes. Then, 2 .mu.l of SuperScript II RT
were added to the sample and incubated at 42.degree. C. for 50
minutes. Sample was then incubated at 70.degree. C. for 15 minutes
and chilled on ice. 2 .mu.l of RNase H were then added and the
sample was incubated at 37.degree. C. for 20 minutes. cDNA was then
used as template for PCR amplification of variable regions of
antibodies. PCR was performed using first-strand cDNA, primers from
Mouse Ig-Primer Set (Novagen, catalog #69831-3) and Platinum Super
Mix High Fidelity (Invitrogen, catalog #12532-016). To amplify
heavy chain variable regions, PCR samples were assembled as
follows: 22.5 .mu.l PCR Super Mix+0.25 .mu.l reverse primer MuIgG
V.sub.H3'-2+1 .mu.l cDNA+1.25 .mu.l of one the forward primers
(VH-A, VH-B) or 0.5 .mu.l of one of the forward primers (VH-C,
VH-D, VH-E, VH-F). To amplify light chain variable regions PCR
samples were assembled as follows: 22.5 .mu.l PCR Super Mix+0.25
.mu.l reverse primer MuIgKV.sub.L-3'-1+1 .mu.l cDNA+1.25 .mu.l of
one the forward primers (VL-A, VL-B) or 0.5 .mu.l of one of the
forward primers (VL-C, VL-D, VL-E, VL-F, VL-G).
[0986] For samples with primers VH-A, VH-B, VL-A and VL-B, the
following PCR cycles were used (40-45 cycles, steps 2 through
4):
1--Denature 94.degree. C. 2 min.
2--Denature 94.degree. C. 30 sec.
3--Anneal 50.degree. C. 30 sec.
4--Extend 68.degree. C. 1 min.
[0987] 5--Final extension 68.degree. C. 5 min. 6--Cool 4.degree. C.
forever
[0988] For samples with primers VH-C through VH-F, and VL-C through
VL-G, the following PCR cycles were used (40-45 cycles, steps 2
through 4):
1--Denature 94.degree. C. 2 min.
2--Denature 94.degree. .degree. C. 30 sec.
3--Anneal 60.degree. C. 30 sec.
4--Extend 68.degree. C. 1 min.
[0989] 5--Final extension 68.degree. C. 5 min. 6--Cool 4.degree. C.
forever
[0990] PCR products were run on 1.2% agarose gel, and bands
migrating at the expected size (400-500 bp) were excised for DNA
extraction. DNA was purified using QIAquick Gel Extraction Kit
(Qiagen, catalog #28704) according to the following protocol: gel
slices were weighed. 3 volumes of buffer QG to 1 volume of gel were
added to each gel slice. Samples were incubated at 50.degree. C.
for 10 minutes until gel slices were completely dissolved, mixing
every 2-3 minutes. One gel volume of isopropanol was then added to
each sample and mixed. Samples were then applied to QIAquick column
and centrifuged for 1 minute at 13000 rpm. To wash, 750 .mu.l of
buffer PE were added to samples and spun for 1 minute at 13000 rpm.
Columns were then centrifuged for an additional minute at 13,000
rpm to completely remove residual ethanol. DNA was eluted by adding
30 .mu.l of H.sub.2O to each column and by spinning 1 minute at
13,000 rpm. Purified PCR products were then sequenced to identify
variable region sequences (Table 9, below).
TABLE-US-00014 TABLE 9 VH and VL Amino Acid Sequences of Rat
Anti-DLL4 Monoclonal Antibodies. SEQ ID Sequence NO: Protein region
123456789012345678901234567890 157 VH 38H12 EVQLVESGGGLVQPGRSLKLSCA
ASGFTFS NYGMYWIRQAPTKGLQWVAFIS HGGGITYY RDSVKGRFTISRDNAKSTLYLQM
DSLRSED TATYHCAALNWELGIDYWGQGV MVTVSS VH 38H12 Residues 31-35 NYGMY
CDR-H1 of SEQ ID NO: 157 VH 38H12 Residues 50-66 FISHGGGITYYRDSVKG
CDR-H2 of SEQ ID NO: 157 VH 38H12 Residues 99-107 LNWELGIDY CDR-H3
of SEQ ID NO: 157 158 VL 38H12 DIQMTQSPASLSASLGETISIECRA SEDIY
SNLAWYQKKSGKSPQLLIYAANR LQDGVPS RFSGSGSGTQYSLKISGMQPEDEG DYFCLQ
GSKFPLTFGSGTKLEIKR VL 38H12 Residues 24-34 RASEDIYSNLA CDR-L1 of
SEQ ID NO: 158 VL 38H12 Residues 50-56 AANRLQD CDR-L2 of SEQ ID NO:
158 VL 38H12 Residues 89-97 LQGSKFPLT CDR-L3 of SEQ ID NO: 158 159
VH 1A11 EVQLVESGGGLVQPGRSMKLSCA ASGFTFR NFPMAWVRQAPTRGLEWVATISS
SDGTTYY RDSVKGRFTISRDNAKSTLYLQV NSLRSED TATYYCSRGYYNSPFAYWGQGT
LVTVSS VH 1A11 Residues 31-35 NFPMA CDR-H1 of SEQ ID NO: 159 VH
1A11 Residues 50-66 TISSSDGTTYYRDSVKG CDR-H2 of SEQ ID NO: 159 VH
1A11 Residues 99-107 GYYNSPFAY CDR-H3 of SEQ ID NO: 159 160 VL 1A11
DIQMTQSPASLSASLGETVTIECR ASEDIY SNLAWYQQKPGNSPQLLIFDTNN LADGVPS
RFSGSGSGTQSSLKINSLQSEDVA SYFCQQ YNNYPPTFGGGTKLELKR VL 1A11 Residues
24-34 RASEDIYSNLA CDR-L1 of SEQ ID NO: 160 VL 1A11 Residues 50-56
DTNNLAD CDR-L2 of SEQ ID NO: 160 VL 1A11 Residues 89-97 QQYNNYPPT
CDR-L3 of SEQ ID NO: 160 161 VH 37D10 AVQLVESGGGLVQPKESLKISCA
ASGFTFS NAAMYWVRLAPGKGLEWVARIR TKPNNYAT YYAESVKGRFTISRDDSKSMVYV
QMDNLKT EDTAMYYCTAAPWRDSYAHVY WGQGVMVTV SS VH 37D10 Residues 31-35
NAAMY CDR-H1 of SEQ ID NO: 161 VH 37D10 Residues 50-68
RIRTKPNNYATYYAESVKG CDR-H2 of SEQ ID NO: 161 VH 37D10 Residues
101-111 APWRDSYAHVY CDR-H3 of SEQ ID NO: 161 162 VL 37D10
DIQMTQSPPVLSASVGDRVTLSCK ASQNIH KNLDWYQQKHGDAPKLLIYYTD HLQTGVPS
RFSGSGSATDYTLTISSLQPEDVA TYYCYQ YNGGPFTFGSGTKLEIKR VL 37D10
Residues 24-34 KASQNIHKNLD CDR-L1 of SEQ ID NO: 162 VL 37D10
Residues 50-56 YTDHLQT CDR-L2 of SEQ ID NO: 162 VL 37D10 Residues
89-97 YQYNGGPFT CDR-L3 of SEQ ID NO: 162 163 VH 32C7
EVQLVESGGGLVQPGRSLKLSCL ASGFPFS SVWMTWIRQAPGKGLEWIATITN SGASTYY
SASVKGRFTISRDNVKSTLYLQM TSLGSED TATYYCTRVGTSFDYWGQGVMV TVSS VH 32C7
Residues 31-35 SVWMT CDR-H1 of SEQ ID NO: 163 VH 32C7 Residues
50-66 TITNSGASTYYSASVKG CDR-H2 of SEQ ID NO: 163 VH 32C7 Residues
99-105 VGTSFDY CDR-H3 of SEQ ID NO: 163 164 VL 32C7
DIQMTQSPASLSASLGETVTIECR ASDDIY NGLAWFQQKPGKSPQLLIYDANT LHTGVPS
RFSGSGSGTQFSLKINSLQSEDVA SYFCQQ FYDYPPYTFGAGTKLELKR VL 32C7
Residues 24-34 RASDDIYNGLA CDR-L1 of SEQ ID NO: 164 VL 32C7
Residues 50-56 DANTLHT CDR-L2 of SEQ ID NO: 164 VL 32C7 Residues
89-98 QQFYDYPPYT CDR-L3 of SEQ ID NO: 164 165 VH 14G1
EVQLQQSGAELAKPGSSVKISCK ASGYTFT NYDISWIKQTNGQGLEYLGYINT GSGGIYS
NEKFKGKATLTVDKSSNTAFMQL SSLTPED TAVYYCVREGNNFDHWGQGVK VTVSS VH 14G1
Residues 31-35 NYDIS CDR-H1 of SEQ ID NO: 165 VH 14G1 Residues
50-66 YINTGSGGIYSNEKFKG CDR-H2 of SEQ ID NO: 165 VH 14G1 Residues
99-105 EGNNFDH CDR-H3 of SEQ ID NO: 165 166 VL 14G1
DTVMTQSPASMSTSVGERVTVNC KASQSVG TIVAWFQQKPGQSPKRLIYLATYR HTGVPD
RFIGSGFGRDFTLTISNVEAEDLA VYYCLQ YGSRPFTFGAGTKLEIKR VL 14G1 Residues
24-34 KASQSVGTIVA CDR-L1 of SEQ ID NO: 166 VL 14G1 Residues 50-56
LATYRHT CDR-L2 of SEQ ID NO: 166 VL 14G1 Residues 89-97 LQYGSRPFT
CDR-L3 of SEQ ID NO: 166 167 VH 14A11 EVQLQQSGPELAKPGSSVKISCKA
SGYTFT NSYISWIKQTTGQGLEYVGYINTG SGGADY NEKFKGKATLTVDKSSRTAFMQL
SSLTPGD SAVYYCAKSILLGSTCYFDYWGQ GVLVTVS S VH 14A11 Residues 31-35
NSYIS CDR-H1 of SEQ ID NO: 167 VH 14A11 Residues 50-66
YINTGSGGADYNEKFKG CDR-H2 of SEQ ID NO: 167 VH 14A11 Residues 99-110
SILLGSTCYFDY CDR-H3 of SEQ ID NO: 167 168 VL 14A11
NTVLTQSPALAVSLGQRVTISCKA SRSVSS PMYSYIYWYQQKPGQQPKLLIYR ASTLASG
VPARFSGSGSGTDFTLNIDPVEAD DIATYF CQQSWSDPFTFGSGTKLEIKR VL 14A11
Residues 23-37 KASRSVSSPMYSYIY CDR-L1 of SEQ ID NO: 168 VL 14A11
Residues 53-59 RASTLAS CDR-L2 of SEQ ID NO: 168 VL 14A11 Residues
92-100 QQSWSDPFT CDR-L3 of SEQ ID NO: 168 169 VH 15D6
EVQLQQSGPELAKPGSSVKISCKA SGYTFT SSYISWIKQTTGQGLEYIGYINTGS GGTDY
NEKFKDKATLTVDKSSRTVFMQL SSLTPGD SAVYYCAKSILLGSTYYLDYWGQ GVMVTVS S
VH 15D6 Residues 31-35 SSYIS CDR-H1 of SEQ ID NO: 169 VH 15D6
Residues 50-66 YINTGSGGTDYNEKFKD CDR-H2 of SEQ ID NO: 169 VH 15D6
Residues 99-110 SILLGSTYYLDY CDR-H3 of SEQ ID NO: 169 170 VL 15D6
DTVLTQSPALAVSLGQRVTISCKA SRSLSS PMYSYIYWYQQKLGQQPRLLIYR ASTLASG
VPARFSGSGSGTDFTLNIDPVEAD DIATYF CQQSWSDPFTFGSGTKLEIKR VL 15D6
Residues 23-37 KASRSLSSPMYSYIY CDR-L1 of SEQ ID NO: 170 VL 15D6
Residues 53-59 RASTLAS CDR-L2 of SEQ ID NO: 170
VL 15D6 Residues 92-100 QQSWSDPFT CDR-L3 of SEQ ID NO: 170
Example 5. Generation of Chimeric Antibodies
[0991] The variable domains of the heavy and light chain of the
anti-DLL4 rat mAbs (Table 9, above) were cloned in-frame to mutant
human IgG1 (L234, 235A) heavy-chain and kappa light-chain constant
regions, respectively. The activities of the resulting chimeric
antibodies were confirmed in FACS-based binding and competition
assays (Table 10, below), and were comparable to their parental rat
mAbs.
TABLE-US-00015 TABLE 10 FACS-Based Binding and Neutralizing
Activity of Recombinant Chimeric Antibodies Containing the Variable
Domains of the Anti-DLL4 Rat mAbs. FACS binding Competition FACS
(EC.sub.50 (IC.sub.50 nM) nM) huNotch-1/DLL4 DLL4 cells cells Human
Mouse Human Mouse Chimera of DLL4 DLL4 DLL4 DLL4 1A11 19.59 0.74
2.338 0.682 38H12 1.468 N/D 1.443 N/D 32C7 3.706 4.114 ND ND 37D10
2.32 0.99 5.951 4.395 14G1 0.994 N/D 4.11 N/D 14A11 1.613 2.139
4.025 1.391 15D6 1.715 1.817 10.48 1.49 N/D = not determined.
Example 6. Humanization of Anti-DLL4 Rat Monoclonal Antibody
1A11
[0992] 1A11 rat anti-DLL4 antibody (Table 9, above) was humanized.
Humanized variant amino acid sequences VH.1, VH.1a, VH.1b, VH.2a,
VL.1, VL.1a, VL.1b, and VL.2a (Table 11, below) were converted to
DNA sequence based on the most homologous human germlines and
synthesized. For the heavy chain variants, human germline heavy
chain acceptor sequences VH3-7 FR1, VH3-7 FR2, VH3-7 FR 3, and JH4
FR4 were used (see, Table 3, above). For light chain variants VL.1,
VL.1a, and VL.1b, human germline light chain acceptor sequences 02
FR1, 02 FR2, 02 FR3, and JK2 FR4 were used (see, Table 3, above).
For light chain variant VL.2a, human germline light chain acceptor
sequences L2 FR1, L2 FR2, L2 FR3, and JK2 FR4 were used (see, Table
4, above). Individual constructs were sequence verified to check
for accuracy. Positive variants were then inoculated into 250 mls
Luria broth plus ampicillin and cultured overnight at 37.degree. C.
DNA was extracted from variant cultures using the Qiagen Hi speed
maxi prep kit (12662).
TABLE-US-00016 TABLE 11 VH and VL Amino Acid Sequences of Humanized
Versions of Rat Anti-DLL4 Monoclonal Antibody 1A11. SEQ Sequence ID
NO: Protein region 123456789012345678901234567890 171 VH.1 1A11
EVQLVESGGGLVQPGGSLRLSCA ASGFTFS NFPMAWVRQAPGKGLEWVATIS SSDGTTYY
RDSVKGRFTISRDNAKNSLYLQM NSLRAED TAVYYCARGYYNSPFAYWGQG TLVTVSS VH.1
1A11 Residues NFPMA CDR-H1 31-35 of SEQ ID NO: 171 VH.1 1A11
Residues TISSSDGTTYYRDSVKG CDR-H2 50-66 of SEQ ID NO: 171 VH.1 1A11
Residues GYYNSPFAY CDR-H3 99-107 of SEQ ID NO: 171 172 VH.1a 1A11
EVQLVESGGGLVQPGGSLRLSCA ASGFTFS NFPMAWVRQAPGKGLEWVATIS SSDGTTYY
RDSVKGRFTISRDNAKSSLYLQM NSLRAED TAVYYCSRGYYNSPFAYWGQGT LVTVSS VH.1a
1A11 Residues NFPMA CDR-H1 31-35 of SEQ ID NO: 172 VH.1a 1A11
Residues TISSSDGTTYYRDSVKG CDR-H2 50-66 of SEQ ID NO: 172 VH.1a
1A11 Residues GYYNSPFAY CDR-H3 99-107 of SEQ ID NO: 172 173 VH.1b
1A11 EVQLVESGGGLVQPGGSLRLSCA ASGFTFS NFPMAWVRQAPGKGLEWVATIS
SSDGTTYY RDSVKGRFTISRDNAKNSLYLQM NSLRAED TAVYYCSRGYYNSPFAYWGQGT
LVTVSS VH.1b 1A11 Residues NFPMA CDR-H1 31-35 of SEQ ID NO: 173
VH.1b 1A11 Residues TISSSDGTTYYRDSVKG CDR-H2 50-66 of SEQ ID NO:
173 VH.1b 1A11 Residues GYYNSPFAY CDR-H3 99-107 of SEQ ID NO: 173
174 VH.2a 1A11 EVQLVESGGGLVQPGGSLRLSCA ASGFTFS
NFPMAWVRQAPGKGLEWVATIS SSDGTTYY RDSVKGRFTISRDNSKSTLYLQM NSLRAED
TAVYYCSRGYYNSPFAYWGQGT LVTVSS VH.2a 1A11 Residues NFPMA CDR-H1
31-35 of SEQ ID NO: 174 VH.2a 1A11 Residues TISSSDGTTYYRDSVKG
CDR-H2 50-66 of SEQ ID NO: 174 VH.2a 1A11 Residues GYYNSPFAY CDR-H3
99-107 of SEQ ID NO: 174 175 VL.1 1A11 DIQMTQSPSSLSASVGDRVTITCR
ASEDIY SNLAWYQQKPGKAPKLLIYDTN NLADGVPS RFSGSGSGTDFTLTISSLQPEDFA
TYYCQQ YNNYPPTFGQGTKLEIKR VL.1 1A11 Residues RASEDIYSNLA CDR-L1
24-34 of SEQ ID NO: 175 VL.1 1A11 Residues DTNNLAD CDR-L2 50-56 of
SEQ ID NO: 175 VL.1 1A11 Residues QQYNNYPPT CDR-L3 89-97 of SEQ ID
NO: 175 176 VL.1a 1A11 DIQMTQSPSSLSASVGDRVTITCR ASEDIY
SNLAWYQQKPGKSPKLLIFDTNN LADGVPS RFSGSGSGTDSTLTISSLQPEDFA TYFCQQ
YNNYPPTFGQGTKLEIKR VL.1a 1A11 Residues RASEDIYSNLA CDR-L1 24-34 of
SEQ ID NO: 176 VL.1a 1A11 Residues DTNNLAD CDR-L2 50-56 of SEQ ID
NO: 176 VL.1a 1A11 Residues QQYNNYPPT CDR-L3 89-97 of SEQ ID NO:
176 177 VL.1b 1A11 DIQMTQSPSSLSASVGDRVTITCR ASEDIY
SNLAWYQQKPGKAPKLLIFDTN NLADGVPS RFSGSGSGTDFTLTISSLQPEDFA TYYCQQ
YNNYPPTFGQGTKLEIKR VL.1b 1A11 Residues RASEDIYSNLA CDR-L1 24-34 of
SEQ ID NO: 177 VL.1b 1A11 Residues DTNNLAD CDR-L2 50-56 of SEQ ID
NO: 177 VL.1b 1A11 Residues QQYNNYPPT CDR-L3 89-97 of SEQ ID NO:
177 178 VL.2A 1A11 EIVMTQSPATLSVSPGERATLSCR ASEDIY
SNLAWYQQKPGQSPRLLIFDTNN LADGVPA RFSGSGSGTESTLTISSLQSEDFA VYFCQQ
YNNYPPTFGQGTKLEIKR VL.2a 1A11 Residues RASEDIYSNLA CDR-L1 24-34 of
SEQ ID NO: 178 VL.2a 1A11 Residues DTNNLAD CDR-L2 50-56 of SEQ ID
NO: 178 VL.2a 1A11 Residues QQYNNYPPT CDR-L3 89-97 of SEQ ID NO:
178
[0993] Humanized antibodies were generated by combining each heavy
chain variant with each light chain variant for a total of 16
variants (Table 12, below). Variants 1-4 each contained VH.1 paired
with each VL variant: VL.1: 0 back mutations, VL.1a: 4 back
mutations, VL.1b: 1 back mutation, and VL.2a: 5 back mutations.
Variants 5-8 each contained VH.1a paired with each VL variant:
VL.1: 2 back mutations, VL.1a: 6 back mutations, VL.1b: 3 back
mutations, and VL.2a: 7 back mutations. Variants 9-12 each
contained VH.1b paired with each VL variant: VL.1: 1 back mutation,
VL.1a: 5 back mutations, VL.1b: 2 back mutations, and VL.2a: 6 back
mutations. Variants 13-16 each contained VH.2a paired with each VL
variant: VL.1: 3 back mutations, VL.1a: 7 back mutations, VL.1b: 4
back mutations, VL.2a: 8 back mutations.
TABLE-US-00017 TABLE 12 Summary of Humanized 1A11 Antibodies
Generated and Back Mutations. Back Mutations in Variable
Regions.sup..dagger. Name VH/VL Combination (VH/VL) h1A11.1
VH.1NL.1 0/0 h1A11.2 VH.1NL.1a 0/A43S, Y49F, F71S, Y87F h1A11.3
VH.1NL.1b 0/Y49F h1A11.4 VH.1NL.2a 0/A435, Y49F, I58V, F71S, Y87F
h1A11.5 VH.1a/VL.1 N765, A93S/0 h1A11.6 VH.1a/VL.1a N76S,
A93S/A43S, Y49F, F71S, Y87F h1A11.7 VH.1a/VL.1b N76S, A93S/Y49F
h1A11.8 VH.1a/VL.2a N76S, A93S/A43S, Y49F, I58V, F71S, Y87F h1A11.9
VH.1bNL.1 A93S/0 h1A11.10 VH.1bNL.1a A93S/A43S, Y49F, F71S, Y87F
h1A11.11 VH.1bNL.1b A93S/Y49F h1A11.12 VH.1bNL.2a A93S/A43S, Y49F,
I58V, F71S, Y87F h1A11.13 VH.2a/VL.1 S49A, N76S, A93S/0 h1A11.14
VH.2a/VL.1a S49A, N76S, A93S,/A43S, Y49F, F71S, Y87F h1A11.15
VH.2a/VL.1b S49A, N76S, A93S/ Y49F h1A11.16 VH.2a/VL.2a S49A, N76S,
A93S/A43S, Y49F, I58V, F71S, Y87F .sup..dagger.Kabat numbering
used.
[0994] All 16 variants were transiently transfected into 50 mls of
HEK 293 6e suspension cell cultures in a ratio of 60% to 40% light
to heavy chain construct. 1 mg/ml PEI was used to transfect the
cells. Cell supernatants were harvested after six days in shaking
flasks, spun down to pellet cells, and filtered through 0.22 .mu.m
filters to separate IgG from culture contaminates. Variant binding
to human DLL4 was initially assessed through a capture binding
ELISA (Example 1.2), which utilized a goat anti human-Fc capture
antibody (Jackson ImmunoResearch, 109-005-008) to capture IgG
within filtered 293 6e cell supernatants (Table 13, below). All 16
variants had very comparable affinities and were purified for
further characterization.
[0995] All 16 variants (h1A11.1-h1A11.16) were batch purified by
adding 1 supernatant volume of protein A IgG binding buffer (Thermo
Scientific 21001) and 1 ml of rProteinA sepharose fast flow beads
(GE Healthcare, 17-1279-04). Supernatants, with beads and buffer
added, were rocked overnight at 4.degree. C., and the day after
beads were collected by gravity over poly prep chromatography
columns (Bio Rad, 731-1550). Once supernatants had passed through
the columns the beads were washed with 10 column volumes of binding
buffer, and IgG was eluted with Immunopure IgG elution buffer
(Pierce, 185 1520) and collected in 1 ml aliquots. Fractions
containing IgG were pooled and dialyzed in PBS overnight at
4.degree. C.
[0996] Purified variants were further characterized for their
affinities for human, murine and cynomolgus DLL4 by binding ELISA
(Example 1.2, Method 2), by Biacore (Example 1.1), and by Flow
Cytometry (FACS). All 16 variants showed comparable affinities to
the parent recombinant antibody 1A11 in all the three assays (Table
13). The humanized variants were then tested for their
functionality with human and murine DLL4 by competition ELISA
(Example 1.3) and by Notch reporter assay (Example 1.6). All 16
variants showed comparable potencies to the parent recombinant
antibody 1A11 in both assays (Table 14, below).
TABLE-US-00018 TABLE 13 Summary of Binding Activities of Humanized
1A11 mAbs. Binding Data Cyno Human DLL4 Mouse DLL4 DLL4 Binding
Binding Binding Binding Binding ELISA FACS ELISA FACS ELISA (EC50,
Biacore (EC50, (EC50, Biacore (EC50, (EC50, mAb nM) (KD, M) nM) nM)
(KD, M) nM) nM) h1A11-1 0.08 1.5 .times. 10.sup.-8 3.13 0.09 3.8
.times. 10.sup.-8 0.21 0.13 h1A11-2 0.08 1.0 .times. 10.sup.-8 2.85
0.09 1.9 .times. 10.sup.-8* 0.21 0.13 h1A11-3 0.07 1.5 .times.
10.sup.-8 3.51 0.08 3.7 .times. 10.sup.-8 0.24 0.16 h1A11-4 0.08
1.6 .times. 10.sup.-8 3.61 0.09 3.9 .times. 10.sup.-8 0.22 0.17
h1A11-5 0.07 0.96 .times. 10.sup.-8 3.61 0.08 2.2 .times. 10.sup.-8
0.21 0.16 h1A11-6 0.08 1.13 .times. 10.sup.-8 3.74 0.10 3.1 .times.
10.sup.-8 0.15 0.13 h1A11-7 0.06 1.3 .times. 10.sup.-8 3.71 0.09
3.5 .times. 10.sup.-8 0.18 0.14 h1A11-8 0.06 1.1 .times. 10.sup.-8
3.34 0.09 2.4 .times. 10.sup.-8 0.18 0.17 h1A11-9 0.07 1.3 .times.
10.sup.-8 3.28 0.09 3.4 .times. 10.sup.-8 0.18 0.15 h1A11-10 0.06
1.3 .times. 10.sup.-8 nt 0.09 3.5 .times. 10.sup.-8 nt 0.15
h1A11-11 0.07 1.2 .times. 10.sup.-8 nt 0.09 2.7 .times. 10.sup.-8
nt 0.15 h1A11-12 0.06 1.4 .times. 10.sup.-8 nt 0.08 3.5 .times.
10.sup.-8 nt 0.16 h1A11-13 0.06 1.4 .times. 10.sup.-8 nt 0.09 3.7
.times. 10.sup.-8 nt 0.16 h1A11-14 0.055 1.3 .times. 10.sup.-8 nt
0.07 2.8 .times. 10.sup.-8 nt 0.14 h1A11-15 0.07 1.4 .times.
10.sup.-8 nt 0.09 3.7 .times. 10.sup.-8 nt 0.13 h1A11-16 0.06 1.5
.times. 10.sup.-8 nt 0.09 3.9 .times. 10.sup.-8 nt 0.12 1A11 0.2
0.9 .times. 10.sup.-8 6.29 0.3 2.4 .times. 10.sup.-8 0.53 0.33
(chimeric) 1A11 (rat 0.6 .times. 10.sup.-8 mAb) nt = not tested
TABLE-US-00019 TABLE 14 Summary of In Vitro Functional Potency of
Humanized 1A11 mAbs. Functional Data Human DLL4 Mouse DLL4
Competition Reporter Competition ELISA Assay ELISA mAb (IC50, nM)
(EC50, nM) (IC50, nM) h1A11-1 0.4 2.53 1.3 h1A11-2 0.3 3.92 0.9
h1A11-3 0.2 2.53 0.9 h1A11-4 0.3 3.28 0.9 h1A11-5 0.3 3.8 0.8
h1A11-6 0.4 1.45 0.8 h1A11-7 0.5 4.84 0.9 h1A11-8 0.35 4.24 0.9
h1A11-9 0.3 3.18 0.9 h1A11-10 0.35 5.88 0.9 h1A11-11 0.4 3.73 0.8
h1A11-12 0.4 2.89 0.9 h1A11-13 0.3 10.42 1 h1A11-14 0.25 4.1 0.7
h1A11-15 0.2 5.4 0.7 h1A11-16 0.3 2.61 0.7 1A11 (chimera) 1.8 5.98
3.5 1A11 (rat 0.5 1.1 hybridoma mAb)
Additional Designs for Humanizing Anti-DLL4 1A11 Antibodies.
[0997] Additional VH and VL designs for humanizing anti-DLL4 rat
monoclonal antibody 1A11 are shown in the table below.
TABLE-US-00020 TABLE 15 Additional VH and VL Designs for Humanizing
1A11 Antibodies. VH or VL Acceptor Framework Design Sequence Back
Mutations.sup..dagger. h1A11VH.2 VH3 CONSENSUS + JH4 0 h1A11VH.2b
VH3 CONSENSUS + JH4 S49A, A93S h1A11VH.3 VH1-46 + JH4 0 (with Q1E
to prevent N- terminal pyroglutamate formation) h1A11VH.3b VH1-46 +
JH4 Y27F, M48V, G49A, (Q1E) A93S h1A11VH.3c VH1-46 + JH4 Y27F,
M48V, G49A, (Q1E) V67F, M69I, T73N, V78L, A93S, and T75I (to avoid
undesirable N- glycosylation signal) h1A11VH.3d VH1-46 + JH4 Y27F,
M48V, G49A, (Q1E) V67F, M69I, V78L, A93S (T73N omitted to avoid
undesirable N- glycosylation signal) h1A11VL.1c O2 + JK2 Y49F, F71S
h1A11VL.2 3-15/L2 + JK2 0 h1A11VL.2b 3-15/L2 + JK2 Y49F h1A11VL.2c
3-15/L2 + JK2 Y49F, F71S .sup..dagger.Kabat numbering used.
Example 7. Humanization of Anti-DLL4 Rat mAb 38H12
[0998] The 38H12 rat anti-DLL4 antibody (Table 9, above) was
humanized. Humanized variant amino acid sequences VH.1, VH.1a,
VH.1b, VH.2a, VL.1, VL.1a, VL.1b, and VL.2a (Table 16, below) were
converted to DNA sequence, based on the most homologous human
germlines, and synthesized. Human germline heavy chain acceptor
sequences VH3-30 FR1, VH3-30 FR2, VH3-30 FR3, and JH3 FR4 (see,
Table 3) were used for generating the humanized heavy chain
variants shown in Table 16. Human germline light chain acceptor
sequences O2 FR1, O2 FR2, O2 FR3, and JK2 FR4 (see, Table 4) were
used for generating the humanized light chain variants shown in
Table 16. Individual constructs were sequence verified to check for
accuracy. Positive variants were then inoculated into 150 mls Luria
broth plus ampicillin and cultured overnight at 37.degree. C. DNA
was extracted from variant cultures using the Qiagen Hi speed maxi
prep kit (12662).
TABLE-US-00021 TABLE 16 VH and VL Amino Acid Sequences of Humanized
Rat Anti-DLL4 Monoclonal Antibody 38H12. SEQ ID Sequence NO:
Protein region 123456789012345678901234567890 179 VH.1 38H12
EVQLVESGGGVVQPGRSLRLSCA ASGFTFS NYGMYWVRQAPGKGLEWVAFI SHGGGITYY
RDSVKGRFTISRDNSKNTLYLQM NSLRAED TAVYYCARLNWELGIDYWGQG TMVTVSS VH.1
38H12 Residues 31-35 of NYGMY CDR-H1 SEQ ID NO: 179 VH.1 38H12
Residues 50-66 of FISHGGGITYYRDSVKG CDR-H2 SEQ ID NO: 179 VH.1
38H12 Residues 99-107 LNWELGIDY CDR-H3 of SEQ ID NO: 179 180 VH.1a
38H12 EVQLVESGGGVVQPGRSLRLSCA ASGFTFS NYGMYWIRQAPGKGLEWVAFIS
HGGGITYY RDSVKGRFTISRDNSKSTLYLQM NSLRAED TAVYHCAALNWELGIDYWGQG
TMVTVSS VH.1a 38H12 Residues 31-35 of NYGMY CDR-H1 SEQ ID NO: 180
VH.1a 38H12 Residues 50-66 of FISHGGGITYYRDSVKG CDR-H2 SEQ ID NO:
180 VH.1a 38H12 Residues 99-107 LNWELGIDY CDR-H3 of SEQ ID NO: 180
181 VH.1b 38H12 EVQLVESGGGVVQPGRSLRLSCA ASGFTFS
NYGMYWVRQAPGKGLEWVAFI SHGGGITYY RDSVKGRFTISRDNSKNTLYLQM NSLRAED
TAVYYCAALNWELGIDYWGQG TMVTVSS VH.1b 38H12 Residues 31-35 of NYGMY
CDR-H1 SEQ ID NO: 181 VH.1b 38H12 Residues 50-66 of
FISHGGGITYYRDSVKG CDR-H2 SEQ ID NO: 181 VH.1b 38H12 Residues 99-107
LNWELGIDY CDR-H3 of SEQ ID NO: 181 182 VH.2a 38H12
EVQLVESGGGLVQPGGSLRLSCA ASGFTFS NYGMYWIRQAPGKGLEWVAFIS HGGGITYY
RDSVKGRFTISRDNSKSTLYLQM NSLRAED TAVYHCAALNWELGIDYWGQG TMVTVSS VH.2a
38H12 Residues 31-35 of NYGMY CDR-H1 SEQ ID NO: 182 VH.2a 38H12
Residues 50-66 of FISHGGGITYYRDSVKG CDR-H2 SEQ ID NO: 182 VH.2a
38H12 Residues 99-107 LNWELGIDY CDR-H3 of SEQ ID NO: 182 183 VL.1
38H12 DIQMTQSPSSLSASVGDRVTITCR ASEDIY SNLAWYQQKPGKAPKLLIYAAN
RLQDGVPS RFSGSGSGTDFTLTISSLQPEDFA TYYCLQ GSKFPLTFGQGTKLEIKR VL.1
38H12 Residues 24-34 of RASEDIYSNLA CDR-L1 SEQ ID NO: 183 VL.1
38H12 Residues 50-56 of AANRLQD CDR-L2 SEQ ID NO: 183 VL.1 38H12
Residues 89-97 of LQGSKFPLT CDR-L3 SEQ ID NO: 183 184 VL.1a 38H12
DIQMTQSPSSLSASVGDRVTITCR ASEDIY SNLAWYQKKPGKSPKLLIYAAN RLQDGVPS
RFSGSGSGTDYTLTISSLQPEDFA TYFCLQ GSKFPLTFGQGTKLEIKR VL.1a 38H12
Residues 24-34 of RASEDIYSNLA CDR-L1 SEQ ID NO: 184 VL.1a 38H12
Residues 50-56 of AANRLQD CDR-L2 SEQ ID NO: 184 VL.1a 38H12
Residues 89-97 of LQGSKFPLT CDR-L3 SEQ ID NO: 184 185 VL.1b 38H12
DIQMTQSPSSLSASVGDRVTITCR ASEDIY SNLAWYQQKPGKAPKLLIYAAN RLQDGVPS
RFSGSGSGTDYTLTISSLQPEDFA TYYCLQ GSKFPLTFGQGTKLEIKR VL.1b 38h12
Residues 24-34 of RASEDIYSNLA CDR-L1 SEQ ID NO: 185 VL.1b 38H12
Residues 50-56 of AANRLQD CDR-L2 SEQ ID NO: 185 VL.1b 38H12
Residues 89-97 of LQGSKFPLT CDR-L3 SEQ ID NO: 185 186 VL.2a 38H12
EIVMTQSPATLSVSPGERATLSCR ASEDIY SNLAWYQKKPGQSPRLLIYAAN RLQDGVPA
RFSGSGSGTEYTLTISSLQSEDFA VYFCLQ GSKFPLTFGQGTKLEIKR VL.2a 38H12
Residues 24-34 of RASEDIYSNLA CDR-L1 SEQ ID NO: 186 VL.2a 38H12
Residues 50-56 of AANRLQD CDR-L2 SEQ ID NO: 186 VL.2a 38H12
Residues 89-97 of LQGSKFPLT CDR-L3 SEQ ID NO: 186
[0999] Humanized antibodies were generated by combining each heavy
chain variant with each light chain variant for a total of 16
variants (Table 17, below). Variants 1-4 each contained VH.1 paired
with each VL variant: VL.1: 0 back mutations, VL.1a: 4 back
mutations, VL.1b: 1 back mutation, and VL.2a: 5 back mutations.
Variants 5-8 each contained VH.1a paired with each VL variant:
VL.1: 4 back mutations, VL.1a: 8 back mutations, VL.1b: 5 back
mutations, and VL.2a: 9 back mutations. Variants 9-12 each
contained VH.1b paired with each VL variant: VL.1: 1 back mutation,
VL.1a: 5 back mutations, VL.1b: 2 back mutations, and VL.2a: 5 back
mutations. Variants 13-16 each contained VH.2a paired with each VL
variant: VL.1: 5 back mutations, VL.1a: 9 back mutations, VL.1b: 6
back mutations, VL.2a:10 back mutations.
TABLE-US-00022 TABLE 17 Summary of Humanized 38H12 Antibodies
Generated and Back Mutations. Back Mutations in Variable
Regions.sup..dagger. Name VH/VL Combination (VH/VL) h38H12.1
VH.1/VL.1 0/0 (Q1E in VH to prevent N-terminal pyroglutamate
formation) h38H12.2 VH.1NL.1a 0/Q38K, A43S, F71Y, Y87F (Q1E in VH
as noted above) h38H12.3 VH.1NL.1b 0/F71Y (Q1E in VH as noted
above) h38H12.4 VH.1NL.2a 0/Q38K, A43S, I58V, F71Y, Y87F (Q1E in VH
as noted above) h38H12.5 VH.1a/VL.1 V37I, N76S, Y91H, R94A/0
h38H12.6 VH.1a/VL.1a V37I, N76S, Y91H, R94A/Q38K, A43S, F71Y, Y87F
h38H12.7 VH.1a/VL.1b V37I, N76S, Y91H, R94A/F71Y h38H12.8
VH.1a/VL.2a V37I, N76S, Y91H, R94A/Q38K, A43S, I58V, F71Y, Y87F
h38H12.9 VH.1bNL.1 R94A/0 h38H12.10 VH.1bNL.1a R94A/Q38K, A43S,
F71Y, Y87F h38H12.11 VH.1bNL.1b R94A/F71Y h38H12.12 VH.1bNL.2a
R94A/Q38K, A43S, I58V, F71Y, Y87F h38H12.13 VH.2a/VL.1 V37I, S49A,
N76S, Y91H, R94A/0 h38H12.14 VH.2a/VL.1a V37I, S49A, N76S, Y91H,
R94A/Q38K, A43S, F71Y, Y87F h38H12.15 VH.2a/VL.1b V37I, S49A, N76S,
Y91H, R94A/F71Y h38H12.16 VH.2a/VL.2a V37I, S49A, N76S, Y91H,
R94A/Q38K, A43S, I58V, F71Y, Y87F .sup..dagger.Kabat numbering
used.
[1000] All 16 variants were transiently transfected into 50 mls of
HEK 293 6e suspension cell cultures in a ratio of 60% to 40% light
to heavy chain construct. 1 mg/ml PEI was used to transfect the
cells. Cell supernatants were harvested after seven days in shaking
flasks, spun down to pellet cells, and filtered through 0.22 .mu.m
filters to separate IgG from culture contaminates. Variant binding
to human DLL4 was initially assessed through a capture binding
ELISA (Example 1.2), which utilized a goat anti human-Fc capture
antibody (Jackson immuno research, 109-005-008) to capture IgG
within filtered 293 6e cell supernatants (see, ELISA binding
EC.sub.50, Table 18, below). Variants containing VH.1 exhibited the
lowest binding affinities as compared to the other variants and
were considered out of the screening. VH.1 is CDR-grafted with no
framework back mutations.
[1001] Good binders (h38H12.5-h38H12.16) were then batch purified
by adding 1 supernatant volume of protein A IgG binding buffer
(Thermo Scientific 21001) and 800 .mu.l of rProteinA sepharose fast
flow beads (GE Healthcare, 17-1279-04). Supernatants, with beads
and buffer added, were stirred at room temperature for 4 hours, and
beads were collected by gravity over poly prep chromatography
columns (Bio Rad, 731-1550). Once supernatants had passed through
the columns the beads were washed with 10 mls binding buffer and
IgG was eluted with Immunopure IgG elution buffer (Pierce, 185
1520) and collected in 1 ml aliquots neutralized with 100 .mu.l 1M
Tris, pH 8.
[1002] Purified variants were further characterized in human
Notch-1 competition ELISAs (Example 1.4), which used a format of
plating Notch-1 Fc onto ELISA plates and pre-incubating
biotinylated human DLL4 plus titrated antibody. Signal was assessed
by free biotinylated DLL4 binding to Notch-1 Fc. Strong binders
inhibited signal at low antibody concentration. h38H12.5 through
h38H12.7 exhibited lower competition potencies as compared to the
other variants (see, Notch Competition ELISA EC.sub.50, Table 18,
below).
[1003] The good binder variants, as determined by binding ELISA,
were assessed by Biacore (Example 1.1) concurrently with cell based
assay screens. The K.sub.D for human DLL4 was similar for all
variants (see, Biacore, K.sub.D in Table 18, below). Variants were
screened in cell-based assays that examined direct binding to human
DLL4 (Example 1.3; FACS binding EC.sub.50 in Table 18, below) and
inhibition of Notch-1 signaling (Example 1.6; Notch Reporter Assay
EC.sub.50 in Table 18, below).
TABLE-US-00023 TABLE 18 Summary of In Vitro Activities Against
Human DLL4 of Humanized 38H12 mAbs. ELISA Notch FACS Notch binding
Competition binding Reporter EC.sub.50 ELISA EC.sub.50 Assay
EC.sub.50 Biacore, Name (nM) EC.sub.50 (nM) (nM) (nM) K.sub.D (nM)
h38H12.1 21.5 17.84 h38H12.2 26.88 h38H12.3 5.57 h38H12.4 20.65
h38H12.5 0.2015 14.81 77.15 7.307 0.401 h38H12.6 0.1584 26.96 12.29
6.317 0.434 h38H12.7 0.1798 12.49 19.06 2.598 0.34 h38H12.8 0.1972
8.315 20.02 5.557 0.397 h38H12.9 0.1155 4.158 3.71 1.436 0.986
h38H12.10 0.1226 3.902 2.489 0.7861 0.578 h38H12.11 0.1264 3.8
2.477 0.6572 0.554 h38H12.12 0.1651 3.228 1.478 1.062 0.819
h38H12.13 0.1534 5.287 2.556 0.7943 0.507 h38H12.14 0.146 5.839
1.04 1.014 0.303 h38H12.15 0.0904 5.714 2.369 0.837 0.355 h38H12.16
0.1696 3.766 2.914 1.185 0.392
Additional Designs for Humanizing Anti-DLL4 38H12 Antibodies.
[1004] Additional VH and VL designs for humanizing anti-DLL4 rat
monoclonal antibody 38H12 are shown in the table below.
TABLE-US-00024 TABLE 19 Additional VH and VL Designs for Humanizing
38H12 Antibodies. Germline Acceptor VH or VL Design Framework
Sequences Back Mutations.sup..dagger. h38H12VH.2 VH3 CONSENSUS +
JH3 0 h38H12VH.2b VH3 CONSENSUS + JH3 S49A, R94A h38H12VH.3 VH1-46
+ JH3 0 (Q1E in VH to prevent N-terminal pyroglutamate formation)
h38H12VH.3b VH1-46 + JH3 Y27F, M48V, G49A, R94A (Q1E in VH as noted
above) h38H12VH.3c VH1-46 + JH3 Y27F, Y37I, M48V, G49A, V67F, M69I,
T73N, V78L, Y91H, R94A (T75I to eliminate undesirable
N-glycosylation signal, Q1E in VH as noted above) h38H12VH.3d
VH1-46 + JH3 Y27F, Y37I, M48V, G49A, V67F, M69I, V78L, Y91H, R94A
(Q1E in VH as noted above) h38H12VL.2 3-15/L2 + JK2 0 h38H12VL.2b
3-15/L2 + JK2 F71Y .sup..dagger.Kabat numbering used.
Example 8. Affinity Maturation of h1A11.1
[1005] Humanized antibody h1A11.1 was used as a template for
affinity maturation. A description of the design of the library is
provided below. Numbering of variable region sequences of
monoclonal antibodies was annotated with Kabat numbering (as
described above; or see worldwide website
bioinf.org.uk/abs/#kabatnum) and was used in generating the
libraries described below.
Three libraries were made as described below. H1+H2 library
(doping: 76080808):
[1006] Doped 11 residues at 30, 31, 32, 35, 50, 52, 52a, 55, 56,
57, and 58.
[1007] Toggle between germline and h1A11 sequence at position
76(V/I).
[1008] A 10.sup.9 library sampled mutants with 4 or fewer mutated
residues at least 3.7 times.
[1009] The majority of the library with mutants carrying 4 to 6
residues was mutated by doping.
H3 library (doping: 70101010)
[1010] Doped 8 residues @ 95, 96, 97, 98, 99, 100, 100a, and
102.
[1011] Toggle between germline and h1A11 sequence at position
93(A/S) and 101 (D/A).
[1012] A 10.sup.9 library sampled mutants with 4 or fewer mutated
residues at least 4.7 times.
[1013] The majority of the library with mutants carrying 4 to 5
residues was mutated by doping.
LC library (doping: 70101010):
[1014] Doped 9 residues at 28, 30, 31, 50, 53, 92, 93, 94, and
96.
[1015] Toggle between germline and h1A11 sequence at 7 positions
27(Q/E), 43(A/S), 49(Y/F), 52(S/N), 71(F/S), 87(Y/F), and
91(S/Y).
[1016] A 10.sup.9 library sampled mutants with 4 or fewer mutated
residues at least 1 time.
[1017] The majority of the library with mutants carrying 4 to 6
residues was mutated by doping. [1018] rHC library: recombine
outputs of H1+H2 and H3 libraries. [1019] rHCLC library: recombine
outputs of H1+H2, H3, and LC libraries.
[1020] Both VH and VL framework germlining reduced predicted
immunogenicity. The most desirable germlining mutation in the
h1A11VL.1a was the S43A and S71F Used codons specified here for
residues that were doped:
[1021] If a proline is to be doped, the doping oligo will have
C.sub.(5-85-5-5)C.sub.(5-85-5-5)S codon regardless of the original
codon in the antibody sequence. These codons were selected based on
the following criteria: [1022] 1. increase non-synonymous mutation
[1023] 2. increase coverage of more amino acids when mutated [1024]
3. uses high frequency codons [1025] 4. avoid SSS and WWW codons.
Doping order was A-C-G-T A.sub.(85-5-5-5), A.sub.(70-10-10-10)
C.sub.(5-85-5-5), C.sub.(10-70-10-10) G.sub.(5-5-85-5),
G.sub.(10-10-70-10) T.sub.(5-5-5-85), T.sub.(10-10-10-70)
TABLE-US-00025 [1025] Alanine (A): GCN
G.sub.(10-10-70-10)C.sub.(10-70-10-10)S
G.sub.(5-5-85-5)C.sub.(5-85-5-5)S Threonine (T): ACN
A.sub.(70-10-10-10)C.sub.(10-70-10-10)S
A.sub.(85-5-5-5)C.sub.(5-85-5-5)S Proline (P): CCN
C.sub.(10-70-10-10)C.sub.(10-70-10-10)S
C.sub.(5-85-5-5)C.sub.(5-85-5-5)S Serine (S): If TCN
T.sub.(10-10-10-70)C.sub.(10-70-10-10)S
T.sub.(5-5-5-85)C.sub.(5-85-5-5)S If AGY
A.sub.(70-10-10-10)G.sub.(10-10-70-10)C.sub.(10-70-10-10)
A.sub.(85-5-5-5)G.sub.(5-5-85-5)C.sub.(5-85-5-5) Valine (V): GTN
G.sub.(10-10-70-10)T.sub.(10-10-10-70)S
G.sub.(5-5-85-5)T.sub.(5-5-5-85)S Glycine (G): GGN
G.sub.(10-10-70-10)G.sub.(10-10-70-10)S
G.sub.(5-5-85-5)G.sub.(5-5-85-5)S Leucine (L): If CTN
C.sub.(10-70-10-10)T.sub.(10-10-10-70)S
C.sub.(5-85-5-5)T.sub.(5-5-5-85)S If TTR
T.sub.(10-10-10-70)T.sub.(10-10-10-70)G.sub.(10-10-70-10)
T.sub.(5-5-5-85)T.sub.(5-5-5-85)G.sub.(5-5-85-5) Arginine (R): If
CGN C.sub.(10-70-10-10)G.sub.(10-10-70-10)S
C.sub.(5-85-5-5)G.sub.(5-5-85-5)S If AGR
A.sub.(70-10-10-10)G.sub.(10-10-70-10)G.sub.(10-10-70-10)
A.sub.(85-5-5-5)G.sub.(5-5-85-5)G.sub.(5-5-85-5) Methionine (M):
ATG A.sub.(70-10-10-10)T.sub.(10-10-10-70)G.sub.(10-10-70-10)
A.sub.(85-5-5-5)T.sub.(5-5-5-85)G.sub.(5-5-85-5) Tryptophan (W):
TGG T.sub.(10-10-10-70)G.sub.(10-10-70-10)G.sub.(10-10-70-10)
T.sub.(5-5-5-85)G.sub.(5-5-85-5)G.sub.(5-5-85-5) Pheylalanine (F):
TTY T.sub.(10-10-10-70)T.sub.(10-10-10-70)C.sub.(10-70-10-10)
T.sub.(5-5-5-85)T.sub.(5-5-5-85)C.sub.(5-85-5-5) Isoleucine (I):
need two oligos 50% ATY
A.sub.(70-10-10-10)T.sub.(10-10-10-70)C.sub.(10-70-10-10)
A.sub.(85-5-5-5)T.sub.(5-5-5-85)C.sub.(5-85-5-5) 50% ATA
A.sub.(70-10-10-10)T.sub.(10-10-10-70)A.sub.(70-10-10-10)
A.sub.(85-5-5-5)T.sub.(5-5-5-85)A.sub.(85-5-5-5) Tyrosine (Y): TAY
T.sub.(10-10-10-70)A.sub.(70-10-10-10)C.sub.(10-70-10-10)
T.sub.(5-5-5-85)A.sub.(85-5-5-5)C.sub.(5-85-5-5) Histidine (H): CAY
C.sub.(10-70-10-10)A.sub.(70-10-10-10)C.sub.(10-70-10-10)
C.sub.(5-85-5-5)A.sub.(85-5-5-5)C.sub.(5-85-5-5) Glutamine (Q): CAR
C.sub.(10-70-10-10)A.sub.(70-10-10-10)G.sub.(10-10-70-10)
C.sub.(5-85-5-5)A.sub.(85-5-5-5)G.sub.(5-5-85-5) Asparagines (N):
AAY A.sub.(70-10-10-10)A.sub.(70-10-10-10)C.sub.(10-70-10-10)
A.sub.(85-5-5-5)A.sub.(85-5-5-5)C.sub.(5-85-5-5) Lysine (K): AAR
A.sub.(70-10-10-10)A.sub.(70-10-10-10)G.sub.(10-10-70-10)
A.sub.(85-5-5-5)A.sub.(85-5-5-5)G.sub.(5-5-85-5) Aspartic Acid (D):
GAY G.sub.(10-10-70-10)A.sub.(70-10-10-10)C.sub.(10-70-10-10)
G.sub.(5-5-85-5)A.sub.(85-5-5-5)C.sub.(5-85-5-5) Glutamic acid (E):
GAR G.sub.(10-10-70-10)A.sub.(70-10-10-10)G.sub.(10-10-70-10)
G.sub.(5-5-85-5)A.sub.(85-5-5-5)G.sub.(5-5-85-5) Cysteine (C): TGY
always NNS
[1026] The h1A11.1 libraries were transformed into yeast cells and
displayed on the cell surface to be selected against low
concentration of biotinylated DLL4 extracellular domain by magnetic
then fluorescence activated cell sorting. Selection for improved
on-rate or off-rate or both were carried out, and antibody protein
sequences of affinity-modulated hu1A11 clones (Tables 20 and 21,
below) were recovered from yeast cells for converting back to IgG
format for further characterization (see, summary of clones in
Table 22). Table 23 lists the amino acids observed during the
affinity maturation selection in both framework regions (FR) and
CDRs.
TABLE-US-00026 TABLE 20 VH sequences of Affinity Matured Humanized
1A11.1 Clones SEQ ID Sequence NO: Protein region
123456789012345678901234567890 187 h1A11VH.1 EVQLVESGGGLVQPGGSLRLSC
VH AASGFTFSNFPMAWVRQAPGKG LEWVATISSSDGTTYYRDSVKG
RFTISRDNAKNSLYLQMNSLRAE DTAVYYCARGYYNSPFAYWGQ GTLVTVSS h1A11VH.1
CDR- Residues NFPMA VH H1 31-35 of SEQ ID NO: 187 h1A11VH.1 CDR-
Residues TISSSDGTTYYRDSVKG VH H2 50-66 of SEQ ID NO: 187 h1A11VH.1
CDR- Residues GYYNSPFAY VH H3 99-107 of SEQ ID NO: 187 188 h1A11.A6
EVQLVESGGGLVQPGGSLRLSC VH AASGFTFRHFPMAWVRQAPGKG
LEWVATISSSDAWPSYRDSVKG RFTISRDNAKNSLYLQMNSLRAE
DTAVYYCSRGYYNSPFAYWGQ GTLVTVSS h1A11.A6 CDR- Residues HFPMA VH H1
31-35 of SEQ ID NO: 188 h1A11.A6 CDR- Residues TISSSDAWPSYRDSVKG VH
H2 50-66 of SEQ ID NO: 188 h1A11.A6 CDR- Residues GYYNSPFAY VH H3
99-107 of SEQ ID NO: 188 189 h1A11.A8 EVQLVESGGGLVQPGGSLRLSC VH
AASGFTFGNFPMSWVRQAPGKG LEWVASISSSDSWATIGDSVKGR
FTISRDNAKNSLYLQMNSLRAED TAVYYCSRGYYNSPFAYWGQG TLVTVSS h1A11.A8 CDR-
Residues NFPMS VH H1 31-35 of SEQ ID NO: 189 h1A11.A8 CDR- Residues
SISSSDSWATIGDSVKG VH H2 50-66 of SEQ ID NO: 189 h1A11.A8 CDR-
Residues GYYNSPFAY VH H3 99-107 of SEQ ID NO: 189 190 h1A11.C6
EVQLVESGGGLVQPGGSLRLSC VH AASGFTFRNFPMAWVRQAPGKG
LEWVATISSSDGWPTYRDSVKG RFTISRDNAKSSLYLQMNSLRAE
DTAVYYCSRGYYNSPFAYWGQ GTLVTVSS h1A11.C6 CDR- Residues NFPMA VH H1
31-35 of SEQ ID NO: 190 h1A11.C6 CDR- Residues TISSSDGWPTYRDSVKG VH
H2 50-66 of SEQ ID NO: 190 h1A11.C6 CDR- Residues GYYNSPFAY VH H3
99-107 of SEQ ID NO: 190 191 h1A11.A11 EVQLVESGGGLVQPGGSLRLSC VH
AASGFTFRHFPMAWVRQAPGKG LEWVATISSSDDWPNYRDSVKG
RFTISRDNAKSSLYLQMNSLRAE DTAVYYCSRGYYNSPFAYWGQ GTLVTVSS h1A11.A11
CDR- Residues HFPMA VH H1 31-35 of SEQ ID NO: 191 h1A11.A11 CDR-
Residues TISSSDDWPNYRDSVKG VH H2 50-66 of SEQ ID NO: 191 h1A11.A11
CDR- Residues GYYNSPFAY VH H3 99-107 of SEQ ID NO: 191 192 h1A11.B5
EVQLVESGGGLVQPGGSLRLSC VH AASGFTFRYFPMSWVRQAPGKG
LEWVASISGSDGWASYRDSVKG RFTISRDNAKNSLYLQMNSLRAE
DTAVYYCARGYYNSPFASWGQ GTLVTVSS h1A11.B5 CDR- Residues YFPMS VH H1
31-35 of SEQ ID NO: 192 h1A11.B5 CDR- Residues SISGSDGWASYRDSVKG VH
H2 50-66 of SEQ ID NO: 192 h1A11.B5 CDR- Residues GYYNSPFAS VH H3
99-107 of SEQ ID NO: 192 193 h1A11.E12 EVQLVESGGGLVQPGGSLRLSC VH
AASGFTFRYFPMAWVRQAPGKG LEWVATISGSDEWPNYRDSVKG
RFTISRDNAKNSLYLQMNSLRAE DTAVYYCARGYYNSPFAFWGQ GTLVTVSS H1A11.E12
CDR- Residues YFPMA VH H1 31-35 of SEQ ID NO: 193 h1A11.E12 CDR-
Residues TISGSDEWPNYRDSVKG VH H2 50-66 of SEQ ID NO: 193 h1A11.E12
CDR- Residues GYYNSPFAF VH H3 99-107 of SEQ ID NO: 193 194 h1A11.G3
EVQLVESGGGLVQPGGSLRLSC VH AASGFTFRYFPMSWVRQAPGKG
LEWVASISGSDGWASYRDSVKG RFTISRDNAKNSLYLQMNSLRAE
DTAVYYCARGYYNSPFAYWGQ GTLVTVSS h1A11.G3 CDR- Residues YFPMS VH H1
31-35 of SEQ ID NO: 194 h1A11.G3 CDR- Residues SISGSDGWASYRDSVKG VH
H2 50-66 of SEQ ID NO: 194 h1A11.G3 CDR- Residues GYYNSPFAY VH H3
99-107 of SEQ ID NO: 194 195 h1A11.F5 EVQLVESGGGLVQPGGSLRLSC VH
AASGFTFRHFPMAWVRQAPGKG LEWVATISSSDAWPSYRDSVKG
RFTISRDNAKNSLYLQMNSLRAE DTAVYYCARGYYNSPFAYWGQ GTLVTVSS h1A11.F5
CDR- Residues HFPMA VH H1 31-35 of SEQ ID NO: 195 h1A11.F5 CDR-
Residues TISSSDAWPSYRDSVKG VH H2 50-66 of SEQ ID NO: 195 h1A11.F5
CDR- Residues GYYNSPFAY VH H3 99-107 of SEQ ID NO: 195 196 h1A11.H2
EVQLVESGGGLVQPGGSLRLSC VH AASGFTFGNFPMSWVRQAPGKG
LEWVASISSSDSWATIGDSVKGR FTISRDNAKNSLYLQMNSLRAED
TAVYYCARGYYNSPFAFWGQG TLVTVSS h1A11.H2 CDR- Residues NFPMS VH H1
31-35 of SEQ ID NO: 196 h1A11.H2 CDR- Residues SISSSDSWATIGDSVKG VH
H2 50-66 of SEQ ID NO: 196 h1A11.H2 CDR- Residues GYYNSPFAF VH H3
99-107 of SEQ ID NO: 196
TABLE-US-00027 TABLE 21 VL Sequences of Affinity Matured Humanized
1A11.1 Clones. SEQ ID Sequence NO: Protein region
123456789012345678901234567890 197 h1A11VL.1
DIQMTQSPSSLSASVGDRVTITC VL RASEDIYSNLAWYQQKPGKAPK
LLIYDTNNLADGVPSRFSGSGSG TDFTLTISSLQPEDFATYYCQQY NNYPPTFGQGTKLEIK
h1A11VL.1 CDR- Residues RASEDIYSNLA VL L1 24-34 of SEQ ID NO: 197
h1A11VL.1 CDR- Residues DTNNLAD VL L2 50-56 of SEQ ID NO: 197
h1A11VL.1 CDR- Residues QQYNNYPPT VL L3 89-97 of SEQ ID NO: 197 198
h1A11.A2 DIQMTQSPSSLSASVGDRVTITC VL RASQDIYINLAWYQQKPGKSPK
LLIFDTNDLADGVPSRFSGSGSG TDFTLTISSLQPEDFATYYCQQY DYVPPTFGQGTKLEIK
h1A11.A2 CDR- Residues RASQDIYINLA VL L1 24-34 of SEQ ID NO: 198
h1A11.A2 CDR- Residues DTNDLAD VL L2 50-56 of SEQ ID NO: 198
h1A11.A2 CDR- Residues QQYDYVPPT VL L3 89-97 of SEQ ID NO: 198 199
h1A11.A2 DIQMTQSPSSLSASVGDRVTITC VL RASQDIYYNLAWYQQKPGKSPK
LLIFDTSSLADGVPSRFSGSGSGT DFTLTISSLQPEDFATYFCQQYD WYPPTFGQGTKLEIK
h1A11.A12 CDR- Residues RASQDIYYNLA VL L1 24-34 of SEQ ID NO: 199
h1A11.A12 CDR- Residues DTSSLAD VL L2 50-56 of SEQ ID NO: 199
h1A11.A12 CDR- Residues QQYDWYPPT VL L3 89-97 of SEQ ID NO: 199 200
h1A11.A7 DIQMTQSPSSLSASVGDRVTITC VL RASQDIYINLAWYQQKPGKAPK
LLIFDTSDLADGVPSRFSGSGSGT DFTLTISSLQPEDFATYYCQQYD YYPPTFGQGTKLEIK
h1A11.A7 CDR- Residues RASQDIYINLA VL L1 24-34 of SEQ ID NO: 200
h1A11.A7 CDR- Residues DTSDLAD VL L2 50-56 of SEQ ID NO: 200
h1A11.A7 CDR- Residues QQYDYYPPT VL L3 89-97 of SEQ ID NO: 200 201
h1A11.B4 DIQMTQSPSSLSASVGDRVTITC VL RASQDIYYNLAWYQQKPGKAPK
LLIFDTNILADGVPSRFSGSGSGT DFTLTISSLQPEDFATYFCQQYD YVPPTFGQGTKLEIK
h1A11.B4 CDR- Residues RASQDIYYNLA VL L1 24-34 of SEQ ID NO: 201
h1A11.B4 CDR- Residues DTNILAD VL L2 50-56 of SEQ ID NO: 201
h1A11.B4 CDR- Residues QQYDYVPPT VL L3 89-97 of SEQ ID NO: 201 202
h1A11.B5 DIQMTQSPSSLSASVGDRVTITC VL RASQDIWNNLAWYQQKPGKSP
KLLIFDTSYLADGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQ YDWYPPTFGQGTKLEIK
h1A11.B5 CDR- Residues RASQDIWNNLA VL L1 24-34 of SEQ ID NO: 202
h1A11.B5 CDR- Residues DTSYLAD VL L2 50-56 of SEQ ID NO: 202
h1A11.B5 CDR- Residues QQYDWYPPT VL L3 89-97 of SEQ ID NO: 202 203
h1A11.E12 DIQMTQSPSSLSASVGDRVTITC VL RASQEIYRNLAWYQQKPGKSPK
LLIFDTSVLADGVPSRFSGSGSGT DSTLTISSLQPEDFATYYCQQYT YYPPTFGQGTKLEIK
h1A11.E12 CDR- Residues RASQEIYRNLA VL L1 24-34 of SEQ ID NO: 203
h1A11.E12 CDR- Residues DTSVLAD VL L2 50-56 of SEQ ID NO: 203
h1A11.E12 CDR- Residues QQYTYYPPT VL L3 89-97 of SEQ ID NO: 203
TABLE-US-00028 TABLE 22 Summary of Affinity Matured h1A11.1
Converted Clones. Clone name VH VL h1A11.A6 h1A11.A6 VH h1A11VL.1
h1A11.C6 h1A11.C6 VH h1A11VL.1 h1A11.A11 h1A11.A11 VH h1A11VL.1
h1A11.A8 h1A11.A8 VH h1A11VL.1 h1A11.B4 h1A11VH.1 h1A11.B4 VL
h1A11.A7 h1A11VH.1 h1A11.A7 VL h1A11.A12 h1A11VH.1 h1A11.A12 VL
h1A11.A2 h1A11VH.1 h1A11.A2 VL h1A11.B5 h1A11.B5 VH h1A11.B5 VL
h1A11.E12 h1A11.E12 VH h1A11.E12 VL h1A11.G3 h1A11.G3 VH h1A11.E12
VL h1A11.F5 h1A11.F5 VH h1A11.E12 VL h1A11.H2 h1A11.H2 VH h1A11.E12
VL
TABLE-US-00029 TABLE 23 Amino acids observed during affinity
maturation selections of h1A11.1 in framework regions (FRs) and
each of the CDRs for VH (SEQ ID NO: 204) and VL (SEQ ID NO: 205)
regions. SEQ ID NO: 204 VH
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNFPMAWVRQAPGKGLEWVATISSSDGTTYYRDSVNG
RYY S S G SSASIG NH T A AFDN GS EWST KA D PA T F D L Q C
RFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGYYNSPFAYWGQGTLVTVSS SL D DF S SEQ
ID NO: 205 VL
DIQMTQSPSSLSASVGDRVTITCRASEDIYSNLAWYQQKPGKAPKLLIYDTNNLAD TQE WN S F
NS T EI D DT Q NR T M V G E
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPPTEGQGTKLEIK S F SDWV P TY
YI F P
[1027] The hu1A11.1 affinity matured clones (Table 22) were
expressed, purified, and further characterized in vitro. Their
antigen binding affinities were determined by Biacore technology as
described in Example 1.1, and are shown in Tables 24 and 25
(below). Their activities of binding to cell-bound DLL4 and
inhibiting cell-bound DLL4-induced Notch activation were further
examined using methods described in Examples 1.3 and 1.6, and are
summarized in Table 26 (below).
TABLE-US-00030 TABLE 24 Biacore Kinetics of Affinity-Matured
Humanized 1A11.1 Antibodies Binding to Human and Cynomolgus Monkey
DLL4. Kinetics on Biacore Human DLL4 Cyno DLL4 k.sub.a k.sub.d
K.sub.D k.sub.a k.sub.d K.sub.D Clone (M.sup.-1s.sup.-1) (s.sup.-1)
(nM) (M.sup.-1s.sup.-1) (s.sup.-1) (nM) h1A11.A6 1.82 .times.
10.sup.+5 6.62 .times. 10.sup.-6 3.6 .times. 10.sup.-11 1.71
.times. 10.sup.+5 2.10 .times. 10.sup.-5 1.2 .times. 10.sup.-10
h1A11.C6 1.78 .times. 10.sup.+5 7.18 .times. 10.sup.-6 4.0 .times.
10.sup.-11 1.71 .times. 10.sup.+5 3.12 .times. 10.sup.-5 1.8
.times. 10.sup.-10 h1A11-G3 1.09 .times. 10.sup.+5 9.39 .times.
10.sup.-6 8.7 .times. 10.sup.-11 9.90 .times. 10.sup.+4 1.73
.times. 10.sup.-5 1.7 .times. 10.sup.-10 h1A11-F5 1.31 .times.
10.sup.+5 9.82 .times. 10.sup.-6 7.5 .times. 10.sup.-11 1.18
.times. 10.sup.+5 2.00 .times. 10.sup.-5 1.7 .times. 10.sup.-10
h1A11.A8 1.74 .times. 10.sup.+5 1.08 .times. 10.sup.-5 6.2 .times.
10.sup.-11 1.60 .times. 10.sup.+5 2.40 .times. 10.sup.-5 1.5
.times. 10.sup.-10 h1A11-A11 1.83 .times. 10.sup.+5 2.66 .times.
10.sup.-5 1.5 .times. 10.sup.-10 1.70 .times. 10.sup.+5 3.35
.times. 10.sup.-5 2.0 .times. 10.sup.-10 h1A11- 1.49 .times.
10.sup.+5 3.26 .times. 10.sup.-5 2.2 .times. 10.sup.-10 1.37
.times. 10.sup.+5 3.84 .times. 10.sup.-5 2.8 .times. 10.sup.-10 E12
h1A11-H2 1.43 .times. 10.sup.+5 3.85 .times. 10.sup.-5 2.7 .times.
10.sup.-10 1.31 .times. 10.sup.+5 4.60 .times. 10.sup.-5 3.5
.times. 10.sup.-10 h1A11-B5 1.30 .times. 10.sup.+5 1.34 .times.
10.sup.-4 1.0 .times. 10.sup.-9 1.17 .times. 10.sup.+5 1.78 .times.
10.sup.-4 1.5 .times. 10.sup.-9 h1A11-A2 1.42 .times. 10.sup.+5
4.21 .times. 10.sup.-4 3.0 .times. 10.sup.-9 1.34 .times. 10.sup.+5
5.27 .times. 10.sup.-4 3.9 .times. 10.sup.-9 h1A11-B4 1.57 .times.
10.sup.+5 8.23 .times. 10.sup.-4 5.2 .times. 10.sup.-9 1.43 .times.
10.sup.+5 9.90 .times. 10.sup.-4 6.9 .times. 10.sup.-9 h1A11-A7
1.70 .times. 10.sup.+5 9.73 .times. 10.sup.-4 5.7 .times. 10.sup.-9
1.58 .times. 10.sup.+5 1.24 .times. 10.sup.-3 7.8 .times. 10.sup.-9
h1A11-A12 1.73 .times. 10.sup.+5 1.32 .times. 10.sup.-3 7.6 .times.
10.sup.-9 1.58 .times. 10.sup.+5 1.62 .times. 10.sup.-3 1.0 .times.
10.sup.-8 h1A11-1 1.58 .times. 10.sup.+5 2.12 .times. 10.sup.-3 1.3
.times. 10.sup.-8 1.44 .times. 10.sup.+5 2.55 .times. 10.sup.-3 1.8
.times. 10.sup.-8
TABLE-US-00031 TABLE 25 Biacore Kinetics of Affinity-Matured
Humanized 1A11.1 Antibodies Binding to Mouse and Rat DLL4. Kinetics
on Biacore Mouse DLL4 Rat DLL4 k.sub.a k.sub.d K.sub.D k.sub.a
k.sub.d K.sub.D Clone (M.sup.-1s.sup.-1) (s.sup.-1) (nM)
(M.sup.-1s.sup.-1) (s.sup.-1) (nM) h1A11.A6 1.98 .times. 10.sup.+5
3.12 .times. 10.sup.-5 1.6 .times. 10.sup.-10 1.29 .times.
10.sup.+5 7.72 .times. 10.sup.-4 6.0 .times. 10.sup.-9 h1A11.C6
2.03 .times. 10.sup.+5 2.34 .times. 10.sup.-5 1.2 .times.
10.sup.-10 1.69 .times. 10.sup.+5 3.05 .times. 10.sup.-3 1.8
.times. 10.sup.-8 h1A11-G3 1.17 .times. 10.sup.+5 4.04 .times.
10.sup.-5 3.5 .times. 10.sup.-10 1.18 .times. 10.sup.+5 1.01
.times. 10.sup.-3 8.6 .times. 10.sup.-9 h1A11-F5 1.43 .times.
10.sup.+5 3.97 .times. 10.sup.-5 2.8 .times. 10.sup.-10 1.16
.times. 10.sup.+5 6.79 .times. 10.sup.-4 5.8 .times. 10.sup.-9
h1A11.A8 1.87 .times. 10.sup.+5 3.27 .times. 10.sup.-5 1.8 .times.
10.sup.-10 1.39 .times. 10.sup.+5 6.50 .times. 10.sup.-3 4.7
.times. 10.sup.-8 h1A11.A11 1.98 .times. 10.sup.+5 3.54 .times.
10.sup.-5 1.8 .times. 10.sup.-10 1.16 .times. 10.sup.+5 1.02
.times. 10.sup.-3 8.8 .times. 10.sup.-9 h1A11- 1.56 .times.
10.sup.+5 5.44 .times. 10.sup.-5 3.5 .times. 10.sup.-10 1.08
.times. 10.sup.+5 1.75 .times. 10.sup.-4 1.6 .times. 10.sup.-9 E12
h1A11-H2 1.54 .times. 10.sup.+5 5.07 .times. 10.sup.-5 3.3 .times.
10.sup.-10 1.78 .times. 10.sup.+5 2.83 .times. 10.sup.-3 1.6
.times. 10.sup.-8 h1A11-B5 1.45 .times. 10.sup.+5 1.66 .times.
10.sup.-4 1.2 .times. 10.sup.-9 9.82 .times. 10.sup.+4 3.97 .times.
10.sup.-2 4.1 .times. 10.sup.-7 h1A11.A2 1.81 .times. 10.sup.+5
9.04 .times. 10.sup.-4 5.0 .times. 10.sup.-9 NB NB NB h1A11-B4 4.79
.times. 10.sup.+5 2.51 .times. 10.sup.-3 5.2 .times. 10.sup.-9 NB
NB NB h1A11.A7 poor binding 1.3 .times. 10.sup.-8 NB NB NB
h1A11.A12 poor binding 1.6 .times. 10.sup.-8 NB NB NB h1A11.1 1.56
.times. 10.sup.+5 4.98 .times. 10.sup.-3 3.2 .times. 10.sup.-8 NB
NB NB NB = no observable binding
TABLE-US-00032 TABLE 26 In Vitro Activities Against Cell-Bound DLL4
of Affinity-Matured Humanized 1A11.1 Antibodies. Inhibition of
Notch Direct Binding Activation via DLL4 to DLL4 cells, Notch
reporter cells, FACS (nM) assay (nM) Human Mouse Human Mouse DLL4
DLL4 DLL4 DLL4 h1A11.A6 2.227 0.636 0.746 1.168 h1A11.C6 2.452
0.517 0.894 1.188 h1A11-G3 3.592 1.397 1.845 2.353 h1A11-F5 1.171
0.460 0.484 0.649 h1A11.A8 3.160 0.744 1.331 1.247 h1A11.A11 2.480
0.500 0.904 1.175 h1A11-E12 0.996 1.615 0.208 0.266 h1A11-H2 1.977
0.420 0.856 0.586 h1A11.A2 2.375 0.634 3.681 0.854 h1A11-B4 2.145
0.665 3.280 1.079 h1A11.A7 2.174 0.625 2.920 1.788 h1A11.A12 1.768
0.568 1.662 0.832
Example 9. Molecular Identity and Physicochemical Properties of Rat
Hybridoma Antibodies
[1028] The identity of monoclonal antibodies specific to DLL4 was
determined by mass spectrometry as described below.
Mass Spectrometry Analysis of h1A11.1
[1029] The light chain molecular weight of 23,501 Daltons matched
well with the theoretical value. The heavy chain molecular weights
matched well with the theoretical values. The observed molecular
weights were 50,190 Daltons; 50,352 Daltons; and 50,514 Daltons,
with the difference corresponding to 162 Daltons as the result of
different glycosylation.
Mass Spectrometry Analysis of h38H12.11
[1030] The light chain molecular weight of 23,408 Daltons matched
well with the theoretical value. The heavy chain molecular weights
matched well with the theoretical values. The observed molecular
weights were 50,368 Dalton; 50,530 Daltons; and 50,692 Daltons;
with the difference corresponding to 162 Daltons as the result of
different glycosylation.
[1031] The solubilities of the antibodies were estimated by
polyethylene glycol (PEG) 3000 precipitation. They were also
directly determined, i.e., real solubility, by concentrating the
antibodies in a specific solution and/or buffer with Amicon
centrifugal filters and then observed for any precipitation at
25.degree. C. and 5.degree. C. Stability was inferred by near
ultra-violet circular (UV-CD) and differential scanning calorimetry
(DSC). Stability to freezing and thawing and at elevated
temperatures (accelerated stability) was assessed by size exclusion
chromatography (SEC). The details of the techniques were described
in Example 1.7, and the results are described below.
Real Solubility Screening Results for 1A11
[1032] For a series of 1A11 clones, including hu1A11.1, hu1A11.3,
hu1A11.9, hu1A11.11, and 1A11 recombinant, 2 mg of each were
concentrated with Amicon centrifugal filters to above 60 mg/ml. No
precipitation or cloudiness was observed at 25.degree. C. or after
storage for 1 day at 5.degree. C. The concentrations of each were
63 mg/ml for 1A11.1, 76 mg/ml for 1A11.3, 63 mg/ml for 1A11.9, 69
mg/ml for 1A11.11, and 76 mg/ml for chimeric 1A11.
Example 10. Anti-DLL4 Antibody Epitope Grouping by Biacore
Technology
[1033] Epitope grouping was performed with the use of Biacore 2000,
3000, and T100 instruments. Antibodies of interest were directly
immobilized on the CMS chip surface via amino coupling. Flow cell
one with similarly immobilized irrelevant IgG served as a reference
surface. First, immobilized monoclonal antibodies (mAbs) were
allowed to bind recombinant antigen (at concentrations of at least
200 nM) for 120 seconds at 50 .mu.l/min. Then, another antibody was
injected at 50 .mu.l/ml for 120-240 seconds to monitor its ability
to bind to the antigen that is already bound to the immobilized
mAbs. The absence of additional binding response on the sensogram
constituted overlap in the epitopes of the two mAbs (the one
immobilized on the chip and the one introduced in liquid phase).
Orientation of the assay was then switched in such a way that the
antibody that was in a liquid phase was immobilized and vice versa.
The pairs of mAbs that did not allow for additional antibody
binding in both orientations of the assay were grouped as truly
overlapping in these experiments. The resulting grouping of mAbs
with overlapping epitopes is shown in Table 27, below.
TABLE-US-00033 TABLE 27 Anti-DLL4 Antibody Epitope Grouping By
BIAcore Technology. 1st Immobilized Injection 2nd Injection
antibody huDLL4 38H12 15D6 13E4 1A11 14G1 14A11 37D10 38H12 + - + +
+ + + + 15D6 + + - - - - - 13E4 + + - - - + - 1A11 + + - - - - -
14G1 + + + + + - - - 14A11 + + - - - - - "+" indicates binding; "-"
indicates no binding
Example 11. Activities of DLL4 Antibodies in Endothelial Cell
Sprouting Assay In Vitro
[1034] Fibrin gel beads sprouting assay was carried out to examine
the in vitro angiogenesis activity of HUVEC (passage 2-3, Lonza) as
described (Nakatsu, M. N. et al. 2003 Microvasc. Res. 66, 102-112).
Briefly, fibrinogen solution was reconstituted with aprotinin (4
units/ml) and thrombin (50 units/ml). Cytodex 3 beads (Amersham
Pharmacia Biotech) were coated with 350-400 HUVECs per bead for
over night. About 20 HUVEC-coated beads were imbedded in the fibrin
clot per well of a 96-well tissue culture plate. Conditioned medium
derived from normal human fibroblasts (NHLF, Lonza) at 80%
confluence was plated on top of the gel. DLL4 antibody and control
antibody KLH at 15 .mu.g/ml were added onto the well. At day 10 and
12, images were taken with inverted microscope and Nikon CCD
camera. Table 28 summarizes the activities of some DLL4 antibodies
to enhance endothelial cell sprouting in vitro. (Nakatsu et al.,
"Angiogenic sprouting and capillary lumen formation modeled by
human umbilical vein endothelial cells (HUVEC) in fibrin gels: the
role of fibroblasts and Angiopoietin-1," Microvasc. Res. 66,
102-112 (2003)).
TABLE-US-00034 TABLE 28 Activities of DLL4 antibodies to stimulate
endothelial cell sprouting. Stimulate HUVEC Tested antibody
sprouting 38H12 rat mAb Yes 1A11 rat mAb Yes h1A11.1 Yes 40B10 rat
mAb Not observed 32C7 rat mAb Not observed
Example 12. Rodent PK Assessment of Hybridoma-Derived
Antibodies
[1035] To assess pharmacokinetics properties of anti-DLL4
antibodies, SCID-Beige mice (n=3 per antibody) were administered a
single intraperitoneal (IP) dose of antibody at either 1, 5, 10, or
30 mg/kg concentration, depending on cross-reactivity of antibody
to murine DLL4. Longitudinal serum samples (5 .mu.l of whole blood
diluted 1:50 in HBS-EP+ buffer per time point) were collected from
each animal over 21 days. Serum concentrations were determined
using a DLL4-specific Biacore platform. Briefly, human DLL4 was
immobilized to a sensorchip and samples were injected over the
flowcell at 5 .mu.l per minute for 5 minutes with the resulting
binding levels measured and compared to standards. Serum
concentration time profiles were used to estimate the
pharmacokinetic parameters of C.sub.max (peak serum concentration),
CL (clearance), and t.sub.1/2 (antibody half-life), summarized in
Table 29, below.
TABLE-US-00035 TABLE 29 Pharmacokinetic Parameters of Anti-DLL4
Antibodies in SCID-Beige Mice Following a Single IP Dose. Dose Cmax
CL t.sub.1/2 Antibody (mg/kg) (.mu.g/mL) (mL/hr/kg) (d) 38H12 rat
mAb 5 30.2 0.3 20-29 h1A11.1 30 163 0.44 11.3 h1A11.1 10 49.9 0.50
9.9 h1A11.1 5 11.0 1.78 6.3 h1A11.1 1 3.1 2.16 4.4
Example 13. DLL4 Antibody Treatment Inhibited Tumor Growth In
Vivo
[1036] The effect of anti-DLL4 antibodies on tumor growth was
evaluated on subcutaneous Calu-6 xenograft tumors implanted in
SCID-Beige mice. Briefly, 2.times.10.sup.6 cells were inoculated
subcutaneously into the right hind flank of female SCID-Beige mice.
Tumors were allowed to establish for 14-18 days, at which point
tumor volume was determined using electronic caliper measurements.
Tumor size was calculated using the formula: L.times.W.sup.2/2.
Mice were allocated into treatment groups (n=10 per group) so that
each cohort of animals had equivalent mean tumor volume prior to
initiation of therapy (typically between 180 and 250 mm.sup.3).
Animals were then dosed intraperitoneally either twice a week for
two weeks (total of 4 doses) or weekly for four weeks (total of 4
doses) with anti-DLL4 antibodies. Tumor volume was measured on
average twice a week for the duration of the experiment until the
mean tumor volume in each group reached an endpoint of
.gtoreq.2,000 mm.sup.3. Results are shown in Table 30, below.
TABLE-US-00036 TABLE 30 Efficacy of Anti-DLL4 Antibodies in the
Calu-6 Human Non-Small Cell Lung Cancer Subcutaneous Xenograft
Model. Dose, Route, Treatment Regimen % T/C.sup.a % ILS.sup.b 1A11
rat mAb 30 mg/kg, IP, 37** 89** 2X/weekX2 1A11 rat mAb 10 mg/kg,
IP, 47** 39** 2X/weekX2 1A11 rat mAb 5 mg/kg, IP, 43** 57**
2X/weekX2 14A11 rat mAb 10 mg/kg, IP, 37** 57** 2X/weekX2 40B10 rat
mAb 30 mg/kg, IP, 29** 89** 2X/weekX2 32C7 rat mAb 30 mg/kg, IP,
65* 28* 2X/weekX2 14A11 chimera 10 mg/kg, IP, 32** 114** q7dX4 15D6
chimera 10 mg/kg, IP, 47** 57** q7dX4 40B10 chimera 10 mg/kg, IP,
43** 73** q7dX4 32C7 chimera 10 mg/kg, 71* 18* IP, q7dX4 h1A11.1 10
mg/kg, IP, 34** 75** q7dX4 h1A11.1 5 mg/kg, IP, q7dX4 31** 80**
h1A11.1 1 mg/kg, IP, q7dX4 43** 36** h1A11.1 0.5 mg/kg, IP, 62**
25* q7dX4 .sup.a% T/C = mean tumor volume of treatment group/tumor
volume of treatment control group x 100. P values (as indicated by
asterisks) were derived from Student's T test comparison of
treatment group versus treatment control group. Based on day
25/26/27 measurements. .sup.b% ILS = (T - C)/C x 100, where T =
median time to endpoint of treatment group and C = median time to
endpoint of treatment control group. P values (as indicated by
asterisks) were derived from Kaplan Meier log-rank comparison of
treatment group versus treatment control group. Based on an
endpoint of 2000 mm.sup.3. *p < 0.05; **p < 0.001
INCORPORATION BY REFERENCE
[1037] The contents of all cited references (including literature
references, patents, patent applications, and websites) that maybe
cited throughout this application are hereby expressly incorporated
by reference in their entirety for any purpose, as are the
references cited therein.
EQUIVALENTS
[1038] The invention may be embodied in other specific forms
without departing from the spirit or essential characteristics
thereof. The foregoing embodiments are therefore to be considered
in all respects illustrative rather than limiting of the invention
described herein. Scope of the invention is thus indicated by the
appended claims rather than by the foregoing description, and all
changes that come within the meaning and range of equivalency of
the claims are therefore intended to be embraced herein.
Sequence CWU 1
1
2061685PRTHomo sapiens 1Met Ala Ala Ala Ser Arg Ser Ala Ser Gly Trp
Ala Leu Leu Leu Leu1 5 10 15Val Ala Leu Trp Gln Gln Arg Ala Ala Gly
Ser Gly Val Phe Gln Leu 20 25 30Gln Leu Gln Glu Phe Ile Asn Glu Arg
Gly Val Leu Ala Ser Gly Arg 35 40 45Pro Cys Glu Pro Gly Cys Arg Thr
Phe Phe Arg Val Cys Leu Lys His 50 55 60Phe Gln Ala Val Val Ser Pro
Gly Pro Cys Thr Phe Gly Thr Val Ser65 70 75 80Thr Pro Val Leu Gly
Thr Asn Ser Phe Ala Val Arg Asp Asp Ser Ser 85 90 95Gly Gly Gly Arg
Asn Pro Leu Gln Leu Pro Phe Asn Phe Thr Trp Pro 100 105 110Gly Thr
Phe Ser Leu Ile Ile Glu Ala Trp His Ala Pro Gly Asp Asp 115 120
125Leu Arg Pro Glu Ala Leu Pro Pro Asp Ala Leu Ile Ser Lys Ile Ala
130 135 140Ile Gln Gly Ser Leu Ala Val Gly Gln Asn Trp Leu Leu Asp
Glu Gln145 150 155 160Thr Ser Thr Leu Thr Arg Leu Arg Tyr Ser Tyr
Arg Val Ile Cys Ser 165 170 175Asp Asn Tyr Tyr Gly Asp Asn Cys Ser
Arg Leu Cys Lys Lys Arg Asn 180 185 190Asp His Phe Gly His Tyr Val
Cys Gln Pro Asp Gly Asn Leu Ser Cys 195 200 205Leu Pro Gly Trp Thr
Gly Glu Tyr Cys Gln Gln Pro Ile Cys Leu Ser 210 215 220Gly Cys His
Glu Gln Asn Gly Tyr Cys Ser Lys Pro Ala Glu Cys Leu225 230 235
240Cys Arg Pro Gly Trp Gln Gly Arg Leu Cys Asn Glu Cys Ile Pro His
245 250 255Asn Gly Cys Arg His Gly Thr Cys Ser Thr Pro Trp Gln Cys
Thr Cys 260 265 270Asp Glu Gly Trp Gly Gly Leu Phe Cys Asp Gln Asp
Leu Asn Tyr Cys 275 280 285Thr His His Ser Pro Cys Lys Asn Gly Ala
Thr Cys Ser Asn Ser Gly 290 295 300Gln Arg Ser Tyr Thr Cys Thr Cys
Arg Pro Gly Tyr Thr Gly Val Asp305 310 315 320Cys Glu Leu Glu Leu
Ser Glu Cys Asp Ser Asn Pro Cys Arg Asn Gly 325 330 335Gly Ser Cys
Lys Asp Gln Glu Asp Gly Tyr His Cys Leu Cys Pro Pro 340 345 350Gly
Tyr Tyr Gly Leu His Cys Glu His Ser Thr Leu Ser Cys Ala Asp 355 360
365Ser Pro Cys Phe Asn Gly Gly Ser Cys Arg Glu Arg Asn Gln Gly Ala
370 375 380Asn Tyr Ala Cys Glu Cys Pro Pro Asn Phe Thr Gly Ser Asn
Cys Glu385 390 395 400Lys Lys Val Asp Arg Cys Thr Ser Asn Pro Cys
Ala Asn Gly Gly Gln 405 410 415Cys Leu Asn Arg Gly Pro Ser Arg Met
Cys Arg Cys Arg Pro Gly Phe 420 425 430Thr Gly Thr Tyr Cys Glu Leu
His Val Ser Asp Cys Ala Arg Asn Pro 435 440 445Cys Ala His Gly Gly
Thr Cys His Asp Leu Glu Asn Gly Leu Met Cys 450 455 460Thr Cys Pro
Ala Gly Phe Ser Gly Arg Arg Cys Glu Val Arg Thr Ser465 470 475
480Ile Asp Ala Cys Ala Ser Ser Pro Cys Phe Asn Arg Ala Thr Cys Tyr
485 490 495Thr Asp Leu Ser Thr Asp Thr Phe Val Cys Asn Cys Pro Tyr
Gly Phe 500 505 510Val Gly Ser Arg Cys Glu Phe Pro Val Gly Leu Pro
Pro Ser Phe Pro 515 520 525Trp Val Ala Val Ser Leu Gly Val Gly Leu
Ala Val Leu Leu Val Leu 530 535 540Leu Gly Met Val Ala Val Ala Val
Arg Gln Leu Arg Leu Arg Arg Pro545 550 555 560Asp Asp Gly Ser Arg
Glu Ala Met Asn Asn Leu Ser Asp Phe Gln Lys 565 570 575Asp Asn Leu
Ile Pro Ala Ala Gln Leu Lys Asn Thr Asn Gln Lys Lys 580 585 590Glu
Leu Glu Val Asp Cys Gly Leu Asp Lys Ser Asn Cys Gly Lys Gln 595 600
605Gln Asn His Thr Leu Asp Tyr Asn Leu Ala Pro Gly Pro Leu Gly Arg
610 615 620Gly Thr Met Pro Gly Lys Phe Pro His Ser Asp Lys Ser Leu
Gly Glu625 630 635 640Lys Ala Pro Leu Arg Leu His Ser Glu Lys Pro
Glu Cys Arg Ile Ser 645 650 655Ala Ile Cys Ser Pro Arg Asp Ser Met
Tyr Gln Ser Val Cys Leu Ile 660 665 670Ser Glu Glu Arg Asn Glu Cys
Val Ile Ala Thr Glu Val 675 680 68522058DNAHomo sapiens 2atggcggcag
cgtcccggag cgcctctggc tgggcgctac tgctgctggt ggcactttgg 60cagcagcgcg
cggccggctc cggcgtcttc cagctgcagc tgcaggagtt catcaacgag
120cgcggcgtac tggccagtgg gcggccttgc gagcccggct gccggacttt
cttccgcgtc 180tgccttaagc acttccaggc ggtcgtctcg cccggaccct
gcaccttcgg gaccgtctcc 240acgccggtat tgggcaccaa ctccttcgct
gtccgggacg acagtagcgg cggggggcgc 300aaccctctcc aactgccctt
caatttcacc tggccgggta ccttctcgct catcatcgaa 360gcttggcacg
cgccaggaga cgacctgcgg ccagaggcct tgccaccaga tgcactcatc
420agcaagatcg ccatccaggg ctccctagct gtgggtcaga actggttatt
ggatgagcaa 480accagcaccc tcacaaggct gcgctactct taccgggtca
tctgcagtga caactactat 540ggagacaact gctcccgcct gtgcaagaag
cgcaatgacc acttcggcca ctatgtgtgc 600cagccagatg gcaacttgtc
ctgcctgccc ggttggactg gggaatattg ccaacagcct 660atctgtcttt
cgggctgtca tgaacagaat ggctactgca gcaagccagc agagtgcctc
720tgccgcccag gctggcaggg ccggctgtgt aacgaatgca tcccccacaa
tggctgtcgc 780cacggcacct gcagcactcc ctggcaatgt acttgtgatg
agggctgggg aggcctgttt 840tgtgaccaag atctcaacta ctgcacccac
cactccccat gcaagaatgg ggcaacgtgc 900tccaacagtg ggcagcgaag
ctacacctgc acctgtcgcc caggctacac tggtgtggac 960tgtgagctgg
agctcagcga gtgtgacagc aacccctgtc gcaatggagg cagctgtaag
1020gaccaggagg atggctacca ctgcctgtgt cctccgggct actatggcct
gcattgtgaa 1080cacagcacct tgagctgcgc cgactccccc tgcttcaatg
ggggctcctg ccgggagcgc 1140aaccaggggg ccaactatgc ttgtgaatgt
ccccccaact tcaccggctc caactgcgag 1200aagaaagtgg acaggtgcac
cagcaacccc tgtgccaacg ggggacagtg cctgaaccga 1260ggtccaagcc
gcatgtgccg ctgccgtcct ggattcacgg gcacctactg tgaactccac
1320gtcagcgact gtgcccgtaa cccttgcgcc cacggtggca cttgccatga
cctggagaat 1380gggctcatgt gcacctgccc tgccggcttc tctggccgac
gctgtgaggt gcggacatcc 1440atcgatgcct gtgcctcgag tccctgcttc
aacagggcca cctgctacac cgacctctcc 1500acagacacct ttgtgtgcaa
ctgcccttat ggctttgtgg gcagccgctg cgagttcccc 1560gtgggcttgc
cgcccagctt cccctgggtg gccgtctcgc tgggtgtggg gctggcagtg
1620ctgctggtac tgctgggcat ggtggcagtg gctgtgcggc agctgcggct
tcgacggccg 1680gacgacggca gcagggaagc catgaacaac ttgtcggact
tccagaagga caacctgatt 1740cctgccgccc agcttaaaaa cacaaaccag
aagaaggagc tggaagtgga ctgtggcctg 1800gacaagtcca actgtggcaa
acagcaaaac cacacattgg actataatct ggccccaggg 1860cccctggggc
gggggaccat gccaggaaag tttccccaca gtgacaagag cttaggagag
1920aaggcgccac tgcggttaca cagtgaaaag ccagagtgtc ggatatcagc
gatatgctcc 1980cccagggact ccatgtacca gtctgtgtgt ttgatatcag
aggagaggaa tgaatgtgtc 2040attgccacgg aggtataa 20583330PRTHomo
sapiens 3Ala Ser Thr Lys Gly Pro Ser Val Phe Phe Leu Ala Pro Ser
Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro Lys Ser Cys
Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150
155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Glu Glu225 230 235 240Met Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265
270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys 325 3304330PRTHomo sapiens 4Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Lys Val
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105
110Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys 130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu225 230
235 240Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 325 3305106PRTHomo sapiens 5Thr Val Ala Ala
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln1 5 10 15Leu Lys Ser
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 20 25 30Pro Arg
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 35 40 45Gly
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 50 55
60Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys65
70 75 80His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
Pro 85 90 95Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100
1056105PRTHomo sapiens 6Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe
Pro Pro Ser Ser Glu1 5 10 15Glu Leu Gln Ala Asn Lys Ala Thr Leu Val
Cys Leu Ile Ser Asp Phe 20 25 30Tyr Pro Gly Ala Val Thr Val Ala Trp
Lys Ala Asp Ser Ser Pro Val 35 40 45Lys Ala Gly Val Glu Thr Thr Thr
Pro Ser Lys Gln Ser Asn Asn Lys 50 55 60Tyr Ala Ala Ser Ser Tyr Leu
Ser Leu Thr Pro Glu Gln Trp Lys Ser65 70 75 80His Arg Ser Tyr Ser
Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 85 90 95Lys Thr Val Ala
Pro Thr Glu Cys Ser 100 10574PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(3)..(3)Any amino acid
7Phe Gly Xaa Gly189PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptideMOD_RES(2)..(9)Any amino acid 8Cys Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 595PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 9Leu Glu Trp Ile Gly1
5104PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(3)..(3)Any amino acid 10Trp Gly Xaa
Gly11130PRTHomo sapiens 11Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser 20 25 301214PRTHomo sapiens 12Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val Ala1 5 101332PRTHomo sapiens
13Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln1
5 10 15Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
Arg 20 25 301411PRTHomo sapiens 14Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser1 5 101530PRTHomo sapiens 15Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser 20 25 301614PRTHomo sapiens 16Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser1 5
101732PRTHomo sapiens 17Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr Leu Gln1 5 10 15Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys Ala Arg 20 25 301811PRTHomo sapiens 18Trp Gly Gln
Gly Thr Leu Val Thr Val Ser Ser1 5 101930PRTHomo sapiens 19Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 20 25
302014PRTHomo sapiens 20Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met Gly1 5 102132PRTHomo sapiens 21Arg Val Thr Met Thr Arg Asp
Thr Ser Thr Ser Thr Val Tyr Met Glu1 5 10 15Leu Ser Ser Leu Arg Ser
Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 302211PRTHomo sapiens
22Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser1 5 102330PRTHomo
sapiens 23Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro
Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser 20 25 302414PRTHomo sapiens 24Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val Ala1 5 102532PRTHomo sapiens 25Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln1 5 10 15Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25
302611PRTHomo sapiens 26Trp Gly Gln Gly Thr Met Val Thr Val Ser
Ser1 5 102730PRTHomo sapiens 27Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser 20 25 302814PRTHomo sapiens 28Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser1 5 102932PRTHomo
sapiens 29Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
Leu Gln1 5 10 15Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys Ala Arg
20 25 303011PRTHomo sapiens 30Trp Gly Gln Gly Thr Met Val Thr Val
Ser Ser1 5 103130PRTHomo sapiens 31Glu Val Thr Leu Arg Glu Ser Gly
Pro Ala Leu Val Lys Pro Thr Gln1 5 10 15Thr Leu Thr Leu Thr Cys Thr
Phe Ser Gly Phe Ser Leu Ser 20 25 303214PRTHomo sapiens 32Trp Ile
Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Ala1 5 103332PRTHomo
sapiens 33Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Val
Leu Thr1 5 10 15Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr
Cys Ala Arg 20 25 303411PRTHomo sapiens 34Trp Gly Gln Gly Thr Thr
Val Thr Val Ser Ser1 5 103530PRTHomo sapiens 35Glu Val Thr Leu Lys
Glu Ser Gly Pro Val Leu Val Lys Pro Thr Glu1 5 10 15Thr Leu Thr Leu
Thr Cys Thr Val Ser Gly Phe Ser Leu Ser 20 25 303614PRTHomo sapiens
36Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Ala1 5
103732PRTHomo sapiens 37Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Ser
Gln Val Val Leu Thr1 5 10 15Met Thr Asn Met Asp Pro Val Asp Thr Ala
Thr Tyr Tyr Cys Ala Arg 20 25 303811PRTHomo sapiens 38Trp Gly Gln
Gly Thr Thr Val Thr Val Ser Ser1 5 103930PRTHomo sapiens 39Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25
304014PRTHomo sapiens 40Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val Gly1 5 104132PRTHomo sapiens 41Arg Phe Thr Ile Ser Arg Asp
Asp Ser Lys Asn Ser Leu Tyr Leu Gln1 5 10 15Met Asn Ser Leu Lys Thr
Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 304211PRTHomo sapiens
42Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser1 5 104330PRTHomo
sapiens 43Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser 20 25 304414PRTHomo sapiens 44Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val Ser1 5 104532PRTHomo sapiens 45Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln1 5 10 15Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25
304611PRTHomo sapiens 46Trp Gly Gln Gly Thr Thr Val Thr Val Ser
Ser1 5 104730PRTHomo sapiens 47Glu Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Gly Thr Phe Ser 20 25 304814PRTHomo sapiens 48Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly1 5 104932PRTHomo
sapiens 49Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
Met Glu1 5 10 15Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
Cys Ala Arg 20 25 305011PRTHomo sapiens 50Trp Gly Gln Gly Thr Thr
Val Thr Val Ser Ser1 5 105130PRTHomo sapiens 51Glu Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 20 25 305214PRTHomo sapiens
52Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly1 5
105332PRTHomo sapiens 53Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser
Thr Ala Tyr Met Glu1 5 10 15Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala
Val Tyr Tyr Cys Ala Arg 20 25 305411PRTHomo sapiens 54Trp Gly Gln
Gly Thr Thr Val Thr Val Ser Ser1 5 105530PRTHomo sapiens 55Glu Val
Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr
Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser 20 25
305614PRTHomo sapiens 56Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu
Trp Ile Gly1 5 105732PRTHomo sapiens 57Arg Val Thr Ile Ser Val Asp
Thr Ser Lys Asn Gln Phe Ser Leu Lys1 5 10 15Leu Ser Ser Val Thr Ala
Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 305811PRTHomo sapiens
58Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser1 5 105930PRTHomo
sapiens 59Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Gly Ser Ile
Ser 20 25 306014PRTHomo sapiens 60Trp Ile Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Ile Gly1 5 106132PRTHomo sapiens 61Arg Val Thr Ile
Ser Val Asp Thr Ser Lys Asn Ser Phe Tyr Leu Gln1 5 10 15Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25
306211PRTHomo sapiens 62Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser1 5 106330PRTHomo sapiens 63Glu Val Gln Leu Gln Glu Ser Gly Pro
Gly Leu Val Lys Pro Gly Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val
Ser Gly Gly Ser Ile Ser 20 25 306414PRTHomo sapiens 64Trp Ile Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly1 5 106532PRTHomo
sapiens 65Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Tyr
Leu Lys1 5 10 15Leu Ser Ser Val Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys Ala Arg 20 25 306611PRTHomo sapiens 66Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser1 5 106730PRTHomo sapiens 67Glu Val Gln Leu Val
Gln Ser Gly Thr Glu Val Lys Lys Pro Gly Glu1 5 10 15Ser Leu Lys Ile
Ser Cys Lys Val Ser Gly Gly Ser Ile Ser 20 25 306814PRTHomo sapiens
68Trp Ile Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Ile Gly1 5
106932PRTHomo sapiens 69Gln Val Thr Ile Ser Val Asp Thr Ser Phe Asn
Thr Phe Phe Leu Gln1 5 10 15Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala
Met Tyr Tyr Cys Ala Arg 20 25 307011PRTHomo sapiens 70Trp Gly Gln
Gly Thr Met Val Thr Val Ser Ser1 5 107130PRTHomo sapiens 71Glu Val
Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln1 5 10 15Thr
Leu Thr Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser 20 25
307214PRTHomo sapiens 72Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu
Trp Ile Gly1 5 107332PRTHomo sapiens 73Arg Val Thr Ile Ser Val Asp
Thr Ser Lys Asn Gln Phe Val Leu Thr1 5 10 15Met Thr Asn Met Asp Pro
Val Asp Thr Ala Thr Tyr Tyr Cys Ala Arg 20 25 307411PRTHomo sapiens
74Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser1 5 107530PRTHomo
sapiens 75Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Lys Ser
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Arg 20 25 307614PRTHomo sapiens 76Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val Ala1 5 107732PRTHomo sapiens 77Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln1 5 10 15Leu Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys 20 25
307811PRTHomo sapiens 78Trp Gly Gln Gly Thr Met Val Thr Val Ser
Ser1 5 107930PRTHomo sapiens 79Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Val Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Gly 20 25 308014PRTHomo sapiens 80Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala1 5 108132PRTHomo
sapiens 81Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
Leu Gln1 5 10 15Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys Ala Lys 20 25 308211PRTHomo sapiens 82Trp Gly Gln Gly Thr Met
Val Thr Val Ser Ser1 5 108323PRTHomo sapiens 83Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys 208415PRTHomo sapiens 84Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile Tyr1 5 10 158532PRTHomo sapiens 85Gly Val
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr1 5 10 15Leu
Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 20 25
308610PRTHomo sapiens 86Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys1 5
108723PRTHomo sapiens 87Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu
Ser Val Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys
208815PRTHomo sapiens 88Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg
Leu Leu Ile Tyr1 5 10 158932PRTHomo sapiens 89Gly Ile Pro Ala Arg
Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr1 5 10 15Leu Thr Ile Ser
Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys 20 25 309010PRTHomo
sapiens 90Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys1 5 109123PRTHomo
sapiens 91Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser
Leu Gly1 5 10 15Glu Arg Ala Thr Ile Asn Cys 209215PRTHomo sapiens
92Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr1 5 10
159332PRTHomo sapiens 93Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr1 5 10 15Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp
Val Ala Val Tyr Tyr Cys 20 25 309411PRTHomo sapiens 94Phe Gly Gly
Gly Thr Lys Val Glu Ile Lys Arg1 5 109523PRTHomo sapiens 95Glu Ile
Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly1 5 10 15Glu
Arg Ala Thr Leu Ser Cys 209615PRTHomo sapiens 96Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr1 5 10 159732PRTHomo sapiens
97Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr1
5 10 15Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr
Cys 20 25 309811PRTHomo sapiens 98Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys Arg1 5 109923PRTHomo sapiens 99Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys 2010015PRTHomo sapiens 100Trp Tyr Gln Gln Lys Pro Glu Lys Ala
Pro Lys Ser Leu Ile Tyr1 5 10 1510132PRTHomo sapiens 101Gly Val Pro
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr1 5 10 15Leu Thr
Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 20 25
3010211PRTHomo sapiens 102Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
Arg1 5 1010323PRTHomo sapiens 103Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Val Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
2010415PRTHomo sapiens 104Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile Tyr1 5 10 1510532PRTHomo sapiens 105Gly Val Pro Ser
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr1 5 10 15Leu Thr Ile
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 20 25
3010611PRTHomo sapiens 106Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
Arg1 5 1010722PRTHomo sapiens 107Ser Tyr Glu Leu Thr Gln Pro Pro
Ser Val Ser Val Ser Pro Gly Gln1 5 10 15Thr Ala Ser Ile Thr Cys
2010815PRTHomo sapiens 108Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro
Val Leu Val Ile Tyr1 5 10 1510932PRTHomo sapiens 109Gly Ile Pro Glu
Arg Phe Ser Gly Ser Asn Ser Gly Asp Thr Ala Thr1 5 10 15Leu Thr Ile
Ser Gly Thr Gln Pro Met Asp Glu Ala Asp Tyr Tyr Cys 20 25
3011010PRTHomo sapiens 110Phe Gly Tyr Gly Thr Lys Val Thr Val Leu1
5 1011122PRTHomo sapiens 111Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val
Ser Val Ser Pro Gly Gln1 5 10 15Thr Ala Ser Ile Thr Cys
2011215PRTHomo sapiens 112Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro
Val Leu Val Ile Tyr1 5 10 1511332PRTHomo sapiens 113Gly Ile Pro Glu
Arg Phe Ser Gly Ser Asn Ser Gly Asp Thr Ala Thr1 5 10 15Leu Thr Ile
Ser Gly Thr Gln Pro Met Asp Glu Ala Asp Tyr Tyr Cys 20 25
3011410PRTHomo sapiens 114Gly Gly Gly Thr Lys Leu Thr Val Leu Gly1
5 1011521PRTHomo sapiens 115Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser
Val Ser Pro Gly Gln Thr1 5 10 15Ala Ser Ile Thr Cys 2011615PRTHomo
sapiens 116Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile
Tyr1 5 10 1511732PRTHomo sapiens 117Gly Ile Pro Glu Arg Phe Ser Gly
Ser Asn Ser Gly Asp Thr Ala Thr1 5 10 15Leu Thr Ile Ser Gly Thr Gln
Pro Met Asp Glu Ala Asp Tyr Tyr Cys 20 25 3011810PRTHomo sapiens
118Gly Gly Gly Thr Lys Leu Thr Val Leu Gly1 5 1011922PRTHomo
sapiens 119Leu Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro
Gly Gln1 5 10 15Thr Ala Ser Ile Thr Cys 2012015PRTHomo sapiens
120Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr1 5
10 1512132PRTHomo sapiens 121Gly Ile Pro Glu Arg Phe Ser Gly Ser
Asn Ser Gly Asp Thr Ala Thr1 5 10 15Leu Thr Ile Ser Gly Thr Gln Thr
Met Asp Glu Ala Asp Tyr Leu Cys 20 25 3012211PRTHomo sapiens 122Phe
Gly Gly Gly Thr Lys Val Thr Val Leu Gly1 5 1012323PRTHomo sapiens
123Glu Tyr Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys1
5 10 15Glu Lys Val Thr Ile Thr Cys 2012415PRTHomo sapiens 124Trp
Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Val Ile Tyr1 5 10
1512532PRTHomo sapiens 125Gly Val Pro Ser Arg Phe Ser Gly Ser Asn
Ser Gly Asp Asp Ala Thr1 5 10 15Leu Thr Ile Asn Ser Leu Glu Ala Glu
Asp Ala Ala Thr Tyr Tyr Cys 20 25 3012611PRTHomo sapiens 126Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys Arg1 5 1012723PRTHomo sapiens
127Glu Tyr Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly1
5 10 15Glu Arg Ala Thr Leu Ser Cys 2012815PRTHomo sapiens 128Trp
Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Leu Val Ile Tyr1 5 10
1512932PRTHomo sapiens 129Asp Ile Pro Ala Arg Phe Ser Gly Ser Asn
Ser Gly Asp Glu Ala Thr1 5 10 15Leu Thr Ile Ser Ser Leu Gln Ser Glu
Asp Phe Ala Val Tyr Tyr Cys 20 25 3013011PRTHomo sapiens 130Phe Gly
Gln Gly Thr Arg Leu Glu Ile Lys Arg1 5 1013123PRTHomo sapiens
131Asp Tyr Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly1
5 10 15Glu Arg Ala Thr Ile Asn Cys 2013215PRTHomo sapiens 132Trp
Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Val Ile Tyr1 5 10
1513332PRTHomo sapiens 133Gly Ile
Pro Asp Arg Phe Ser Gly Ser Asn Ser Gly Asp Asp Ala Thr1 5 10 15Leu
Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys 20 25
3013411PRTHomo sapiens 134Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
Arg1 5 1013522PRTHomo sapiens 135Leu Pro Val Leu Thr Gln Pro Pro
Ser Val Ser Val Ser Pro Gly Gln1 5 10 15Thr Ala Ser Ile Thr Cys
2013615PRTHomo sapiens 136Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro
Val Leu Val Ile Tyr1 5 10 1513732PRTHomo sapiens 137Gly Ile Pro Glu
Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr1 5 10 15Leu Thr Ile
Ser Gly Thr Gln Thr Met Asp Glu Ala Asp Tyr Leu Cys 20 25
3013810PRTHomo sapiens 138Phe Gly Gly Gly Thr Lys Val Thr Val Leu1
5 1013922PRTHomo sapiens 139Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val
Ser Val Ser Pro Gly Gln1 5 10 15Thr Ala Ser Ile Thr Cys
2014015PRTHomo sapiens 140Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro
Val Leu Val Ile Tyr1 5 10 1514132PRTHomo sapiens 141Gly Ile Pro Glu
Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr1 5 10 15Leu Thr Ile
Ser Gly Thr Gln Thr Met Asp Glu Ala Asp Tyr Leu Cys 20 25
3014210PRTHomo sapiens 142Phe Gly Gly Gly Thr Lys Leu Thr Val Leu1
5 1014330PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMOD_RES(30)..(30)Ser, Arg or Gly 143Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Xaa 20 25
3014414PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 144Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val Ala1 5 1014532PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptideMOD_RES(11)..(11)Asn or
SerMOD_RES(31)..(31)Ala or Ser 145Arg Phe Thr Ile Ser Arg Asp Asn
Ala Lys Xaa Ser Leu Tyr Leu Gln1 5 10 15Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys Xaa Arg 20 25 3014611PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 146Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser1 5 1014723PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 147Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys 2014815PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(9)..(9)Ala or SerMOD_RES(15)..(15)Phe or Tyr 148Trp
Tyr Gln Gln Lys Pro Gly Lys Xaa Pro Lys Leu Leu Ile Xaa1 5 10
1514932PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMOD_RES(15)..(15)Phe or Ser 149Gly Val Pro Ser
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Xaa Thr1 5 10 15Leu Thr Ile
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 20 25
3015010PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 150Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys1 5
101515PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(1)..(1)Asn, His or TyrMOD_RES(5)..(5)Ala
or Ser 151Xaa Phe Pro Met Xaa1 515217PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(1)..(1)Thr or SerMOD_RES(4)..(4)Ser or
GlyMOD_RES(7)..(7)Gly, Ala, Asp, Ser or GluMOD_RES(8)..(8)Thr or
TrpMOD_RES(9)..(9)Thr, Pro or AlaMOD_RES(10)..(10)Tyr, Ser, Thr or
AsnMOD_RES(11)..(11)Tyr or IleMOD_RES(12)..(12)Arg or Gly 152Xaa
Ile Ser Xaa Ser Asp Xaa Xaa Xaa Xaa Xaa Xaa Asp Ser Val Lys1 5 10
15Gly1539PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(9)..(9)Tyr, Phe or Ser 153Gly Tyr Tyr Asn
Ser Pro Phe Ala Xaa1 515411PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(4)..(4)Glu or
GlnMOD_RES(5)..(5)Asp or GluMOD_RES(7)..(7)Tyr or
TrpMOD_RES(8)..(8)Ser, Ile, Tyr, Asn or Arg 154Arg Ala Ser Xaa Xaa
Ile Xaa Xaa Asn Leu Ala1 5 101557PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptideMOD_RES(3)..(3)Asn or
SerMOD_RES(4)..(4)Asn, Asp, Ser, Ile, Tyr or Val 155Asp Thr Xaa Xaa
Leu Ala Asp1 51569PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptideMOD_RES(4)..(4)Asn, Asp or
ThrMOD_RES(5)..(5)Asn, Tyr or TrpMOD_RES(6)..(6)Tyr or Val 156Gln
Gln Tyr Xaa Xaa Xaa Pro Pro Thr1 5157118PRTRattus sp. 157Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1 5 10 15Ser
Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25
30Gly Met Tyr Trp Ile Arg Gln Ala Pro Thr Lys Gly Leu Gln Trp Val
35 40 45Ala Phe Ile Ser His Gly Gly Gly Ile Thr Tyr Tyr Arg Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr
Leu Tyr65 70 75 80Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala
Thr Tyr His Cys 85 90 95Ala Ala Leu Asn Trp Glu Leu Gly Ile Asp Tyr
Trp Gly Gln Gly Val 100 105 110Met Val Thr Val Ser Ser
115158108PRTRattus sp. 158Asp Ile Gln Met Thr Gln Ser Pro Ala Ser
Leu Ser Ala Ser Leu Gly1 5 10 15Glu Thr Ile Ser Ile Glu Cys Arg Ala
Ser Glu Asp Ile Tyr Ser Asn 20 25 30Leu Ala Trp Tyr Gln Lys Lys Ser
Gly Lys Ser Pro Gln Leu Leu Ile 35 40 45Tyr Ala Ala Asn Arg Leu Gln
Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Gln
Tyr Ser Leu Lys Ile Ser Gly Met Gln Pro65 70 75 80Glu Asp Glu Gly
Asp Tyr Phe Cys Leu Gln Gly Ser Lys Phe Pro Leu 85 90 95Thr Phe Gly
Ser Gly Thr Lys Leu Glu Ile Lys Arg 100 105159118PRTRattus sp.
159Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1
5 10 15Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asn
Phe 20 25 30Pro Met Ala Trp Val Arg Gln Ala Pro Thr Arg Gly Leu Glu
Trp Val 35 40 45Ala Thr Ile Ser Ser Ser Asp Gly Thr Thr Tyr Tyr Arg
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Ser Thr Leu Tyr65 70 75 80Leu Gln Val Asn Ser Leu Arg Ser Glu Asp
Thr Ala Thr Tyr Tyr Cys 85 90 95Ser Arg Gly Tyr Tyr Asn Ser Pro Phe
Ala Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser
115160108PRTRattus sp. 160Asp Ile Gln Met Thr Gln Ser Pro Ala Ser
Leu Ser Ala Ser Leu Gly1 5 10 15Glu Thr Val Thr Ile Glu Cys Arg Ala
Ser Glu Asp Ile Tyr Ser Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Asn Ser Pro Gln Leu Leu Ile 35 40 45Phe Asp Thr Asn Asn Leu Ala
Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Gln
Ser Ser Leu Lys Ile Asn Ser Leu Gln Ser65 70 75 80Glu Asp Val Ala
Ser Tyr Phe Cys Gln Gln Tyr Asn Asn Tyr Pro Pro 85 90 95Thr Phe Gly
Gly Gly Thr Lys Leu Glu Leu Lys Arg 100 105161122PRTRattus sp.
161Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Glu1
5 10 15Ser Leu Lys Ile Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn
Ala 20 25 30Ala Met Tyr Trp Val Arg Leu Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ala Arg Ile Arg Thr Lys Pro Asn Asn Tyr Ala Thr Tyr
Tyr Ala Glu 50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp
Ser Lys Ser Met65 70 75 80Val Tyr Val Gln Met Asp Asn Leu Lys Thr
Glu Asp Thr Ala Met Tyr 85 90 95Tyr Cys Thr Ala Ala Pro Trp Arg Asp
Ser Tyr Ala His Val Tyr Trp 100 105 110Gly Gln Gly Val Met Val Thr
Val Ser Ser 115 120162108PRTRattus sp. 162Asp Ile Gln Met Thr Gln
Ser Pro Pro Val Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Leu
Ser Cys Lys Ala Ser Gln Asn Ile His Lys Asn 20 25 30Leu Asp Trp Tyr
Gln Gln Lys His Gly Asp Ala Pro Lys Leu Leu Ile 35 40 45Tyr Tyr Thr
Asp His Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Ala Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Val Ala Thr Tyr Tyr Cys Tyr Gln Tyr Asn Gly Gly Pro Phe
85 90 95Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg 100
105163116PRTRattus sp. 163Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Arg1 5 10 15Ser Leu Lys Leu Ser Cys Leu Ala Ser
Gly Phe Pro Phe Ser Ser Val 20 25 30Trp Met Thr Trp Ile Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Ala Thr Ile Thr Asn Ser Gly
Ala Ser Thr Tyr Tyr Ser Ala Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Val Lys Ser Thr Leu Tyr65 70 75 80Leu Gln Met Thr
Ser Leu Gly Ser Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95Thr Arg Val
Gly Thr Ser Phe Asp Tyr Trp Gly Gln Gly Val Met Val 100 105 110Thr
Val Ser Ser 115164109PRTRattus sp. 164Asp Ile Gln Met Thr Gln Ser
Pro Ala Ser Leu Ser Ala Ser Leu Gly1 5 10 15Glu Thr Val Thr Ile Glu
Cys Arg Ala Ser Asp Asp Ile Tyr Asn Gly 20 25 30Leu Ala Trp Phe Gln
Gln Lys Pro Gly Lys Ser Pro Gln Leu Leu Ile 35 40 45Tyr Asp Ala Asn
Thr Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Ser65 70 75 80Glu
Asp Val Ala Ser Tyr Phe Cys Gln Gln Phe Tyr Asp Tyr Pro Pro 85 90
95Tyr Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg 100
105165116PRTRattus sp. 165Glu Val Gln Leu Gln Gln Ser Gly Ala Glu
Leu Ala Lys Pro Gly Ser1 5 10 15Ser Val Lys Ile Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Asn Tyr 20 25 30Asp Ile Ser Trp Ile Lys Gln Thr
Asn Gly Gln Gly Leu Glu Tyr Leu 35 40 45Gly Tyr Ile Asn Thr Gly Ser
Gly Gly Ile Tyr Ser Asn Glu Lys Phe 50 55 60Lys Gly Lys Ala Thr Leu
Thr Val Asp Lys Ser Ser Asn Thr Ala Phe65 70 75 80Met Gln Leu Ser
Ser Leu Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Val Arg Glu
Gly Asn Asn Phe Asp His Trp Gly Gln Gly Val Lys Val 100 105 110Thr
Val Ser Ser 115166108PRTRattus sp. 166Asp Thr Val Met Thr Gln Ser
Pro Ala Ser Met Ser Thr Ser Val Gly1 5 10 15Glu Arg Val Thr Val Asn
Cys Lys Ala Ser Gln Ser Val Gly Thr Ile 20 25 30Val Ala Trp Phe Gln
Gln Lys Pro Gly Gln Ser Pro Lys Arg Leu Ile 35 40 45Tyr Leu Ala Thr
Tyr Arg His Thr Gly Val Pro Asp Arg Phe Ile Gly 50 55 60Ser Gly Phe
Gly Arg Asp Phe Thr Leu Thr Ile Ser Asn Val Glu Ala65 70 75 80Glu
Asp Leu Ala Val Tyr Tyr Cys Leu Gln Tyr Gly Ser Arg Pro Phe 85 90
95Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys Arg 100
105167121PRTRattus sp. 167Glu Val Gln Leu Gln Gln Ser Gly Pro Glu
Leu Ala Lys Pro Gly Ser1 5 10 15Ser Val Lys Ile Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Asn Ser 20 25 30Tyr Ile Ser Trp Ile Lys Gln Thr
Thr Gly Gln Gly Leu Glu Tyr Val 35 40 45Gly Tyr Ile Asn Thr Gly Ser
Gly Gly Ala Asp Tyr Asn Glu Lys Phe 50 55 60Lys Gly Lys Ala Thr Leu
Thr Val Asp Lys Ser Ser Arg Thr Ala Phe65 70 75 80Met Gln Leu Ser
Ser Leu Thr Pro Gly Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala Lys Ser
Ile Leu Leu Gly Ser Thr Cys Tyr Phe Asp Tyr Trp Gly 100 105 110Gln
Gly Val Leu Val Thr Val Ser Ser 115 120168111PRTRattus sp. 168Asn
Thr Val Leu Thr Gln Ser Pro Ala Leu Ala Val Ser Leu Gly Gln1 5 10
15Arg Val Thr Ile Ser Cys Lys Ala Ser Arg Ser Val Ser Ser Pro Met
20 25 30Tyr Ser Tyr Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Gln Pro
Lys 35 40 45Leu Leu Ile Tyr Arg Ala Ser Thr Leu Ala Ser Gly Val Pro
Ala Arg 50 55 60Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn
Ile Asp Pro65 70 75 80Val Glu Ala Asp Asp Ile Ala Thr Tyr Phe Cys
Gln Gln Ser Trp Ser 85 90 95Asp Pro Phe Thr Phe Gly Ser Gly Thr Lys
Leu Glu Ile Lys Arg 100 105 110169121PRTRattus sp. 169Glu Val Gln
Leu Gln Gln Ser Gly Pro Glu Leu Ala Lys Pro Gly Ser1 5 10 15Ser Val
Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Ser 20 25 30Tyr
Ile Ser Trp Ile Lys Gln Thr Thr Gly Gln Gly Leu Glu Tyr Ile 35 40
45Gly Tyr Ile Asn Thr Gly Ser Gly Gly Thr Asp Tyr Asn Glu Lys Phe
50 55 60Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Arg Thr Val
Phe65 70 75 80Met Gln Leu Ser Ser Leu Thr Pro Gly Asp Ser Ala Val
Tyr Tyr Cys 85 90 95Ala Lys Ser Ile Leu Leu Gly Ser Thr Tyr Tyr Leu
Asp Tyr Trp Gly 100 105 110Gln Gly Val Met Val Thr Val Ser Ser 115
120170111PRTRattus sp. 170Asp Thr Val Leu Thr Gln Ser Pro Ala Leu
Ala Val Ser Leu Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Lys Ala Ser
Arg Ser Leu Ser Ser Pro Met 20 25 30Tyr Ser Tyr Ile Tyr Trp Tyr Gln
Gln Lys Leu Gly Gln Gln Pro Arg 35 40 45Leu Leu Ile Tyr Arg Ala Ser
Thr Leu Ala Ser Gly Val Pro Ala Arg 50 55 60Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Asn Ile Asp Pro65 70 75 80Val Glu Ala Asp
Asp Ile Ala Thr Tyr Phe Cys Gln Gln Ser Trp Ser 85 90 95Asp Pro Phe
Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg 100 105
110171118PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 171Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Asn Phe 20 25 30Pro Met Ala Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Ser Ser Asp
Gly Thr Thr Tyr Tyr Arg Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95Ala Arg Gly Tyr Tyr Asn Ser Pro Phe Ala Tyr Trp Gly Gln Gly
Thr 100 105 110Leu Val Thr Val Ser Ser 115172118PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
172Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn
Phe 20 25 30Pro Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ala Thr Ile Ser Ser Ser Asp Gly Thr Thr Tyr Tyr Arg
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Ser Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ser Arg Gly Tyr Tyr Asn Ser Pro Phe
Ala Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser
115173118PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 173Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Asn Phe 20 25 30Pro Met Ala Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Ser Ser Asp
Gly Thr Thr Tyr Tyr Arg Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ser Arg Gly
Tyr Tyr Asn Ser Pro Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110Leu
Val Thr Val Ser Ser 115174118PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 174Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Phe 20 25 30Pro Met Ala
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Thr
Ile Ser Ser Ser Asp Gly Thr Thr Tyr Tyr Arg Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Ser Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ser Arg Gly Tyr Tyr Asn Ser Pro Phe Ala Tyr Trp Gly Gln Gly
Thr 100 105 110Leu Val Thr Val Ser Ser 115175108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
175Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asp Ile Tyr Ser
Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Asp Thr Asn Asn Leu Ala Asp Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Tyr Asn Asn Tyr Pro Pro 85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu
Ile Lys Arg 100 105176108PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 176Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Glu Asp Ile Tyr Ser Asn 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile 35 40 45Phe Asp
Thr Asn Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Ser Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Tyr Asn Asn Tyr Pro Pro
85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100
105177108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 177Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Glu Asp Ile Tyr Ser Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Phe Asp Thr Asn Asn Leu Ala
Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Tyr Asn Asn Tyr Pro Pro 85 90 95Thr Phe Gly
Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105178108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
178Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly1
5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Glu Asp Ile Tyr Ser
Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Leu
Leu Ile 35 40 45Phe Asp Thr Asn Asn Leu Ala Asp Gly Val Pro Ala Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Ser Thr Leu Thr Ile Ser
Ser Leu Gln Ser65 70 75 80Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln
Tyr Asn Asn Tyr Pro Pro 85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu
Ile Lys Arg 100 105179118PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 179Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30Gly Met Tyr
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Phe
Ile Ser His Gly Gly Gly Ile Thr Tyr Tyr Arg Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Leu Asn Trp Glu Leu Gly Ile Asp Tyr Trp Gly Gln Gly
Thr 100 105 110Met Val Thr Val Ser Ser 115180118PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
180Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn
Tyr 20 25 30Gly Met Tyr Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ala Phe Ile Ser His Gly Gly Gly Ile Thr Tyr Tyr Arg
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Ser Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr His Cys 85 90 95Ala Ala Leu Asn Trp Glu Leu Gly Ile
Asp Tyr Trp Gly Gln Gly Thr 100 105 110Met Val Thr Val Ser Ser
115181118PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 181Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Asn Tyr 20 25 30Gly Met Tyr Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Phe Ile Ser His Gly Gly
Gly Ile Thr Tyr Tyr Arg Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala Leu
Asn Trp Glu Leu Gly Ile Asp Tyr Trp Gly Gln Gly Thr 100 105 110Met
Val Thr Val Ser Ser 115182118PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 182Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30Gly Met Tyr
Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Phe
Ile Ser His Gly Gly Gly Ile Thr Tyr Tyr Arg Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Ser Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr His Cys
85 90 95Ala Ala Leu Asn Trp Glu Leu Gly Ile Asp Tyr Trp Gly Gln Gly
Thr 100 105 110Met Val Thr Val Ser Ser 115183108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
183Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asp Ile Tyr Ser
Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Ala Ala Asn Arg Leu Gln Asp Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln
Gly Ser Lys Phe Pro Leu 85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu
Ile Lys Arg 100 105184108PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 184Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Glu Asp Ile Tyr Ser Asn 20 25 30Leu Ala Trp
Tyr Gln Lys Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile 35 40 45Tyr Ala
Ala Asn Arg Leu Gln Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Phe Cys Leu Gln Gly Ser Lys Phe Pro Leu
85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100
105185108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 185Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Glu Asp Ile Tyr Ser Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Asn Arg Leu Gln
Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Leu Gln Gly Ser Lys Phe Pro Leu 85 90 95Thr Phe Gly
Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105186108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
186Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly1
5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Glu Asp Ile Tyr Ser
Asn 20 25 30Leu Ala Trp Tyr Gln Lys Lys Pro Gly Gln Ser Pro Arg Leu
Leu Ile 35 40 45Tyr Ala Ala Asn Arg Leu Gln Asp Gly Val Pro Ala Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser
Ser Leu Gln Ser65 70 75 80Glu Asp Phe Ala Val Tyr Phe Cys Leu Gln
Gly Ser Lys Phe Pro Leu 85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu
Ile Lys Arg 100 105187118PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 187Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Phe 20 25 30Pro Met Ala
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Thr
Ile Ser Ser Ser Asp Gly Thr Thr Tyr Tyr Arg Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Gly Tyr Tyr Asn Ser Pro Phe Ala Tyr Trp Gly Gln Gly
Thr 100 105 110Leu Val Thr Val Ser Ser 115188118PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
188Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg His
Phe 20 25 30Pro Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ala Thr Ile Ser Ser Ser Asp Ala Trp Pro Ser Tyr Arg
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ser Arg Gly Tyr Tyr Asn Ser Pro Phe
Ala Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser
115189118PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 189Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Gly Asn Phe 20 25 30Pro Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ser Ile Ser Ser Ser Asp
Ser Trp Ala Thr Ile Gly Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ser Arg Gly
Tyr Tyr Asn Ser Pro Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110Leu
Val Thr Val Ser Ser 115190118PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 190Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asn Phe 20 25 30Pro Met Ala
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Thr
Ile Ser Ser Ser Asp Gly Trp Pro Thr Tyr Arg Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Ser Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ser Arg Gly Tyr Tyr Asn Ser Pro Phe Ala Tyr Trp Gly Gln Gly
Thr 100 105 110Leu Val Thr Val Ser Ser 115191118PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
191Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg His
Phe 20 25 30Pro Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ala Thr Ile Ser Ser Ser Asp Asp Trp Pro Asn Tyr Arg
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Ser Ser Leu Tyr65 70
75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ser Arg Gly Tyr Tyr Asn Ser Pro Phe Ala Tyr Trp Gly Gln
Gly Thr 100 105 110Leu Val Thr Val Ser Ser 115192118PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
192Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Tyr
Phe 20 25 30Pro Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ala Ser Ile Ser Gly Ser Asp Gly Trp Ala Ser Tyr Arg
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Tyr Tyr Asn Ser Pro Phe
Ala Ser Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser
115193118PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 193Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Arg Tyr Phe 20 25 30Pro Met Ala Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Gly Ser Asp
Glu Trp Pro Asn Tyr Arg Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly
Tyr Tyr Asn Ser Pro Phe Ala Phe Trp Gly Gln Gly Thr 100 105 110Leu
Val Thr Val Ser Ser 115194118PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 194Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Tyr Phe 20 25 30Pro Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ser
Ile Ser Gly Ser Asp Gly Trp Ala Ser Tyr Arg Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Gly Tyr Tyr Asn Ser Pro Phe Ala Tyr Trp Gly Gln Gly
Thr 100 105 110Leu Val Thr Val Ser Ser 115195118PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
195Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg His
Phe 20 25 30Pro Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ala Thr Ile Ser Ser Ser Asp Ala Trp Pro Ser Tyr Arg
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Tyr Tyr Asn Ser Pro Phe
Ala Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser
115196118PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 196Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Gly Asn Phe 20 25 30Pro Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ser Ile Ser Ser Ser Asp
Ser Trp Ala Thr Ile Gly Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly
Tyr Tyr Asn Ser Pro Phe Ala Phe Trp Gly Gln Gly Thr 100 105 110Leu
Val Thr Val Ser Ser 115197107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 197Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Glu Asp Ile Tyr Ser Asn 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Asp
Thr Asn Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Asn Tyr Pro Pro
85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100
105198107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 198Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Asp Ile Tyr Ile Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Lys Ser Pro Lys Leu Leu Ile 35 40 45Phe Asp Thr Asn Asp Leu Ala
Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Tyr Asp Tyr Val Pro Pro 85 90 95Thr Phe Gly
Gln Gly Thr Lys Leu Glu Ile Lys 100 105199107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
199Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Tyr Tyr
Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu
Leu Ile 35 40 45Phe Asp Thr Ser Ser Leu Ala Asp Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln
Tyr Asp Trp Tyr Pro Pro 85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu
Ile Lys 100 105200107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 200Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Asp Ile Tyr Ile Asn 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Phe Asp
Thr Ser Asp Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Tyr Tyr Pro Pro
85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100
105201107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 201Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Asp Ile Tyr Tyr Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Phe Asp Thr Asn Ile Leu Ala
Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Phe Cys Gln Gln Tyr Asp Tyr Val Pro Pro 85 90 95Thr Phe Gly
Gln Gly Thr Lys Leu Glu Ile Lys 100 105202107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
202Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Trp Asn
Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu
Leu Ile 35 40 45Phe Asp Thr Ser Tyr Leu Ala Asp Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Tyr Asp Trp Tyr Pro Pro 85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu
Ile Lys 100 105203107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 203Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Glu Ile Tyr Arg Asn 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile 35 40 45Phe Asp
Thr Ser Val Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Ser Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Thr Tyr Tyr Pro Pro
85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100
105204118PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMOD_RES(30)..(30)Ser, Arg, Asn, Gly, Lys, Thr
or LeuMOD_RES(31)..(31)Asn, Tyr, His, Ser or
AlaMOD_RES(32)..(32)Phe or TyrMOD_RES(35)..(35)Ala, Ser or
ThrMOD_RES(50)..(50)Thr, Ser or AlaMOD_RES(53)..(53)Ser or
GlyMOD_RES(56)..(56)Gly, Ser, Ala, Glu, Asp, Phe, Gln or
CysMOD_RES(57)..(57)Thr, Ser, Phe or TrpMOD_RES(58)..(58)Thr, Ala,
Asp, Ser or ProMOD_RES(59)..(59)Tyr, Ser, Asn, Thr, Ala or
AspMOD_RES(60)..(60)Tyr or IleMOD_RES(61)..(61)Arg or
GlyMOD_RES(77)..(77)Asn or SerMOD_RES(78)..(78)Ser or
LeuMOD_RES(89)..(89)Glu or AspMOD_RES(106)..(106)Ala or
AspMOD_RES(107)..(107)Tyr, Phe or Ser 204Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Xaa Xaa Xaa 20 25 30Pro Met Xaa Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Xaa Ile
Ser Xaa Ser Asp Xaa Xaa Xaa Xaa Xaa Xaa Asp Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Xaa Xaa Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Xaa Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Gly Tyr Tyr Asn Ser Pro Phe Xaa Xaa Trp Gly Gln Gly
Thr 100 105 110Leu Val Thr Val Ser Ser 115205107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideMOD_RES(26)..(26)Ser or ThrMOD_RES(27)..(27)Glu or
GlnMOD_RES(28)..(28)Asp, Glu or ThrMOD_RES(30)..(30)Tyr, Trp, Glu,
Asp or AsnMOD_RES(31)..(31)Ser, Asn, Ile, Thr, Arg, Met or
GlyMOD_RES(43)..(43)Ala or SerMOD_RES(49)..(49)Tyr or
PheMOD_RES(53)..(53)Asn, Ser, Asp, Gln, Thr, Val or
GluMOD_RES(71)..(71)Phe or SerMOD_RES(87)..(87)Tyr or
PheMOD_RES(91)..(91)Tyr or SerMOD_RES(92)..(92)Asn, Asp, Thr or
TyrMOD_RES(93)..(93)Asn, Trp, Tyr, Ile, Phe or
ProMOD_RES(94)..(94)Tyr or ValMOD_RES(97)..(97)Thr or Pro 205Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Arg Ala Xaa Xaa Xaa Ile Xaa Xaa Asn
20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Xaa Pro Lys Leu Leu
Ile 35 40 45Xaa Asp Thr Asn Xaa Leu Ala Asp Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Xaa Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Xaa Cys Gln Gln Xaa
Xaa Xaa Xaa Pro Pro 85 90 95Xaa Phe Gly Gln Gly Thr Lys Leu Glu Ile
Lys 100 1052066PRTArtificial SequenceDescription of Artificial
Sequence Synthetic 6xHis tag 206His His His His His His1 5
* * * * *