Therapeutic DLL4 binding proteins

• US 9,115,195 B2
• Filed: 03/01/2011
• Issued: 08/25/2015
• Est. Priority Date: 03/02/2010
• Status: Active Grant

• Current Assignee: Abbvie Incorporated
• Original Assignee: Abbvie Incorporated
• Inventors: Chen, Ming-Jiu, Hsieh, Chung-Ming, Gu, Jijie, Morgan-Lappe, Susan E., Li, Yingchun
• Primary Examiner(s): Goddard, Laura B
• Assistant Examiner(s): Natarajan, Meera
• Application Number:
  US13/037,932
• Publication Number:
  US 20110217237A1
• Time in Patent Office: 1,638 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 39/3955 against proteinaceous materials, e.g. enzymes, hormones, lymphokines
• CPC Class Codes: A61K 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 16/28 against receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/73 Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
• CPC Class Codes: C07K 2317/76 Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• CPC Class Codes: C07K 2317/92 Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

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.

Claims

1. A binding protein comprising an antigen-binding domain capable of binding DLL4, wherein the antigen-binding domain comprises a set of six complementarity determining region (CDR) sequences:

CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, wherein;

(a) CDR-H1 is selected from the group consisting of;

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;

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);

(b) CDR-H2 is selected from the group consisting of;

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;

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);

(c) CDR-H3 is selected from the group consisting of;

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;

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);

(d) CDR-L1 is selected from the group consisting of;

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;

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);

(e) CDR-L2 is selected from group consisting of;

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;

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

(f) CDR-L3 is selected from the group consisting of;

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;

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).

2. The binding protein according to claim 1, wherein the binding protein comprises a CDR set of three CDRs selected from the group of variable domain CDR sets consisting of:

VH1A11 CDR Set, comprising;

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, comprising;

CDR-L1;

residues 24-34 of SEQ ID NO;

160

CDR-L2;

residues 50-56 of SEQ ID NO;

160 and

CDR-L3;

residues 89-97 of SEQ ID NO;

160

VH.1 1A11 CDR Set, comprising;

CDR-H1;

residues 31-35 of SEQ ID NO;

171

CDR-H2;

residues 50-66 of SEQ ID NO;

171 and

CDR-H3;

residues 99-107 of SEQ ID NO;

171

VH.1a 1A11 CDR Set, comprising;

CDR-H1;

residues 31-35 of SEQ ID NO;

172

CDR-H2;

residues 50-66 of SEQ ID NO;

172 and

CDR-H3;

residues 99-107 of SEQ ID NO;

172

VH.1b 1A11 CDR Set, comprising;

CDR-H1;

residues 31-35 of SEQ ID NO;

173

CDR-H2;

residues 50-66 of SEQ ID NO;

173 and

CDR-H3;

residues 99-107 of SEQ ID NO;

173

VH.2a 1A11 CDR Set, comprising;

CDR-H1;

residues 31-35 of SEQ ID NO;

174

CDR-H2;

residues 50-66 of SEQ ID NO;

174 and

CDR-H3;

residues 99-107 of SEQ ID NO;

174

VL.1 1A11 CDR Set, comprising;

CDR-L1;

residues 24-34 of SEQ ID NO;

175

CDR-L2;

residues 50-56 of SEQ ID NO;

175 and

CDR-L3;

residues 89-97 of SEQ ID NO;

175

VL.1a 1A11 CDR Set, comprising;

CDR-L1;

residues 24-34 of SEQ ID NO;

176

CDR-L2;

residues 50-56 of SEQ ID NO;

176 and

CDR-L3;

residues 89-97 of SEQ ID NO;

176

VL.1b 1A11 CDR Set, comprising;

CDR-L1;

residues 24-34 of SEQ ID NO;

177

CDR-L2;

residues 50-56 of SEQ ID NO;

177 and

CDR-L3;

residues 89-97 of SEQ ID NO;

177

VL.2a 1A11 CDR Set, comprising;

CDR-L1;

residues 24-34 of SEQ ID NO;

178

CDR-L2;

residues 50-56 of SEQ ID NO;

178 and

CDR-L3;

residues 89-97 of SEQ ID NO;

178

VH h1A11VH.1 CDR Set, comprising;

CDR-H1;

residues 31-35 of SEQ ID NO;

187

CDR-H2;

residues 50-66 of SEQ ID NO;

187 and

CDR-H3;

residues 99-107 of SEQ ID NO;

187

VH h1A11.A6 CDR Set, comprising;

CDR-H1;

residues 31-35 of SEQ ID NO;

188

CDR-H2;

residues 50-66 of SEQ ID NO;

188 and

CDR-H3;

residues 99-107 of SEQ ID NO;

188

VH h1A11.A8 CDR Set, comprising;

CDR-H1;

residues 31-35 of SEQ ID NO;

189

CDR-H2;

residues 50-66 of SEQ ID NO;

189 and

CDR-H3;

residues 99-107 of SEQ ID NO;

189

VH h1A11.C6 CDR Set, comprising;

CDR-H1;

residues 31-35 of SEQ ID NO;

190

CDR-H2;

residues 50-66 of SEQ ID NO;

190 and

CDR-H3;

residues 99-107 of SEQ ID NO;

190

VH h1A11.A11 CDR Set, comprising;

CDR-H1;

residues 31-35 of SEQ ID NO;

191

CDR-H2;

residues 50-66 of SEQ ID NO;

191 and

CDR-H3;

residues 99-107 of SEQ ID NO;

191

VH h1A11.B5 CDR Set, comprising;

CDR-H1;

residues 31-35 of SEQ ID NO;

192

CDR-H2;

residues 50-66 of SEQ ID NO;

192 and

CDR-H3;

residues 99-107 of SEQ ID NO;

192

VH h1A11.E12 CDR Set, comprising;

CDR-H1;

residues 31-35 of SEQ ID NO;

193

CDR-H2;

residues 50-66 of SEQ ID NO;

193 and

CDR-H3;

residues 99-107 of SEQ ID NO;

193

VH h1A11.G3 CDR Set, comprising;

CDR-H1;

residues 31-35 of SEQ ID NO;

194

CDR-H2;

residues 50-66 of SEQ ID NO;

194 and

CDR-H3;

residues 99-107 of SEQ ID NO;

194

VH h1A11.F5 CDR Set, comprising;

CDR-H1;

residues 31-35 of SEQ ID NO;

195

CDR-H2;

residues 50-66 of SEQ ID NO;

195 and

CDR-H3;

residues 99-107 of SEQ ID NO;

195

VH h1A11.H2 CDR Set, comprising;

CDR-H1;

residues 31-35 of SEQ ID NO;

196

CDR-H2;

residues 50-66 of SEQ ID NO;

196 and

CDR-H3;

residues 99-107 of SEQ ID NO;

196

VL h1A11VL.1 CDR Set, comprising;

CDR-L1;

residues 24-34 of SEQ ID NO;

197

CDR-L2;

residues 50-56 of SEQ ID NO;

197 and

CDR-L3;

residues 89-97 of SEQ ID NO;

197

VL h1A11.A2 CDR Set, comprising;

CDR-L1;

residues 24-34 of SEQ ID NO;

198

CDR-L2;

residues 50-56 of SEQ ID NO;

198 and

CDR-L3;

residues 89-97 of SEQ ID NO;

198

VL h1A11.A12 CDR Set, comprising;

CDR-L1;

residues 24-34 of SEQ ID NO;

199

CDR-L2;

residues 50-56 of SEQ ID NO;

199 and

CDR-L3;

residues 89-97 of SEQ ID NO;

199

VL h1A11.A7 CDR Set, comprising;

CDR-L1;

residues 24-34 of SEQ ID NO;

200

CDR-L2;

residues 50-56 of SEQ ID NO;

200 and

CDR-L3;

residues 89-97 of SEQ ID NO;

200

VL h1A11.B4 CDR Set, comprising;

CDR-L1;

residues 24-34 of SEQ ID NO;

201

CDR-L2;

residues 50-56 of SEQ ID NO;

201 and

CDR-L3;

residues 89-97 of SEQ ID NO;

201

VL h1A11.B5 CDR Set, comprising;

CDR-L1;

residues 24-34 of SEQ ID NO;

202

CDR-L2;

residues 50-56 of SEQ ID NO;

202 and

CDR-L3;

residues 89-97 of SEQ ID NO;

202 and

VL h1A11.E12 CDR Set, comprising;

CDR-L1;

residues 24-34 of SEQ ID NO;

203

CDR-L2;

residues 50-56 of SEQ ID NO;

203 and

CDR-L3;

residues 89-97 of SEQ ID NO;

203.

3. The binding protein according to claim 2, comprising at least two variable domain CDR sets.

4. The binding protein according to claim 3, wherein the at least two variable domain CDR sets are selected from the group consisting of:

VH1A11 CDR Set and VL 1A11 CDR Set,

VH.1 1A11 CDR Set and VL.1 1A11 CDR Set,

VH.1 1A11 CDR Set and VL.1a 1A11 CDR Set,

VH.1 1A11 CDR Set and VL.1b 1A11 CDR Set,

VH.1 1A11 CDR Set and VL.2a 1A11 CDR Set,

VH.1a 1A11 CDR Set and VL.1 1A11 CDR Set,

VH.1a 1A11 CDR Set and VL.1a 1A11 CDR Set,

VH.1a 1A11 CDR Set and VL.1b 1A11 CDR Set,

VH.1a 1A11 CDR Set and VL.2a 1A11 CDR Set,

VH.1b 1A11 CDR Set and VL.1 1A11 CDR Set,

VH.1b 1A11 CDR Set and VL.1a 1A11 CDR Set,

VH.1b 1A11 CDR Set and VL.1b 1A11 CDR Set,

VH.1b 1A11 CDR Set and VL.2a 1A11 CDR Set,

VH.2a 1A11 CDR Set and VL.1 1A11 CDR Set,

VH.2a 1A11 CDR Set and VL.1a 1A11 CDR Set,

VH.2a 1A11 CDR Set and VL.1b 1A11 CDR Set,

VH.2a 1A11 CDR Set and VL.2a 1A11 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.

5. The binding protein according to claim 1, further comprising a human acceptor framework sequence.

6. The binding protein according to claim 5, wherein the human acceptor framework comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:

6-34 and SEQ ID NOs;

35-98.

7. The binding protein according to claim 5, wherein the binding protein comprises at least one acceptor framework sequence selected from the group consisting of:

(a) 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-X30 (SEQ ID NO;

143), wherein X30 is S, R, or G;

(b) heavy chain framework-2 (H-FR2);

W-V-R-Q-A-P-G-K-G-L-E-W-V-A (SEQ ID NO;

144);

(c) heavy chain framework-3 (H-FR3);

R-F-T-I-S-R-D-N-A-K-X11-S-L-Y-L-Q-M-N-S-L-R-A-E-D-T-A-V-Y-Y-C-X31-R (SEQ ID NO;

145), wherein;

X11 is N or S; and

X31 is A or S;

(d) heavy chain framework-4 (H-FR4);

W-G-Q-G-T-L-V-T-V-S-S (SEQ ID NO;

146);

(e) 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);

(f) light chain framework-2 (L-FR2);

W-Y-Q-Q-K-P-G-K-X₉-P-K-L-L-I-X15 (SEQ ID NO;

148), wherein;

X9 is A or S; and

X15 is F or Y;

(g) light chain framework-3 (L-FR3);

G-V-P-S-R-F-S-G-S-G-S-G-T-D-X15-T-L-T-I-S-S-L-Q-P-E-D-F-A-T-Y-Y-C (SEQ ID NO;

149), wherein;

X15 is F or S; and

(h) light chain framework-4 (L-FR4);

F-G-Q-G-T-K-L-E-I-K (SEQ ID NO;

150).

8. The binding protein of claim 2, wherein the antigen-binding domain comprises variable domain sequences selected from the group consisting of:

SEQ ID NOs;

159 and 160;

SEQ ID NOs;

171 and 175;

SEQ ID NOs;

171 and 176;

SEQ ID NOs;

171 and 177;

SEQ ID NOs;

171 and 178;

SEQ ID NOs;

172 and 175;

SEQ ID NOs;

172 and 176;

SEQ ID NOs;

172 and 177;

SEQ ID NOs;

172 and 178;

SEQ ID NOs;

173 and 175;

SEQ ID NOs;

173 and 176;

SEQ ID NOs;

173 and 178;

SEQ ID NOs;

174 and 175;

SEQ ID NOs;

174 and 176;

SEQ ID NOs;

174 and 177;

SEQ ID NOs;

174 and 178;

SEQ ID NOs;

188 and 197;

SEQ ID NOs;

190 and 197;

SEQ ID NOs;

189 and 197;

SEQ ID NOs;

187 and 197;

SEQ ID NOs;

187 and 201;

SEQ ID NOs;

187 and 200;

SEQ ID NOs;

187 and 199;

SEQ ID NOs;

187 and 198;

SEQ ID NOs;

192 and 202;

SEQ ID NOs;

193 and 203;

SEQ ID NOs;

194 and 203;

SEQ ID NOs;

195 and 203; and

SEQ ID NOs;

196 and 203.

9. The binding protein according to claim 5, wherein the framework sequence of the binding protein comprises at least one amino acid substitution relative to a human germline acceptor framework at a 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.

10. The binding protein according to claim 5, 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.

11. The binding protein of claim 1, wherein the antigen-binding domain comprises variable domain sequences selected from the group consisting of:

SEQ ID NOs;

159 and 160;

SEQ ID NOs;

171 and 175;

SEQ ID NOs;

171 and 176;

SEQ ID NOs;

171 and 177;

SEQ ID NOs;

171 and 178;

SEQ ID NOs;

172 and 175;

SEQ ID NOs;

172 and 176;

SEQ ID NOs;

172 and 177;

SEQ ID NOs;

172 and 178;

SEQ ID NOs;

173 and 175;

SEQ ID NOs;

173 and 176;

SEQ ID NOs;

173 and 177;

SEQ ID NOs;

173 and 178;

SEQ ID NOs;

174 and 175;

SEQ ID NOs;

174 and 176;

SEQ ID NOs;

174 and 177;

SEQ ID NOs;

174 and 178; and

SEQ ID NOs;

187 and 197.

12. The binding protein according to claim 1, wherein the binding protein is capable of blocking DLL4 interaction with a Notch protein.

13. The binding protein according to claim 1, 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.

14. The binding protein according to claim 1, wherein the binding protein is capable of modulating a biological function of DLL4.

15. The binding protein according to claim 1, wherein the binding protein is capable of neutralizing a biological function of DLL4.

16. The binding protein according to claim 1, wherein the binding protein is capable of inhibiting VEGFR2 activity, VEGFR1 activity, or both.

17. The binding protein according to claim 1, wherein the binding protein is capable of diminishing the ability of DLL4 to bind to its receptor.

18. The binding protein according to claim 1, wherein the binding protein is capable of inhibiting normal angiogenesis.

19. The binding protein according to claim 1, wherein the binding protein has an on rate constant (K_on) to DLL4 selected from the group consisting of:

at least about 10²M<sup>−

1</sup>s<sup>−

1</sup>;

at least about 10³M<sup>−

1</sup>s<sup>−

1</sup>;

at least about 10⁴M<sup>−

1</sup>s<sup>−

1</sup>;

at least about 10⁵M<sup>−

1</sup>s<sup>−

1</sup>; and

at least about 10⁶M<sup>−

1</sup>s<sup>−

1</sup>, as measured by surface plasmon resonance.

20. The binding protein according to claim 1, wherein the binding protein has an off rate constant (K_off) to DLL4 selected from the group consisting of:

at most about 10<sup>−

3</sup>s<sup>−

1</sup>;

at most about 10<sup>−

4</sup>s<sup>−

1</sup>;

at most about 10<sup>−

5</sup>s<sup>−

1</sup>; and

at most about 10<sup>−

6</sup>s<sup>−

1</sup>, as measured by surface plasmon resonance.

21. The binding protein according to claim 1, wherein the binding protein has a dissociation constant (K_D) to DLL4 selected from the group consisting of:

at most about 10<sup>−

7</sup> M;

at most about 10<sup>−

8</sup> M;

at most about 10<sup>−

9</sup> M;

at most about 10<sup>−

10</sup> M;

at most about 10<sup>−

11</sup> M;

at most about 10<sup>−

12</sup> M; and

at most about 10<sup>−

13</sup> M.

22. A binding protein construct comprising the binding protein of claim 1, the binding protein construct further comprising a linker polypeptide or an immunoglobulin constant domain.

23. The binding protein construct according to claim 22, wherein the binding protein is 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′

)₂,

an Fv,

a disulfide-linked Fv,

an scFv,

a single domain antibody,

a diabody,

a multispecific antibody,

a dual specific antibody,

a dual variable domain immunoglobulin (DVD-Ig) binding protein, and

a bispecific antibody.

24. The binding protein construct according to claim 22, wherein the binding protein 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.

25. The binding protein construct according to claim 22, 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.

26. A binding protein conjugate comprising the binding protein construct of claim 22, binding protein conjugate further comprising an agent selected from the group consisting of:

an imaging agent, a therapeutic agent, a cytotoxic agent, and an immunoadhesion molecule.

27. The binding protein conjugate according to claim 26, wherein the 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.

28. The binding protein conjugate according to claim 26, wherein the imaging agent is a radiolabel selected from the group consisting of:

³H, ¹⁴C, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, and ¹⁵³Sm.

29. The binding protein conjugate according to claim 26, wherein the 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.

30. The binding protein construct according to claim 22, wherein the binding protein possesses a human glycosylation pattern.

31. The binding protein conjugate according to claim 26, wherein the binding protein possesses a human glycosylation pattern.

32. The binding protein according to claim 1, wherein the binding protein exists as a crystal.

33. The binding protein construct according to claim 22, wherein the binding protein construct exists as a crystal.

34. The binding protein conjugate according to claim 26, wherein the binding protein construct exists as a crystal.

35. The binding protein according to claim 32, wherein the crystal is a carrier-free pharmaceutical controlled release crystal.

36. The binding protein construct according to claim 33, wherein the crystal is a carrier-free pharmaceutical controlled release crystal.

37. The binding protein conjugate according to claim 34, wherein the crystal is a carrier-free pharmaceutical controlled release crystal.

38. The binding protein according to claim 32, wherein the binding protein crystal has a greater half-life in vivo than the soluble counterpart of the binding protein.

39. The binding protein construct according to claim 33, wherein the binding protein construct crystal has a greater half-life in vivo than the soluble counterpart of the binding protein construct.

40. The binding protein conjugate according to claim 34, wherein the binding protein conjugate crystal has a greater half-life in vivo than the soluble counterpart of the binding protein conjugate.

41. The binding protein according to claim 32, wherein the binding protein crystal retains biological activity of the non-crystal form of the binding protein.

42. The binding protein construct according to claim 33, wherein the binding protein construct crystal retains biological activity of the non-crystal form of the binding protein construct.

43. The binding protein conjugate according to claim 34, wherein the binding protein conjugate crystal retains biological activity of the non-crystal form of the binding protein conjugate.

44. An isolated nucleic acid encoding the binding protein amino acid sequence of claim 1.

45. A vector comprising the isolated nucleic acid according to claim 44.

46. The vector according to claim 45, wherein the vector is selected from the group consisting of:

pcDNA, pTT, pTT3, pEFBOS, pBV, NV, and pBJ.

47. A host cell comprising the vector of claim 45.

48. The host cell according to claim 47, wherein said host cell is a prokaryotic cell.

49. The host cell according to claim 48, wherein the host cell is an Escherichia coli cell.

50. The host cell according to claim 47, wherein said host cell is a eukaryotic cell.

51. The host cell according to claim 50, wherein said eukaryotic cell is selected from the group consisting of:

a protist cell, an animal cell, a plant cell, and a fungal cell.

52. The host cell according to claim 51, wherein said eukaryotic cell is an animal cell selected from the group consisting of:

a mammalian cell, an avian cell, and an insect cell.

53. The host cell according to claim 52, wherein said mammalian cell is a CHO cell.

54. The host cell according to claim 52, wherein said mammalian cell is a COS cell.

55. The host cell according to claim 51, wherein the fungal cell is a Saccharomyces cerevisiae cell.

56. The host cell according to claim 52, wherein said insect cell is an Sf9 cell,

57. A method of producing a binding protein that binds human DLL4, comprising culturing the host cell of claim 47 in a culture medium under conditions sufficient to produce a binding protein that binds human DLL4.

58. A binding protein produced according to the method of claim 57.

59. A composition for the release of a binding protein, the composition comprising:

(a) a formulation, wherein said formulation comprises a crystallized binding protein of claim 32, and an ingredient; and

(b) at least one polymeric carrier.

60. The composition according to claim 59, 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-hydroxybutryate), 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.

61. The composition according to claim 59, wherein said ingredient is selected from the group consisting of albumin, sucrose, trehalose, lactitol, gelatin, hydroxypropyl-β

-cyclodextrin, methoxypolyethylene glycol and polyethylene glycol.

62. A method for treating a mammal comprising the step of administering to the mammal an effective amount of the composition of claim 59.

63. A pharmaceutical composition comprising the binding protein of claim 1, and a pharmaceutically acceptable carrier.

64. The pharmaceutical composition of claim 63, further comprising at least one additional agent for treating a disorder in which DLL4 activity is detrimental.

65. The pharmaceutical composition of claim 64, 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 BcI2/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.

66. A method for reducing human DLL4 activity comprising contacting human DLL4 with the binding protein of claim 1 such that human DLL4 activity is reduced.

67. 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 the binding protein of claim 1 such that human DLL4 activity in the human subject is reduced.

68. A method for treating a subject for a disease or a disorder in which DLL4 activity is detrimental by administering to the subject the binding protein of claim 2 such that treatment is achieved.

69. The method of claim 68, 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.

70. The method according to claim 69, wherein the disorder is a primary and metastatic cancer.

71. The method according to claim 69, wherein the urinary tract cancer is selected from the group consisting of renal cancer, bladder cancer, and urothelium cancer.

72. The method according to claim 69, wherein the female genital tract cancer is selected from the group consisting of cervical cancer, uterine cancer, ovarian cancer, choriocarcinoma, and gestational trophoblastic disease.

73. The method according to claim 69, wherein the male genital tract cancer is selected from the group consisting of prostate cancer, seminal vesicles cancer, testicular cancer, and germ cell tumor.

74. The method according to claim 69, wherein the endocrine gland cancer is selected from the group consisting of thyroid cancer, adrenal cancer, and pituitary gland cancer.

75. The method according to claim 69, wherein the sarcoma is selected from the group consisting of a bone sarcoma, a soft tissue sarcoma, and Kaposi'"'"'s sarcoma.

76. The method according to claim 69, 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.

77. The method according to claim 69, 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.

78. The method according to claim 69, wherein the ocular neovascularization is selected from the group consisting of diabetic blindness, a retinopathy, an age-related macular degeneration, and a rubeosis.

79. The method according to claim 68, 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.

80. A method of treating a patient suffering from a disorder in which DLL4 is detrimental comprising the step of administering the DLL4 binding protein of claim 1 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-lg;

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 receptorantibody;

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.

81. The binding protein according to claim 1, wherein the binding protein is an antibody.

82. The antibody according to claim 81, wherein the antibody is selected from the group consisting of a monoclonal antibody, a full-length tetrameric immunoglobulin, an IgG molecule, an IgG1 molecule, a chimeric antibody, a CDR-grafted antibody, a humanized antibody, and an affinity matured antibody.

83. The antibody according to claim 82, wherein the antibody is a monoclonal antibody.

84. The binding protein according to claim 8, 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.

87. The binding protein according to claim 1, wherein the binding protein is a dual variable domain immunoglobulin (DVD-Ig) binding protein.

85. A binding protein comprising a heavy chain variable domain sequence and a light chain variable domain sequence that together form a functional binding site for DLL4, wherein the variable domains comprise CDRs 1-3 from SEQ ID NO:

187 and CDRs 1-3 from SEQ ID NO;

197.

86. The binding protein of claim 85, wherein the variable domains comprise SEQ ID NO:

187 and SEQ ID NO;

197.

90. The binding protein of claim 85, wherein the binding protein is an antibody.

91. The binding protein of claim 86, wherein the binding protein is an antibody.

88. A DVD-Ig binding protein comprising a heavy chain variable domain sequence and a light chain variable domain sequence that together form a functional binding site for DLL4, wherein the variable domains comprise CDRs 1-3 from SEQ ID NO:

187 and CDRs 1-3 from SEQ ID NO;

197.

89. The DVD-Ig binding protein of claim 88, wherein the variable domains comprise SEQ ID NO:

187 and SEQ ID NO;

197.