WO2012092539A2 - Antibodies to dll4 and uses thereof - Google Patents

Antibodies to dll4 and uses thereof Download PDF

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Publication number
WO2012092539A2
WO2012092539A2 PCT/US2011/068044 US2011068044W WO2012092539A2 WO 2012092539 A2 WO2012092539 A2 WO 2012092539A2 US 2011068044 W US2011068044 W US 2011068044W WO 2012092539 A2 WO2012092539 A2 WO 2012092539A2
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seq id
antibody
variable region
chain variable
dll4
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PCT/US2011/068044
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French (fr)
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WO2012092539A3 (en
Inventor
Jinhong Fan
Yuuichirou KAJIWARA
Gregory Landes
Spencer LIANG
Linda Masat
Jennifer STRATTON
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Takeda Pharmaceutical Company Limited
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Priority to US201061429037P priority
Priority to US201061429035P priority
Priority to US201061429040P priority
Priority to US61/429,042 priority
Priority to US61/429,037 priority
Priority to US61/429,035 priority
Application filed by Takeda Pharmaceutical Company Limited filed Critical Takeda Pharmaceutical Company Limited
Publication of WO2012092539A2 publication Critical patent/WO2012092539A2/en
Publication of WO2012092539A3 publication Critical patent/WO2012092539A3/en

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/30Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/60Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622Single chain antibody (scFv)
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/70Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/70Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/90Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Abstract

The present disclosure relates to antibodies to delta-like ligand 4 (DLL4), including antibodies which bind to human DLL4, and their uses.

Description

ANTIBODIES TO DLL4 AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001 ] This application claims the benefit of U.S. Provisional Application No.

61/429,035, filed December 31 , 2010; U.S. Provisional Application No. 61/429,037, filed December 31 , 2010; U.S. Provisional Application No. 61/429,040, filed December 31 , 2010; and U.S. Provisional Application No. 61/429,042, filed December 31 , 2010, the disclosures of which are incorporated by reference herein in their entirety.

FIELD OF THE DISCLOSURE

[0002] The present disclosure relates to antibodies that bind to delta-like ligand 4 (DLL4) and uses thereof. The present disclosure also relates to methods of modulating human T cell activation, cytokine production and/or differentiation, including with antibodies to DLL4. The present disclosure also relates to methods of treating diseases or disorders in humans, including Th17 cell-mediated diseases or disorders.

BACKGROUND

[0003] The Notch receptor was first identified in Drosophila fruit-fly mutants. Loss- of-function of the Notch receptor produces an embryonic lethal "neurogenic" phenotype where cells of the epidermis switch fate to neural tissue (Moohr, 1919, Genet. 4:252; Poulson, 1937, PNAS 23:133; Poulson, 1940, J. Exp. Zool. 83:271 ). Four mammalian Notch protein receptors have been identified (NOTCH 1 , NOTCH2, NOTCH3, and NOTCH4), and mutations in these receptors result in developmental abnormalities and human pathologies including cancers (Gridley, 1997, Mol. Cell. Neurosci. 9:103; Joutel & Tournier-Lasserve, 1998, Semin. Cell Dev. Biol. 9:619-25).

[0004] The activation and differentiation of cells of the lymphoid system, including T cells, is a highly regulated process that is controlled by many pathways. Of these multiple pathways, Notch receptors and ligands have been shown to play a role in some aspects of T cell development, activation and differentiation (see, e.g., reviews by Radtke et al., Immunity 32:14-27; Yuan et al., Annu. Rev. Immunol. 28:343-365). In addition to the four Notch protein receptors (NOTCH 1 , 2, 3, 4), the Notch pathways consists of five ligands: delta-like ligand 1 (DLL1 ), delta-like ligand 3 (DLL3), delta-like ligand 4 (DLL4), Jagged 1 and Jagged 2. In terms of T cell function and T helper cell differentiation, some studies have examined the effects of the individual ligands. Subpopulations of T cells (e.g., Th1 cells, Th2 cells, and more recently Th17 cells) have been implicated in autoimmune disease (see e.g., Korn et al., 2009, Annu. Rev. Immunol. 27:485-517; Littman and Rudensky, Cell 140:845-858; Weaver et al., 2007, Annu. Rev. Immunol. 25:821 -852). In contrast to the mouse (see, e.g., Mukherjee et al. 2009, J. Immunol. 182:7381-7388; Ito et al., 2009, J. Clin. Invest. 1 19:33-46; Alam et al., Proc. Natl. Acad. Sci USA 107:5943- 5948), the role of the Notch pathway in regulating human T cell differentiation is not well understood.

SUMMARY

[0005] The present disclosure provides antibodies that bind to delta-like ligand 4 (DLL4) and uses thereof.

[0006] The present disclosure provides an isolated delta-like ligand 4 (DLL4) antibody comprising a heavy chain variable region and a light chain variable region, wherein:

i.) the heavy chain variable region comprises HCDR1 (GYSWH, SEQ ID NO: 1 ; DHSITSG, SEQ ID NO: 2; or DHSITSGYS; SEQ ID NO: 3), HCDR2 (YIHYSGYTHYNPSLKS, SEQ ID NO: 4; or IHYSGY, SEQ ID NO: 5) and HCDR3 (SDYSANEGAMAY, SEQ ID NO: 6; or AGSDYSANEGAMAY, SEQ ID NO: 7); and/or the light chain variable region comprises LCDR1 (RASESVDSFGNSFMH , SEQ ID NO: 8; or ESVDSFGNSF, SEQ ID NO: 9), LCDR2 (RTSNLQS, SEQ ID NO: 10; or R) and LCDR3 (QQSNEDPWT, SEQ ID NO: 1 1 );

ii.) the heavy chain variable region comprises HCDR1 (DEYWS, SEQ ID NO: 12;

GGSFND, SEQ ID NO: 13; or GGSFNDEY; SEQ ID NO: 14), HCDR2 (EIHESGKTNYNPSLKP, SEQ ID NO: 15; or IHESGKT, SEQ ID NO: 16) and HCDR3 (LAYRDRWYGAFDV, SEQ ID NO: 17; or ARLAYRDRWYGAFDV, SEQ ID NO: 18); and/or the light chain variable region comprises LCDR1 (QASHDITNYIN, SEQ ID NO: 19; or HDITNY, SEQ ID NO: 20), LCDR2 (HHTSKLQT, SEQ ID NO: 21 ; or H) and LCDR3 (QQFDNLLLT, SEQ ID NO: 22);

iii. ) the heavy chain variable region comprises HCDR1 (GYYMH, SEQ ID NO: 23; GYTFTG, SEQ ID NO: 24; or GYTFTGYY; SEQ ID NO: 25), HCDR2 (WINPNSGGTNYAQ, SEQ ID NO: 26; or INPNSGG, SEQ ID NO: 27) and HCDR3 (GAGVAAYDY, SEQ ID NO: 28; or ARGAGVAAYDY, SEQ ID NO: 29); and/or the light chain variable region comprises LCDR1 (RASQSISSYLN, SEQ ID NO: 30; or QSISSY, SEQ ID NO: 31 ), LCDR2 (AASSLQS, SEQ ID NO: 32; or A) and LCDR3 (QQSYSTPVT, SEQ ID NO: 33);

iv. ) the heavy chain variable region comprises HCDR1 (DYAMS, SEQ ID NO: 34; GFTFSD, SEQ ID NO: 35; or GFTFSDYA; SEQ ID NO: 36), HCDR2 (TISGSGGDTFYADSVKG, SEQ ID NO: 37; or ISGSGGDT, SEQ ID NO: 38) and HCDR3 (DKNRGAYADAFD, SEQ ID NO: 39; or AKDKNRGAYADAFD, SEQ ID NO: 40); and/or the light chain variable region comprises LCDR1 (TLRSDINVGTYRIY, SEQ ID NO: 41 ; or SDINVGTYR, SEQ ID NO: 42), LCDR2 (YKSDTDK, SEQ ID NO: 43; or YKSDTD, SEQ ID NO: 44) and LCDR3 (MVWHKSAPI, SEQ ID NO: 45); or

v.) the heavy chain variable region comprises HCDR1 (DYAMS, SEQ ID NO: 34; GFTFSD, SEQ ID NO: 35; or GFTFSDYA; SEQ ID NO: 36), HCDR2 (TISGSGGDTFYADSVKG, SEQ ID NO: 37; or ISGSGGD, SEQ ID NO: 46) and HCDR3 (DKNRGAYADAFDI, SEQ ID NO: 47; or AKDKNRGAYADAFDI, SEQ ID NO: 48); and/or the light chain variable region comprises LCDR1 (TLRSDINVATYRIY, SEQ ID NO: 49; or SDINVATYR, SEQ ID NO: 50), LCDR2 (YKSDSDK, SEQ ID NO: 51 ; or YKSDSD, SEQ ID NO: 52) and LCDR3 (MVWHKSAPI, SEQ ID NO: 45).

[0007] In some embodiments, which may be used or combined with any of the above or below mentioned disclosure and embodiments, the antibody in part i.) above comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 53, the antibody in part ii.) comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 54, the antibody in part iii.) comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 55, the antibody in part iv.) comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 56, or the antibody in part v.) comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 57.

[0008] In some embodiments, which may be used or combined with any of the above or below mentioned disclosure and embodiments, the antibody in part i.) above comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58, the antibody in part ii.) comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 59, the antibody in part iii.) comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 60, the antibody in part iv.) comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 61 , or the antibody in part v.) comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 62.

[0009] In some embodiments, which may be used or combined with any of the above or below mentioned disclosure and embodiments, the antibody in part i.) above comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 53 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58, the antibody in part ii.) comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 54 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 59, the antibody in part iii.) comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 55 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 60, the antibody in part iv.) comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 56 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 61 , or the antibody in part v.) comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 57 and a light chain variable region comprising the amino acid sequence SEQ ID NO: 62.

[0010] The present disclosure also provides an isolated delta-like ligand 4 (DLL4) antibody that binds to the same epitope that an antibody comprising a heavy chain variable region and a light chain variable region binds to, wherein:

i. ) the heavy chain variable region comprises HCDR1 (GYSWH, SEQ ID NO: 1 ; DHSITSG, SEQ ID NO: 2; or DHSITSGYS; SEQ ID NO: 3), HCDR2

(YIHYSGYTHYNPSLKS, SEQ ID NO: 4; or IHYSGY, SEQ ID NO: 5) and HCDR3 (SDYSANEGAMAY, SEQ ID NO: 6; or AGS DYS AN E GAM AY, SEQ ID NO: 7); and/or the light chain variable region comprises LCDR1 (RASESVDSFGNSFMH , SEQ ID NO: 8; or ESVDSFGNSF, SEQ ID NO: 9), LCDR2 (RTSNLQS, SEQ ID NO: 10; or R) and LCDR3 (QQSNEDPWT, SEQ ID NO: 1 1 );

ii. ) the heavy chain variable region comprises HCDR1 (DEYWS, SEQ ID NO: 12; GGSFND, SEQ ID NO: 13; or GGSFNDEY; SEQ ID NO: 14), HCDR2 (EIHESGKTNYNPSLKP, SEQ ID NO: 15; or IHESGKT, SEQ ID NO: 16) and HCDR3 (LAYRDRWYGAFDV, SEQ ID NO: 17; or ARLAYRDRWYGAFDV, SEQ ID NO: 18); and/or the light chain variable region comprises LCDR1 (QASHDITNYIN, SEQ ID NO: 19; or HDITNY, SEQ ID NO: 20), LCDR2 (HHTSKLQT, SEQ ID NO: 21 ; or H) and LCDR3 (QQFDNLLLT, SEQ ID NO: 22);

iii. ) the heavy chain variable region comprises HCDR1 (GYYMH, SEQ ID NO: 23; GYTFTG, SEQ ID NO: 24; or GYTFTGYY; SEQ ID NO: 25), HCDR2 (WINPNSGGTNYAQ, SEQ ID NO: 26; or INPNSGG, SEQ ID NO: 27) and HCDR3 (GAGVAAYDY, SEQ ID NO: 28; or ARGAGVAAYDY, SEQ ID NO: 29); and/or the light chain variable region comprises LCDR1 (RASQSISSYLN, SEQ ID NO: 30; or QSISSY, SEQ ID NO: 31 ), LCDR2 (AASSLQS, SEQ ID NO: 32; or A) and LCDR3 (QQSYSTPVT, SEQ ID NO: 33);

iv. ) the heavy chain variable region comprises HCDR1 (DYAMS, SEQ ID NO: 34; GFTFSD, SEQ ID NO: 35; or GFTFSDYA; SEQ ID NO: 36), HCDR2

(TISGSGGDTFYADSVKG, SEQ ID NO: 37; or ISGSGGDT, SEQ ID NO: 38) and HCDR3 (DKNRGAYADAFD, SEQ ID NO: 39; or AKDKNRGAYADAFD, SEQ ID NO: 40); and/or the light chain variable region comprises LCDR1 (TLRSDINVGTYRIY, SEQ ID NO: 41 ; or SDINVGTYR, SEQ ID NO: 42), LCDR2 (YKSDTDK, SEQ ID NO: 43; or YKSDTD, SEQ ID NO: 44) and LCDR3 (MVWHKSAPI, SEQ ID NO: 45); or v.) the heavy chain variable region comprises HCDR1 (DYAMS, SEQ ID NO: 34; GFTFSD, SEQ ID NO: 35; or GFTFSDYA; SEQ ID NO: 36), HCDR2 (TISGSGGDTFYADSVKG, SEQ ID NO: 37; or ISGSGGD, SEQ ID NO: 46) and HCDR3 (DKNRGAYADAFDI, SEQ ID NO: 47; or AKD KN RGAYAD AFD I , SEQ ID NO: 48); and/or the light chain variable region comprises LCDR1 (TLRSDINVATYRIY, SEQ ID NO: 49; or SDINVATYR, SEQ ID NO: 50), LCDR2 (YKSDSDK, SEQ ID NO: 51 ; or YKSDSD, SEQ ID NO: 52) and LCDR3 (MVWHKSAPI, SEQ ID NO: 45).

[001 1 ] The present disclosure also provides an isolated delta-like ligand 4 (DLL4) antibody that binds to the same epitope that an antibody comprising: i.) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 53 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58 binds to, ii.) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 54 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 59 binds to, iii.) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 55 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 60 binds to, iv.) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 56 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 61 binds to, or v.) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 57 and a light chain variable region comprising the amino acid sequence SEQ ID NO: 62 binds to.

[0012] The present disclosure also provides isolated delta-like ligand 4 (DLL4) antibody that binds to human DLL4 (SEQ ID NO: 63) and competes with the binding of an antibody comprising a heavy chain variable region and a light chain variable region, wherein:

i.) the heavy chain variable region comprises HCDR1 (GYSWH, SEQ ID NO: 1 ;

DHSITSG, SEQ ID NO: 2; or DHSITSGYS; SEQ ID NO: 3), HCDR2 (YIHYSGYTHYNPSLKS, SEQ ID NO: 4; or IHYSGY, SEQ ID NO: 5) and HCDR3 (SDYSANEGAMAY, SEQ ID NO: 6; or AGS DYS AN E GAM AY, SEQ ID NO: 7); and/or the light chain variable region comprises LCDR1 (RASESVDSFGNSFMH , SEQ ID NO: 8; or ESVDSFGNSF, SEQ ID NO: 9), LCDR2 (RTSNLQS, SEQ ID NO: 10; or R) and LCDR3 (QQSNEDPWT, SEQ ID NO: 1 1 );

ii.) the heavy chain variable region comprises HCDR1 (DEYWS, SEQ ID NO: 12; GGSFND, SEQ ID NO: 13; or GGSFNDEY; SEQ ID NO: 14), HCDR2 (EIHESGKTNYNPSLKP, SEQ ID NO: 15; or IHESGKT, SEQ ID NO: 16) and HCDR3 (LAYRDRWYGAFDV, SEQ ID NO: 17; or ARLAYRDRWYGAFDV, SEQ ID NO: 18); and/or the light chain variable region comprises LCDR1 (QASHDITNYIN, SEQ ID NO: 19; or HDITNY, SEQ ID NO: 20), LCDR2 (HHTSKLQT, SEQ ID NO: 21 ; or H) and LCDR3 (QQFDNLLLT, SEQ ID NO: 22);

iii. ) the heavy chain variable region comprises HCDR1 (GYYMH, SEQ ID NO: 23; GYTFTG, SEQ ID NO: 24; or GYTFTGYY; SEQ ID NO: 25), HCDR2 (WINPNSGGTNYAQ, SEQ ID NO: 26; or INPNSGG, SEQ ID NO: 27) and HCDR3 (GAGVAAYDY, SEQ ID NO: 28; or ARGAGVAAYDY, SEQ ID NO: 29); and/or the light chain variable region comprises LCDR1 (RASQSISSYLN, SEQ ID NO: 30; or QSISSY, SEQ ID NO: 31 ), LCDR2 (AASSLQS, SEQ ID NO: 32; or A) and LCDR3 (QQSYSTPVT, SEQ ID NO: 33);

iv. ) the heavy chain variable region comprises HCDR1 (DYAMS, SEQ ID NO: 34; GFTFSD, SEQ ID NO: 35; or GFTFSDYA; SEQ ID NO: 36), HCDR2

(TISGSGGDTFYADSVKG, SEQ ID NO: 37; or ISGSGGDT, SEQ ID NO: 38) and HCDR3 (DKNRGAYADAFD, SEQ ID NO: 39; or AKDKNRGAYADAFD, SEQ ID NO: 40); and/or the light chain variable region comprises LCDR1 (TLRSDINVGTYRIY, SEQ ID NO: 41 ; or SDINVGTYR, SEQ ID NO: 42), LCDR2 (YKSDTDK, SEQ ID NO: 43; or YKSDTD, SEQ ID NO: 44) and LCDR3 (MVWHKSAPI, SEQ ID NO: 45); or

v. ) the heavy chain variable region comprises HCDR1 (DYAMS, SEQ ID NO: 34; GFTFSD, SEQ ID NO: 35; or GFTFSDYA; SEQ ID NO: 36), HCDR2 (TISGSGGDTFYADSVKG, SEQ ID NO: 37; or ISGSGGD, SEQ ID NO: 46) and HCDR3 (DKNRGAYADAFDI, SEQ ID NO: 47; or AKD KN RGAYAD AFD I , SEQ ID NO: 48); and/or the light chain variable region comprises LCDR1 (TLRSDINVATYRIY, SEQ ID NO: 49; or SDINVATYR, SEQ ID NO: 50), LCDR2 (YKSDSDK, SEQ ID NO: 51 ; or YKSDSD, SEQ ID NO: 52) and LCDR3 (MVWHKSAPI, SEQ ID NO: 45).

[0013] The present disclosure also provides an isolated delta-like ligand 4 (DLL4) antibody that binds to human DLL4 (SEQ ID NO: 63) and competes with the binding of an antibody comprising: i.) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 53 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58, ii.) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 54 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 59, iii.) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 55 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 60, iv.) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 56 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 61 , or v.) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 57 and a light chain variable region comprising the amino acid sequence SEQ ID NO: 62. [0014] The present disclosure provides an isolated delta-like ligand 4 (DLL4) antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 (GYSWH, SEQ ID NO: 1 ; DHSITSG, SEQ ID NO: 2; or DHSITSGYS; SEQ ID NO: 3), HCDR2 (YIHYSGYTHYNPSLKS, SEQ ID NO: 4; or IHYSGY, SEQ ID NO: 5) and HCDR3 (SDYSANEGAMAY, SEQ ID NO: 6; or AGSDYSANEGAMAY, SEQ ID NO: 7); and/or wherein the light chain variable region comprises LCDR1 (RASESVDSFGNSFMH , SEQ ID NO: 8; or ESVDSFGNSF, SEQ ID NO: 9), LCDR2 (RTSNLQS, SEQ ID NO: 10; or R) and LCDR3 (QQSNEDPWT, SEQ ID NO: 1 1 ).

[0015] In some embodiments, which may be used or combined with any of the above or below mentioned disclosure and embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 53.

[0016] In some embodiments, which may be used or combined with any of the above or below mentioned disclosure and embodiments, the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58.

[0017] In some embodiments, which may be used or combined with any of the above or below mentioned disclosure and embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 53 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58.

[0018] The present disclosure also provides an isolated delta-like ligand 4 (DLL4) antibody that binds to the same epitope that an antibody comprising a heavy chain variable region and a light chain variable region binds to, wherein the heavy chain variable region comprises HCDR1 (GYSWH, SEQ ID NO: 1 ; DHSITSG, SEQ ID NO: 2; or DHSITSGYS; SEQ ID NO: 3), HCDR2 (YIHYSGYTHYNPSLKS, SEQ ID NO: 4; or IHYSGY, SEQ ID NO: 5) and HCDR3 (SDYSANEGAMAY, SEQ ID NO: 6; or AGSDYSANEGAMAY, SEQ ID NO: 7); and/or wherein the light chain variable region comprises LCDR1 (RASESVDSFGNSFMH , SEQ ID NO: 8; or ESVDSFGNSF, SEQ ID NO: 9), LCDR2 (RTSNLQS, SEQ ID NO: 10; or R) and LCDR3 (QQSNEDPWT, SEQ ID NO: 1 1 ).

[0019] The present disclosure also provides an isolated delta-like ligand 4 (DLL4) antibody that binds to the same epitope that an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 53 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58 binds to.

[0020] The present disclosure also provides an isolated delta-like ligand 4 (DLL4) antibody that binds to human DLL4 (SEQ ID NO: 63) and competes with the binding of an antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 (GYSWH, SEQ ID NO: 1 ; DHSITSG, SEQ ID NO: 2; or DHSITSGYS; SEQ ID NO: 3), HCDR2 (YIHYSGYTHYNPSLKS, SEQ ID NO: 4; or IHYSGY, SEQ ID NO: 5) and HCDR3 (SDYSANEGAMAY, SEQ ID NO: 6; or AGSDYSANEGAMAY, SEQ ID NO: 7); and/or wherein the light chain variable region comprises LCDR1 (RASESVDSFGNSFMH , SEQ ID NO: 8; or ESVDSFGNSF, SEQ ID NO: 9), LCDR2 (RTSNLQS, SEQ ID NO: 10; or R) and LCDR3 (QQSNEDPWT, SEQ ID NO: 1 1 ).

[0021 ] The present disclosure also provides an isolated delta-like ligand 4 (DLL4) antibody that binds to human DLL4 (SEQ ID NO: 63) and competes with the binding of an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 53 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58.

[0022] In some embodiments, which may be used or combined with any of the above or below mentioned disclosure and embodiments, the heavy chain variable region comprises HCDR1 (GYSWH, SEQ ID NO: 1 ), HCDR2 (YIHYSGYTHYNPSLKS, SEQ ID NO: 4) and HCDR3 (SDYSANEGAMAY, SEQ ID NO: 6); and/or wherein the light chain variable region comprises LCDR1 (RASESVDSFGNSFMH, SEQ ID NO: 8), LCDR2 (RTSNLQS, SEQ ID NO: 10) and LCDR3 (QQSNEDPWT, SEQ ID NO: 1 1 ).

[0023] In some embodiments, which may be used or combined with any of the above or below mentioned disclosure and embodiments, the heavy chain variable region comprises HCDR1 (DHSITSG, SEQ ID NO: 2), HCDR2 (YIHYSGYTHYNPSLKS, SEQ ID NO: 4) and HCDR3 (SDYSANEGAMAY, SEQ ID NO: 6); and/or wherein the light chain variable region comprises LCDR1 (RASESVDSFGNSFMH, SEQ ID NO: 8), LCDR2 (RTSNLQS, SEQ ID NO: 10) and LCDR3 (QQSNEDPWT, SEQ ID NO: 1 1 ).

[0024] In some embodiments, which may be used or combined with any of the above or below mentioned disclosure and embodiments, the heavy chain variable region comprises HCDR1 (DHSITSGYS; SEQ ID NO: 3), HCDR2 (IHYSGY, SEQ ID NO: 5) and HCDR3 (AGSDYSANEGAMAY, SEQ ID NO: 7); and/or wherein the light chain variable region comprises LCDR1 (ESVDSFGNSF, SEQ ID NO: 9), LCDR2 (R) and LCDR3 (QQSNEDPWT, SEQ ID NO: 1 1 ).

[0025] The present disclosure provides an isolated delta-like ligand 4 (DLL4) antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 (DEYWS, SEQ ID NO: 12; GGSFND, SEQ ID NO: 13; or GGSFNDEY; SEQ ID NO: 14), HCDR2 (EIHESGKTNYNPSLKP, SEQ ID NO: 15; or IHESGKT, SEQ ID NO: 16) and HCDR3 (LAYRDRWYGAFDV, SEQ ID NO: 17; or ARLAYRDRWYGAFDV, SEQ ID NO: 18); and/or wherein the light chain variable region comprises LCDR1 (QASHDITNYIN, SEQ ID NO: 19; or HDITNY, SEQ ID NO: 20), LCDR2 (HHTSKLQT, SEQ ID NO: 21 ; or H) and LCDR3 (QQFDNLLLT, SEQ ID NO: 22).

[0026] In some embodiments, which may be used or combined with any of the above or below mentioned disclosure and embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 54.

[0027] In some embodiments, which may be used or combined with any of the above or below mentioned disclosure and embodiments, the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 59.

[0028] In some embodiments, which may be used or combined with any of the above or below mentioned disclosure and embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 54 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 59.

[0029] The present disclosure also provides an isolated delta-like ligand 4 (DLL4) antibody that binds to the same epitope that an antibody comprising a heavy chain variable region and a light chain variable region binds to, wherein the heavy chain variable region comprises HCDR1 (DEYWS, SEQ ID NO: 12; GGSFND, SEQ ID NO: 13; or GGSFNDEY; SEQ ID NO: 14), HCDR2 (EIHESGKTNYNPSLKP, SEQ ID NO: 15; or IHESGKT, SEQ ID NO: 16) and HCDR3 (LAYRDRWYGAFDV, SEQ ID NO: 17; or ARLAYRDRWYGAFDV, SEQ ID NO: 18); and/or wherein the light chain variable region comprises LCDR1 (QASHDITNYIN, SEQ ID NO: 19; or HDITNY, SEQ ID NO: 20), LCDR2 (HHTSKLQT, SEQ ID NO: 21 ; or H) and LCDR3 (QQFDNLLLT, SEQ ID NO: 22).

[0030] The present disclosure also provides an isolated delta-like ligand 4 (DLL4) antibody that binds to the same epitope that an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 54 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 59 binds to.

[0031 ] The present disclosure also provides an isolated delta-like ligand 4 (DLL4) antibody that binds to human DLL4 (SEQ ID NO: 63) and competes with the binding of an antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 (DEYWS, SEQ ID NO: 12; GGSFND, SEQ ID NO: 13; or GGSFNDEY; SEQ ID NO: 14), HCDR2 (EIHESGKTNYNPSLKP, SEQ ID NO: 15; or IHESGKT, SEQ ID NO: 16) and HCDR3 (LAYRDRWYGAFDV, SEQ ID NO: 17; or ARLAYRDRWYGAFDV, SEQ ID NO: 18); and/or wherein the light chain variable region comprises LCDR1 (QASHDITNYIN, SEQ ID NO: 19; or HDITNY, SEQ ID NO: 20), LCDR2 (HHTSKLQT, SEQ ID NO: 21 ; or H) and LCDR3 (QQFDNLLLT, SEQ ID NO: 22). [0032] The present disclosure also provides an isolated delta-like ligand 4 (DLL4) antibody that binds to human DLL4 (SEQ ID NO: 63) and competes with the binding of an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 54 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 59.

[0033] In some embodiments, which may be used or combined with any of the above or below mentioned disclosure and embodiments, the heavy chain variable region comprises HCDR1 (DEYWS, SEQ ID NO: 12), HCDR2 (EIHESGKTNYNPSLKP, SEQ ID NO: 15) and HCDR3 (LAYRDRWYGAFDV, SEQ ID NO: 17); and/or wherein the light chain variable region comprises LCDR1 (QASHDITNYIN, SEQ ID NO: 19), LCDR2 (HTSKLQT, SEQ ID NO: 21 ) and LCDR3 (QQFDNLLLT, SEQ ID NO: 22).

[0034] In some embodiments, which may be used or combined with any of the above or below mentioned disclosure and embodiments, the heavy chain variable region comprises HCDR1 (GGSFND, SEQ ID NO: 13), HCDR2 (EIHESGKTNYNPSLKP, SEQ ID NO: 15) and HCDR3 (LAYRDRWYGAFDV, SEQ ID NO: 17); and/or wherein the light chain variable region comprises LCDR1 (QASHDITNYIN, SEQ ID NO: 19), LCDR2 (HTSKLQT, SEQ ID NO: 21 ) and LCDR3 (QQFDNLLLT, SEQ ID NO: 22).

[0035] In some embodiments, which may be used or combined with any of the above or below mentioned disclosure and embodiments, the heavy chain variable region comprises HCDR1 (GGSFNDEY; SEQ ID NO: 14), HCDR2 (IHESGKT, SEQ ID NO: 16) and HCDR3 (ARLAYRDRWYGAFDV, SEQ ID NO: 18); and/or wherein the light chain variable region comprises LCDR1 (HDITNY, SEQ ID NO: 20), LCDR2 (H) and LCDR3 (QQFDNLLLT, SEQ ID NO: 22).

[0036] The present disclosure provides an isolated delta-like ligand 4 (DLL4) antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 (GYYMH, SEQ ID NO: 23; GYTFTG, SEQ ID NO: 24; or GYTFTGYY; SEQ ID NO: 25), HCDR2 (WINPNSGGTNYAQ, SEQ ID NO: 26; or INPNSGG, SEQ ID NO: 27) and HCDR3 (GAGVAAYDY, SEQ ID NO: 28; or ARGAGVAAYDY, SEQ ID NO: 29); and/or wherein the light chain variable region comprises LCDR1 (RASQSISSYLN, SEQ ID NO: 30; or QSISSY, SEQ ID NO: 31 ), LCDR2 (AASSLQS, SEQ ID NO: 32; or A) and LCDR3 (QQSYSTPVT, SEQ ID NO: 33).

[0037] In some embodiments, which may be used or combined with any of the above or below mentioned disclosure and embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 55. [0038] In some embodiments, which may be used or combined with any of the above or below mentioned disclosure and embodiments, the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 60.

[0039] In some embodiments, which may be used or combined with any of the above or below mentioned disclosure and embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 55 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 60.

[0040] The present disclosure also provides an isolated delta-like ligand 4 (DLL4) antibody that binds to the same epitope that an antibody comprising a heavy chain variable region and a light chain variable region binds to, wherein the heavy chain variable region comprises HCDR1 (GYYMH, SEQ ID NO: 23; GYTFTG, SEQ ID NO: 24; or GYTFTGYY; SEQ ID NO: 25), HCDR2 (WINPNSGGTNYAQ, SEQ ID NO: 26; or INPNSGG, SEQ ID NO: 27) and HCDR3 (GAGVAAYDY, SEQ ID NO: 28; or ARGAGVAAYDY, SEQ ID NO: 29); and/or wherein the light chain variable region comprises LCDR1 (RASQSISSYLN, SEQ ID NO: 30; or QSISSY, SEQ ID NO: 31 ), LCDR2 (AASSLQS, SEQ ID NO: 32; or A) and LCDR3 (QQSYSTPVT, SEQ ID NO: 33).

[0041 ] The present disclosure also provides an isolated delta-like ligand 4 (DLL4) antibody that binds to the same epitope that an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 55 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 60 binds to.

[0042] The present disclosure also provides an isolated delta-like ligand 4 (DLL4) antibody that binds to human DLL4 (SEQ ID NO: 63) and competes with the binding of an antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 (GYYMH, SEQ ID NO: 23; GYTFTG, SEQ ID NO: 24; or GYTFTGYY; SEQ ID NO: 25), HCDR2 (WINPNSGGTNYAQ, SEQ ID NO: 26; or INPNSGG, SEQ ID NO: 27) and HCDR3 (GAGVAAYDY, SEQ ID NO: 28; or ARGAGVAAYDY, SEQ ID NO: 29); and/or wherein the light chain variable region comprises LCDR1 (RASQSISSYLN, SEQ ID NO: 30; or QSISSY, SEQ ID NO: 31 ), LCDR2 (AASSLQS, SEQ ID NO: 32; or A) and LCDR3 (QQSYSTPVT, SEQ ID NO: 33).

[0043] The present disclosure also provides an isolated delta-like ligand 4 (DLL4) antibody that binds to human DLL4 (SEQ ID NO: 63) and competes with the binding of an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 55 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 60.

[0044] In some embodiments, which may be used or combined with any of the above or below mentioned disclosure and embodiments, the heavy chain variable region comprises HCDR1 (GYYMH, SEQ ID NO: 23), HCDR2 (WINPNSGGTNYAQ, SEQ ID NO: 26) and HCDR3 (GAGVAAYDY, SEQ ID NO: 28); and/or wherein the light chain variable region comprises LCDR1 (RASQSISSYLN, SEQ ID NO: 30), LCDR2 (AASSLQS, SEQ ID NO: 32) and LCDR3 (QQSYSTPVT, SEQ ID NO: 33).

[0045] In some embodiments, which may be used or combined with any of the above or below mentioned disclosure and embodiments, the heavy chain variable region comprises HCDR1 (GYTFTG, SEQ ID NO: 24), HCDR2 (WINPNSGGTNYAQ, SEQ ID NO: 26) and HCDR3 (GAGVAAYDY, SEQ ID NO: 28); and/or wherein the light chain variable region comprises LCDR1 (RASQSISSYLN, SEQ ID NO: 30), LCDR2 (AASSLQS, SEQ ID NO: 32) and LCDR3 (QQSYSTPVT, SEQ ID NO: 33).

[0046] In some embodiments, which may be used or combined with any of the above or below mentioned disclosure and embodiments, the heavy chain variable region comprises HCDR1 (GYTFTGYY; SEQ ID NO: 25), HCDR2 (INPNSGG, SEQ ID NO: 27) and HCDR3 (ARGAGVAAYDY, SEQ ID NO: 29); and/or wherein the light chain variable region comprises LCDR1 (QSISSY, SEQ ID NO: 31 ), LCDR2 (A) and LCDR3 (QQSYSTPVT, SEQ ID NO: 33).

[0047] The present disclosure provides an isolated delta-like ligand 4 (DLL4) antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 (DYAMS, SEQ ID NO: 34; GFTFSD, SEQ ID NO: 35; or GFTFSDYA; SEQ ID NO: 36), HCDR2 (TISGSGGDTFYADSVKG, SEQ ID NO: 37; or ISGSGGDT, SEQ ID NO: 38) and HCDR3 (DKNRGAYADAFD, SEQ ID NO: 39; or AKDKNRGAYADAFD, SEQ ID NO: 40); and/or wherein the light chain variable region comprises LCDR1 (TLRSDINVGTYRIY, SEQ ID NO: 41 ; or SDINVGTYR, SEQ ID NO: 42), LCDR2 (YKSDTDK, SEQ ID NO: 43; or YKSDTD, SEQ ID NO: 44) and LCDR3 (MVWHKSAPI, SEQ ID NO: 45).

[0048] In some embodiments, which may be used or combined with any of the above or below mentioned disclosure and embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 56.

[0049] In some embodiments, which may be used or combined with any of the above or below mentioned disclosure and embodiments, the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 61.

[0050] In some embodiments, which may be used or combined with any of the above or below mentioned disclosure and embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 56 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 61. [0051 ] The present disclosure also provides an isolated delta-like ligand 4 (DLL4) antibody that binds to the same epitope that an antibody comprising a heavy chain variable region and a light chain variable region binds to, wherein the heavy chain variable region comprises HCDR1 (DYAMS, SEQ ID NO: 34; GFTFSD, SEQ ID NO: 35; or GFTFSDYA; SEQ ID NO: 36), HCDR2 (TISGSGGDTFYADSVKG, SEQ ID NO: 37; or ISGSGGDT, SEQ ID NO: 38) and HCDR3 (DKNRGAYADAFD, SEQ ID NO: 39; or AKDKNRGAYADAFD, SEQ ID NO: 40); and/or wherein the light chain variable region comprises LCDR1 (TLRSDINVGTYRIY, SEQ ID NO: 41 ; or SDINVGTYR, SEQ ID NO: 42), LCDR2 (YKSDTDK, SEQ ID NO: 43; or YKSDTD, SEQ ID NO: 44) and LCDR3 (MVWHKSAPI, SEQ ID NO: 45).

[0052] The present disclosure also provides an isolated delta-like ligand 4 (DLL4) antibody that binds to the same epitope that an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 56 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 61 binds to.

[0053] The present disclosure also provides an isolated delta-like ligand 4 (DLL4) antibody that binds to human DLL4 (SEQ ID NO: 63) and competes with the binding of an antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 (DYAMS, SEQ ID NO: 34; GFTFSD, SEQ ID NO: 35; or GFTFSDYA; SEQ ID NO: 36), HCDR2 (TISGSGGDTFYADSVKG, SEQ ID NO: 37; or ISGSGGDT, SEQ ID NO: 38) and HCDR3 (DKNRGAYADAFD, SEQ ID NO: 39; or AKDKNRGAYADAFD, SEQ ID NO: 40); and/or wherein the light chain variable region comprises LCDR1 (TLRSDINVGTYRIY, SEQ ID NO: 41 ; or SDINVGTYR, SEQ ID NO: 42), LCDR2 (YKSDTDK, SEQ ID NO: 43; or YKSDTD, SEQ ID NO: 44) and LCDR3 (MVWHKSAPI, SEQ ID NO: 45).

[0054] The present disclosure also provides an isolated delta-like ligand 4 (DLL4) antibody that binds to human DLL4 (SEQ ID NO: 63) and competes with the binding of an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 56 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 61.

[0055] In some embodiments, which may be used or combined with any of the above or below mentioned disclosure and embodiments, the heavy chain variable region comprises HCDR1 (DYAMS, SEQ ID NO: 34), HCDR2 (TISGSGGDTFYADSVKG, SEQ ID NO: 37) and HCDR3 (DKNRGAYADAFD, SEQ ID NO: 39); and/or wherein the light chain variable region comprises LCDR1 (TLRSDINVGTYRIY, SEQ ID NO: 41 ), LCDR2 (YKSDTDK, SEQ ID NO: 43) and LCDR3 (MVWHKSAPI, SEQ ID NO: 45). [0056] In some embodiments, which may be used or combined with any of the above or below mentioned disclosure and embodiments, the heavy chain variable region comprises HCDR1 (GFTFSD, SEQ ID NO: 35), HCDR2 (TISGSGGDTFYADSVKG, SEQ ID NO: 37) and HCDR3 (DKNRGAYADAFD, SEQ ID NO: 39); and/or wherein the light chain variable region comprises LCDR1 (TLRSDINVGTYRIY, SEQ ID NO: 41 ), LCDR2 (YKSDTDK, SEQ ID NO: 43) and LCDR3 (MVWHKSAPI, SEQ ID NO: 45).

[0057] In some embodiments, which may be used or combined with any of the above or below mentioned disclosure and embodiments, the heavy chain variable region comprises HCDR1 (GFTFSDYA; SEQ ID NO: 36), HCDR2 (ISGSGGDT, SEQ ID NO: 38) and HCDR3 (AKDKNRGAYADAFD, SEQ ID NO: 40); and/or wherein the light chain variable region comprises LCDR1 (SDINVGTYR, SEQ ID NO: 42), LCDR2 (YKSDTD, SEQ ID NO: 44) and LCDR3 (MVWHKSAPI, SEQ ID NO: 45).

[0058] The present disclosure provides an isolated delta-like ligand 4 (DLL4) antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 (DYAMS, SEQ ID NO: 34; GFTFSD, SEQ ID NO: 35; or GFTFSDYA; SEQ ID NO: 36), HCDR2 (TISGSGGDTFYADSVKG, SEQ ID NO: 37; or ISGSGGD, SEQ ID NO: 46) and HCDR3 (DKNRGAYADAFDI, SEQ ID NO: 47; or AKDKNRGAYADAFDI, SEQ ID NO: 48); and/or wherein the light chain variable region comprises LCDR1 (TLRSDINVATYRIY, SEQ ID NO: 49; or SDINVATYR, SEQ ID NO: 50), LCDR2 (YKSDSDK, SEQ ID NO: 51 ; or YKSDSD, SEQ ID NO: 52) and LCDR3 (MVWHKSAPI, SEQ ID NO: 45).

[0059] In some embodiments, which may be used or combined with any of the above or below mentioned disclosure and embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 57.

[0060] In some embodiments, which may be used or combined with any of the above or below mentioned disclosure and embodiments, the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 62.

[0061 ] In some embodiments, which may be used or combined with any of the above or below mentioned disclosure and embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 57 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 62.

[0062] The present disclosure also provides an isolated delta-like ligand 4 (DLL4) antibody that binds to the same epitope that an antibody comprising a heavy chain variable region and a light chain variable region binds to, wherein the heavy chain variable region comprises HCDR1 (DYAMS, SEQ ID NO: 34; GFTFSD, SEQ ID NO: 35; or GFTFSDYA; SEQ ID NO: 36), HCDR2 (TISGSGGDTFYADSVKG, SEQ ID NO: 37; or ISGSGGD, SEQ ID NO: 46) and HCDR3 (DKNRGAYADAFDI, SEQ ID NO: 47; or AKDKNRGAYADAFDI, SEQ ID NO: 48); and/or wherein the light chain variable region comprises LCDR1 (TLRSDINVATYRIY, SEQ ID NO: 49; or SDINVATYR, SEQ ID NO: 50), LCDR2 (YKSDSDK, SEQ ID NO: 51 ; or YKSDSD, SEQ ID NO: 52) and LCDR3 (MVWHKSAPI, SEQ ID NO: 45).

[0063] The present disclosure also provides an isolated delta-like ligand 4 (DLL4) antibody that binds to the same epitope that an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 57 and a light chain variable region comprising the amino acid sequence SEQ ID NO: 62 binds to.

[0064] The present disclosure also provides an isolated delta-like ligand 4 (DLL4) antibody that binds to human DLL4 (SEQ ID NO: 63) and competes with the binding of an antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 (DYAMS, SEQ ID NO: 34; GFTFSD, SEQ ID NO: 35; or GFTFSDYA; SEQ ID NO: 36), HCDR2 (TISGSGGDTFYADSVKG, SEQ ID NO: 37; or ISGSGGD, SEQ ID NO: 46) and HCDR3 (DKNRGAYADAFDI, SEQ ID NO: 47; or AKDKNRGAYADAFDI, SEQ ID NO: 48); and/or wherein the light chain variable region comprises LCDR1 (TLRSDINVATYRIY, SEQ ID NO: 49; or SDINVATYR, SEQ ID NO: 50), LCDR2 (YKSDSDK, SEQ ID NO: 51 ; or YKSDSD, SEQ ID NO: 52) and LCDR3 (MVWHKSAPI, SEQ ID NO: 45).

[0065] The present disclosure also provides an isolated delta-like ligand 4 (DLL4) antibody that binds to human DLL4 (SEQ ID NO: 63) and competes with the binding of an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 57 and a light chain variable region comprising the amino acid sequence SEQ ID NO: 62.

[0066] In some embodiments, which may be used or combined with any of the above or below mentioned disclosure and embodiments, the heavy chain variable region comprises HCDR1 (DYAMS, SEQ ID NO: 34), HCDR2 (TISGSGGDTFYADSVKG, SEQ ID NO: 37) and HCDR3 (DKNRGAYADAFDI, SEQ ID NO: 47); and/or wherein the light chain variable region comprises LCDR1 (TLRSDINVATYRIY, SEQ ID NO: 49), LCDR2 (YKSDSDK, SEQ ID NO: 51 ) and LCDR3 (MVWHKSAPI, SEQ ID NO: 45).

[0067] In some embodiments, which may be used or combined with any of the above or below mentioned disclosure and embodiments, the heavy chain variable region comprises HCDR1 (GFTFSD, SEQ ID NO: 35), HCDR2 (TISGSGGDTFYADSVKG, SEQ ID NO: 37) and HCDR3 (DKNRGAYADAFDI, SEQ ID NO: 47); and/or wherein the light chain variable region comprises LCDR1 (TLRSDINVATYRIY, SEQ ID NO: 49), LCDR2 (YKSDSDK, SEQ ID NO: 51 ) and LCDR3 (MVWHKSAPI, SEQ ID NO: 45). [0068] In some embodiments, which may be used or combined with any of the above or below mentioned disclosure and embodiments, the heavy chain variable region comprises HCDR1 (GFTFSDYA; SEQ ID NO: 36), HCDR2 (ISGSGGD, SEQ ID NO: 46) and HCDR3 (AKDKNRGAYADAFDI, SEQ ID NO: 48); and/or wherein the light chain variable region comprises LCDR1 (SDINVATYR, SEQ ID NO: 50), LCDR2 (YKSDSD, SEQ ID NO: 52) and LCDR3 (MVWHKSAPI, SEQ ID NO: 45).

[0069] In some embodiments, which may be used or combined with any of the above or below mentioned disclosure and embodiments, the isolated DLL4 antibody is humanized.

[0070] In some embodiments, which may be used or combined with any of the above or below mentioned disclosure and embodiments, the isolated DLL4 antibody is a full length antibody.

[0071 ] In some embodiments, which may be used or combined with any of the above or below mentioned disclosure and embodiments, the isolated DLL4 antibody is a human IgG.

[0072] In some embodiments, which may be used or combined with any of the above or below mentioned disclosure and embodiments, the isolated DLL4 antibody is an antibody fragment.

[0073] In some embodiments, which may be used or combined with any of the above or below mentioned disclosure and embodiments, the antibody fragment is a Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2 or SCA (single chain antibody).

[0074] In some embodiments, which may be used or combined with any of the above or below mentioned disclosure and embodiments, the isolated DLL4 antibody is bound to a detectable label.

[0075] The present disclosure also provides an immobilized antibody comprising an isolated DLL4 antibody as disclosed herein bound to a solid phase.

[0076] The present disclosure also provides a conjugate comprising an isolated DLL4 antibody as disclosed herein bound to a cytotoxic or non-cytotoxic agent.

[0077] The present disclosure also provides methods for determining the presence of a DLL4 protein by exposing a sample suspected of containing the DLL4 protein to an isolated DLL4 antibody as disclosed herein including, for example, a DLL4 antibody in any of the above or below mentioned disclosure and embodiments, and determining binding of the antibody to the sample.

[0078] The present disclosure also provides a kit comprising an isolated DLL4 antibody as disclosed herein including, for example, a DLL4 antibody in any of the above or below mentioned disclosure and embodiments, and instructions for using the DLL4 antibody to detect the DLL4 protein.

[0079] The present disclosure also provides an isolated nucleic acid sequence encoding an isolated DLL4 antibody as disclosed herein including, for example, a DLL4 antibody in any of the above or below mentioned disclosure and embodiments,.

[0080] The present disclosure also provides vectors comprising the nucleic acid sequences encoding a DLL4 antibody as disclosed herein including, for example, a DLL4 antibody in any of the above or below mentioned disclosure and embodiments,.

[0081 ] The present disclosure also provides host cells comprising a nucleic acid sequence or vector encoding a DLL4 antibody as disclosed herein including, for example, a DLL4 antibody in any of the above or below mentioned disclosure and embodiments,.

[0082] The present disclosure also provides processes of producing a DLL4 antibody by culturing a host cell comprising a nucleic acid sequence encoding a DLL4 antibody as disclosed herein including, for example, a DLL4 antibody in any of the above or below mentioned disclosure and embodiments, so that the nucleic acid sequence is expressed. In some embodiments, the process further comprises recovering the DLL4 antibody from the host cell culture.

[0083] The present disclosure also provides a composition comprising an isolated DLL4 antibody as disclosed herein including, for example, a DLL4 antibody in any of the above or below mentioned disclosure and embodiments, and one or more pharmaceutically acceptable carriers or diluents.

[0084] The present disclosure also provides methods for treating an DLL4- associated disease or disorder in a subject by administering to the subject a therapeutically effective amount of an isolated DLL4 antibody as disclosed herein including, for example, a DLL4 antibody in any of the above or below mentioned disclosure and embodiments.

[0085] In some embodiments, which may be used or combined with any of the above or below mentioned disclosure and embodiments, the disease or disorder is a tumor, a cancer, a cell proliferative disorder, a pathological condition associated with angiogenesis or non-neoplastic disorder.

[0086] In some embodiments, which may be used or combined with any of the above or below mentioned disclosure and embodiments, the methods may further comprise administering another agent such as a chemotherapeutic agent or another therapy before, after or simultaneously with the isolated DLL4 antibody.

[0087] The present disclosure provides methods and materials that relate to delta-like ligand 4 (DLL4), including antibodies to DLL4. [0088] The present disclosure also provides methods of modulating human T cell activation, cytokine production and/or differentiation, including with antibodies to DLL4.

[0089] The present disclosure provides methods of treating Th17 cell-mediated diseases or disorders in a human.

[0090] The present disclosure also provides methods for treating a Th17 cell- mediated disease or disorder comprising administering a therapeutically effective amount of a DLL4 antibody, including a fragment thereof to the human.

[0091 ] The present disclosure also provides methods of treating autoimmune diseases or disorders in a human by administering a therapeutically effective amount of a DLL4 antibody, including a fragment thereof to the human.

[0092] The present disclosure also provides a method of treating lupus in a human by administering a therapeutically effective amount of a DLL4 antibody, including a fragment thereof to the human.

[0093] The present disclosure also provides methods for reducing activation and/or differentiation of human Th17 cells comprising contacting human T cells with a DLL4 antibody, including a fragment thereof.

[0094] The present disclosure also provides methods for reducing cytokine production by human Th17 cells by contacting the human Th17 cells with a DLL4 antibody, including a fragment thereof.

[0095] The present disclosure also provides methods for modulating cytokine production by human Th17 cells by activating the Th17 cells in the presence of a DLL4 antibody, including a fragment thereof.

[0096] In some embodiments, which may be used or combined with any of the above or below mentioned disclosure and embodiments, the antibody fragment is a Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2 or SCA (single chain antibody).

[0097] In some embodiments, which may be used or combined with any of the above or below mentioned disclosure and embodiments, the antibody is humanized or a human antibody.

[0098] In some embodiments, which may be used or combined with any of the above or below mentioned disclosure and embodiments, the production of the cytokine is reduced. In some embodiments, the production of the cytokine is increased.

[0099] In some embodiments, which may be used or combined with any of the above or below mentioned disclosure and embodiments, the cytokine is IL-22, IL-17 or IFN-γ. In some embodiments, the cytokine is TNF-a. [00100] In any or all of the above or below disclosure (e.g., antibodies, uses, or methods) or embodiments utilizing an antibody to DLL4, any DLL4 antibody may be used including, for example, any of the above mentioned antibodies to DLL4.

[00101] In any or all of the above or below disclosure (e.g., antibodies, uses, or methods) or embodiments utilizing an antibody to DLL4, additionally or alternatively a DLL4 antagonist may be used.

BRIEF DESCRIPTION OF THE DRAWINGS

[00102] The foregoing summary, as well as the following detailed description of the disclosure, will be better understood when read in conjunction with the appended figures. For the purpose of illustrating the disclosure, shown in the figures are embodiments which are presently preferred. It should be understood, however, that the disclosure is not limited to the precise arrangements, examples and instrumentalities shown.

[00103] Figure 1 shows the 3.1 C1 heavy and light chain antibody variable region sequences with CDR regions determined by Kabat in bold italicized font text, CDR regions determined by Chothia underlined text, and CDR regions determined by IMGT shown in bracketed text (A); the 12.73 and 12.08 heavy and light chain antibody variable region sequences with CDR regions determined by Kabat in bold italicized font, CDR regions determined by Chothia underlined, and CDR regions determined by IMGT shown in bracketed text (B and C, respectively); and the 12.14 and 12.55 heavy and light chain antibody variable region sequences with CDR regions determined by Kabat in bold italicized font, CDR regions determined by Chothia underlined, and CDR regions determined by IMGT shown in bracketed text (D and E, respectively).

[00104] Figure 2 shows the BA1 (A) and BA2 (B) variable heavy and light chain antibody sequences with CDR regions determined by Kabat in bold italicized font text.

[00105] Figure 3 is a dendrogram showing seven bins from epitope binning assays produced from the hierarchical clustering analysis of Example 3. The table of distances for the various clusters identified in this analysis is shown on the right.

[00106] Figure 4 shows a graphical display of interactions between antibodies in epitope binning assays.

[00107] Figure 5 is a dendrogram showing eight bins from epitope binning assays produced from the hierarchical clustering analysis of Example 3. The table of distances for the various clusters identified in this analysis is shown on the right. MAb pairs that appear to share partial DLL4 binding epitopes ("1 " in the matrix) are also listed.

[00108] Figure 6: Naive (CD45RA+) human CD4 T cells were purified and activated with anti-CD3/anti-CD28 beads in the presence of the indicated cytokines and antibodies in wells previously coated with either DLL4 or PBS. IL-17A expression in conditioned media was measured on day 5 (A). Cells were activated as described in (A) and intracellular cytokine staining performed on day 5 (B). Naive CD4 T cells were labeled with CFSE and activated with immobilized DLL4-coated (thick) or PBS-coated (thick hatched) as described in A. As a control, a portion of CFSE labeled cells were not activated (thin). CFSE dilution profiles were assessed on day 5 of activation (C). Naive (CD45RA+) and memory/activated (CD45RO+) CD4 T cells from 8 individual donors were activated as described in A. IL-17A in conditioned media was measured on day 5. Each connected open and filled circle pair represents an individual donor activated with DLL4-coated (filled) or with PBS-coated (open) wells. Where indicated, fold change relative to PBS condition was calculated (D).

[00109] Figure 7: Naive human CD4 T cells were activated with anti-CD3/anti- CD28 beads in chemically-defined serum-free media (X-Vivo 20) and in the presence of the indicated cytokines and antibodies in wells previously coated with either DLL4 or PBS. IL-17A expression in conditioned media was measured on day 5.

[001 10] Figure 8: Naive human CD4 T cells were activated with anti-CD3/anti- CD28 beads in wells previously coated with DLL4 or PBS without any polarizing cytokines. IL-22, IFN-γ, and IL-4 was examined in conditioned media from day 5 of activation (A). Naive and memory/Ag experienced human CD4 T cells from 8 individual donors were activated with anti-CD3/anti-CD28 beads in the presence of the indicated cytokines and antibodies in wells coated with either DLL4 or PBS. Cytokine expression in conditioned media was measured on day 5. Each connected open and filled circle pair represents an individual donor activated with DLL4-coated (filled) or with PBS-coated (open) wells. Fold change was calculated by dividing values from DLL4-coated wells by PBS-control coated wells for (B) or dividing values from PBS-control wells by DLL4-coated wells for (C). NC indicates changes that were less than 1 .5 fold (B) and (C).

[001 1 1] Figure 9: Naive and memory/activated human CD4 T cells from 8 individual donors were activated with anti-CD3/anti-CD28 beads in the presence of the indicated cytokines and antibodies in wells coated with either DLL4 or PBS. IL-10 expression in conditioned media was measured on day 5 (A). Intracellular cytokine staining from cultures in (A) was performed on day 5 of activation (B).

[001 12] Figure 10: Recombinant DLL4, DIM , Jaggedl , and Jagged2 were immobilized onto wells overnight. A) HELA cells were transfected with a HES1 luciferase reporter construct. The next days, cells were plated onto wells coated with PBS, DLL4, DIM , Jaggedl , or Jagged2. After 24 hours, the amount of luciferase expressed in HELA cells was determined by Steady-Glo. B) Naive human CD4 T cells were activated with anti- CD3/anti-CD28 beads in the presence of TGF-β, I L-1 β, IL-6, and IL-23 in wells previously coated with the indicated protein. Cytokine expression in conditioned media was measured on day 5.

[001 13] Figure 1 1 : The expression of Notchl , Notch2, Notch3, and Notch4 was determined on naive CD4 T cells and on day 5 CD4 T cells activated with anti-CD3/anti- CD28 beads (A). Notchl Fc and Notch2 Fc were immobilized onto plates. Soluble DLL4- biotin at the indicated concentration was added and binding determined to each receptor determined (B). DLL4 antibodies were generated as described in Example 1. To check for neutralization properties, HELA cells were transfected with a HES1 luciferase reporter construct. After transfection, HELA cells were plated onto wells previously coated with recombinant DLL4 protein in the presence of DLL4 antibody at the indicated concentrations. After a 24 hour incubation, luciferase amounts were determined by SteadyGlo™. Percent activation was calculated by dividing the signal obtained for each antibody condition by maximum signal without DLL4 neutralization minus the minimum signal in PBS-coated wells (C). DLL4 antibody was tested for neutralization of DLL4 induced I L-17 production. Naive human CD4 T cells were activated with anti-CD3/anti- CD28 beads in the presence of TGF-β, I L- 1 β , IL-6, and IL-23 in wells previously coated with DLL4. DLL4 antibody or mlgG isotype control was used at 10 μg/ml (D). Notchl or Notch2 Fc protein were immobilized onto the surface of the well. Soluble DLL4-biotin at 200 ng/ml and DLL4 antibody or mlgG isotype control at the indicated concentrations were added to the wells containing Notchl or Notch2 coated proteins and incubated. After washing, DLL4 bound to Notchl or Notch2 was detected by streptavidin HRP (E).

[001 14] Figure 12: PBMCs were activated with 100 ng/ml LPS for 2 days and stained for DLL4 and CD14 (A). Expression of DLL4 was examined on CD83+ cells. Shaded represents IgG isotype control, Thick represents DLL4 expression (B). pDCs and mDCs were purified from whole blood and cultured with 100 ng/ml LPS for 2 days. DLL4 expression was examined on both cell types (C). CD1 c+ mDC1 cells were purified and activated with LPS for 2 days. Naive CD4 T cells were co-cultured with CD1 c+ mDC1 cells for 5 days with the indicated polarizing cytokines and antibodies. DLL4 or relevant isotype control was used at 10 μg/ml. Cytokine production was examined on day 7 (D). Intracellular cytokine staining for IL-22 and IL-17A in T cells activated with TGF-β, Pro, anti- IFN-γ, anti-IL-4 from C was performed (E). Neutralizing Anti-IL10 at 10 μg/ml was added in MLR cultures activated with in a similar fashion as in (D). DLL4 was not added to these cultures. Cytokine expression was analyzed on day 7 of activation (F). DETAILED DESCRI PTION

[001 15] The present disclosure provides antibodies to DLL4 and uses thereof. Such DLL4 antibodies may be useful for the treatment or prevention of disease states associated with expression and/or activity of DLL4 such as increased expression and/or activity or undesired expression and/or activity of DLL4. The DLL4 antibodies, as described herein, bind (e.g., specifically or selectively bind) an epitope of DLL4 that is not recognized by existing DLL4 binding molecules as described herein and exhibit surprising potency in assays of DLL4 binding and function. Such antibodies with unexpected epitope specificity, binding affinity and/or functional activity, including in cell-based assays, are useful for assays, procedures and administration to subjects, including for modulation of Notch receptor signaling. In some embodiments, the antibodies disclosed herein are used for inhibiting Notch activation. In some embodiments, the antibodies disclosed herein are used to treat a tumor, a cancer, and/or a cell proliferative disorder. In some embodiments, the antibodies disclosed herein are used to treat a pathological condition associated with angiogenesis. In some embodiments, the antibodies disclosed herein are used to treat a non-neoplastic disorder.

[001 16] The present disclosure also provides methods and materials that relate to delta-like ligand 4 (DLL4), including antibodies to DLL4, for example, methods of utilizing agents that bind to human DLL4, including agents that bind to and neutralize a DLL4 activity such as neutralizing antibodies to DLL4. Such methods may include using antibodies to DLL4 for modulation of human T cell activation, cytokine production and/or differentiation. Surprisingly, it has been found that DLL4 has a role in regulating human T cell function (e.g., human Th17 cells) and that antibodies to DLL4 may be effective in reducing IL-17, I L-22 and/or I L-10 production, including enhancing TN F-a production. Antibodies to DLL4 may be used for the treatment of Th17 cell-mediated diseases or disorders. Antibodies to DLL4 may be used to treat autoimmune and/or inflammatory diseases or disorders. Antibodies to DLL4 may be used for the treatment of lupus. In any or all of the methods and/or embodiments disclosed herein utilizing an antibody to DLL4, additionally or alternatively a DLL4 antagonist may be used.

[001 17] The present disclosure provides an isolated delta-like ligand 4 (DLL4) antibody, including an antibody fragment (e.g., a Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2 or SCA (single chain antibody)) comprising a heavy chain variable region and a light chain variable region, wherein: i.) the heavy chain variable region comprises HCDR1 (GYSWH, SEQ I D NO: 1 ; DHSITSG, SEQ I D NO: 2; or DHSITSGYS; SEQ I D NO: 3), HCDR2 (YI HYSGYTHYNPSLKS, SEQ I D NO: 4; or I HYSGY, SEQ I D NO: 5) and HCDR3 (SDYSANEGAMAY, SEQ ID NO: 6; or AGSDYSANEGAMAY, SEQ ID NO: 7); and/or wherein the light chain variable region comprises LCDR1 (RASESVDSFGNSFMH , SEQ ID NO: 8; or ESVDSFGNSF, SEQ ID NO: 9), LCDR2 (RTSNLQS, SEQ ID NO: 10; or R) and LCDR3 (QQSNEDPWT, SEQ ID NO: 1 1 ); ii.) the heavy chain variable region comprises HCDR1 (DEYWS, SEQ ID NO: 12; GGSFND, SEQ ID NO: 13; or GGSFNDEY; SEQ ID NO: 14), HCDR2 (EIHESGKTNYNPSLKP, SEQ ID NO: 15; or IHESGKT, SEQ ID NO: 16) and HCDR3 (LAYRDRWYGAFDV, SEQ ID NO: 17; or ARLAYRDRWYGAFDV, SEQ ID NO: 18); and/or wherein the light chain variable region comprises LCDR1 (QASHDITNYIN, SEQ ID NO: 19; or HDITNY, SEQ ID NO: 20), LCDR2 (HHTSKLQT, SEQ ID NO: 21 ; or H) and LCDR3 (QQFDNLLLT, SEQ ID NO: 22); iii.) the heavy chain variable region comprises HCDR1 (GYYMH, SEQ ID NO: 23; GYTFTG, SEQ ID NO: 24; or GYTFTGYY; SEQ ID NO: 25), HCDR2 (WINPNSGGTNYAQ, SEQ ID NO: 26; or INPNSGG, SEQ ID NO: 27) and HCDR3 (GAGVAAYDY, SEQ ID NO: 28; or ARGAGVAAYDY, SEQ ID NO: 29); and/or wherein the light chain variable region comprises LCDR1 (RASQSISSYLN, SEQ ID NO: 30; or QSISSY, SEQ ID NO: 31 ), LCDR2 (AASSLQS, SEQ ID NO: 32; or A) and LCDR3 (QQSYSTPVT, SEQ ID NO: 33); iv.) the heavy chain variable region comprises HCDR1 (DYAMS, SEQ ID NO: 34; GFTFSD, SEQ ID NO: 35; or GFTFSDYA; SEQ ID NO: 36), HCDR2 (TISGSGGDTFYADSVKG, SEQ ID NO: 37; or ISGSGGDT, SEQ ID NO: 38) and HCDR3 (DKNRGAYADAFD, SEQ ID NO: 39; or AKDKNRGAYADAFD, SEQ ID NO: 40); and/or wherein the light chain variable region comprises LCDR1 (TLRSDINVGTYRIY, SEQ ID NO: 41 ; or SDINVGTYR, SEQ ID NO: 42), LCDR2 (YKSDTDK, SEQ ID NO: 43; or YKSDTD, SEQ ID NO: 44) and LCDR3 (MVWHKSAPI, SEQ ID NO: 45); or v.) the heavy chain variable region comprises HCDR1 (DYAMS, SEQ ID NO: 34; GFTFSD, SEQ ID NO: 35; or GFTFSDYA; SEQ ID NO: 36), HCDR2 (TISGSGGDTFYADSVKG, SEQ ID NO: 37; or ISGSGGD, SEQ ID NO: 46) and HCDR3 (DKNRGAYADAFDI, SEQ ID NO: 47; or AKDKNRGAYADAFDI, SEQ ID NO: 48); and/or wherein the light chain variable region comprises LCDR1 (TLRSDINVATYRIY, SEQ ID NO: 49; or SDINVATYR, SEQ ID NO: 50), LCDR2 (YKSDSDK, SEQ ID NO: 51 ; or YKSDSD, SEQ ID NO: 52) and LCDR3 (MVWHKSAPI, SEQ ID NO: 45). In some embodiments, the antibody including, the antibody fragment, in part i.) above comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 53, the antibody in part ii.) comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 54, the antibody in part iii.) comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 55, the antibody in part iv.) comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 56, or the antibody in part v.) comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 57. In some embodiments, the antibody including, the antibody fragment, in part i.) above comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58, the antibody in part ii.) comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 59, the antibody in part iii.) comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 60, the antibody in part iv.) comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 61 , or the antibody in part v.) comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 62. In some embodiments, the antibody including, the antibody fragment, in part i.) above comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 53 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58, the antibody in part ii.) comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 54 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 59, the antibody in part iii.) comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 55 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 60, the antibody in part iv.) comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 56 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 61 , or the antibody in part v.) comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 57 and a light chain variable region comprising the amino acid sequence SEQ ID NO: 62.

[001 18] The present disclosure also provides an isolated delta-like ligand 4 (DLL4) antibody including, an antibody fragment (e.g., a Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2 or SCA (single chain antibody)), that binds to the same epitope that an antibody comprising a heavy chain variable region and a light chain variable region binds to, wherein: i.) the heavy chain variable region comprises HCDR1 (GYSWH, SEQ ID NO: 1 ; DHSITSG, SEQ ID NO: 2; or DHSITSGYS; SEQ ID NO: 3), HCDR2 (YIHYSGYTHYNPSLKS, SEQ ID NO: 4; or IHYSGY, SEQ ID NO: 5) and HCDR3 (S DYSAN EGAMAY, SEQ ID NO: 6; or AGSDYSANEGAMAY, SEQ ID NO: 7); and/or wherein the light chain variable region comprises LCDR1 (RASESVDSFGNSFMH , SEQ ID NO: 8; or ESVDSFGNSF, SEQ I D NO: 9), LCDR2 (RTSNLQS, SEQ ID NO: 10; or R) and LCDR3 (QQSNEDPWT, SEQ ID NO: 1 1 ); ii.) the heavy chain variable region comprises HCDR1 (DEYWS, SEQ ID NO: 12; GGSFND, SEQ ID NO: 13; or GGSFNDEY; SEQ ID NO: 14), HCDR2 (EIHESGKTNYNPSLKP, SEQ ID NO: 15; or IHESGKT, SEQ ID NO: 16) and HCDR3 (LAYRDRWYGAFDV, SEQ ID NO: 17; or ARLAYRDRWYGAFDV, SEQ ID NO: 18); and/or wherein the light chain variable region comprises LCDR1 (QASHDITNYIN, SEQ ID NO: 19; or HDITNY, SEQ ID NO: 20), LCDR2 (HHTSKLQT, SEQ ID NO: 21 ; or H) and LCDR3 (QQFDNLLLT, SEQ ID NO: 22); iii.) the heavy chain variable region comprises HCDR1 (GYYMH, SEQ ID NO: 23; GYTFTG, SEQ ID NO: 24; or GYTFTGYY; SEQ ID NO: 25), HCDR2 (WINPNSGGTNYAQ, SEQ ID NO: 26; or INPNSGG, SEQ ID NO: 27) and HCDR3 (GAGVAAYDY, SEQ ID NO: 28; or ARGAGVAAYDY, SEQ ID NO: 29); and/or wherein the light chain variable region comprises LCDR1 (RASQSISSYLN, SEQ ID NO: 30; or QSISSY, SEQ ID NO: 31 ), LCDR2 (AASSLQS, SEQ ID NO: 32; or A) and LCDR3 (QQSYSTPVT, SEQ ID NO: 33); iv.) the heavy chain variable region comprises HCDR1 (DYAMS, SEQ ID NO: 34; GFTFSD, SEQ ID NO: 35; or GFTFSDYA; SEQ ID NO: 36), HCDR2 (TISGSGGDTFYADSVKG, SEQ ID NO: 37; or ISGSGGDT, SEQ ID NO: 38) and HCDR3 (DKNRGAYADAFD, SEQ ID NO: 39; or AKDKNRGAYADAFD, SEQ ID NO: 40); and/or wherein the light chain variable region comprises LCDR1 (TLRSDINVGTYRIY, SEQ ID NO: 41 ; or SDINVGTYR, SEQ ID NO: 42), LCDR2 (YKSDTDK, SEQ ID NO: 43; or YKSDTD, SEQ ID NO: 44) and LCDR3 (MVWHKSAPI, SEQ ID NO: 45); or v.) the heavy chain variable region comprises HCDR1 (DYAMS, SEQ ID NO: 34; GFTFSD, SEQ ID NO: 35; or GFTFSDYA; SEQ ID NO: 36), HCDR2 (TISGSGGDTFYADSVKG, SEQ ID NO: 37; or ISGSGGD, SEQ ID NO: 46) and HCDR3 (DKNRGAYADAFDI, SEQ ID NO: 47; or AKDKNRGAYADAFDI, SEQ ID NO: 48); and/or wherein the light chain variable region comprises LCDR1 (TLRSDINVATYRIY, SEQ ID NO: 49; or SDINVATYR, SEQ ID NO: 50), LCDR2 (YKSDSDK, SEQ ID NO: 51 ; or YKSDSD, SEQ ID NO: 52) and LCDR3 (MVWHKSAPI, SEQ ID NO: 45).

[001 19] The present disclosure also provides an isolated delta-like ligand 4 (DLL4) antibody including, an antibody fragment (e.g., a Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2 or SCA (single chain antibody)), that binds to the same epitope that an antibody comprising: i.) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 53 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58 binds to, ii.) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 54 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 59 binds to, iii.) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 55 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 60 binds to, iv.) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 56 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 61 binds to, or v.) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 57 and a light chain variable region comprising the amino acid sequence SEQ ID NO: 62 binds to.

[00120] The present disclosure also provides isolated delta-like ligand 4 (DLL4) antibody including, an antibody fragment (e.g., a Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2 or SCA (single chain antibody)), that binds to human DLL4 (SEQ ID NO: 63) and competes with the binding of an antibody comprising a heavy chain variable region and a light chain variable region, wherein: i.) the heavy chain variable region comprises HCDR1 (GYSWH, SEQ ID NO: 1 ; DHSITSG, SEQ ID NO: 2; or DHSITSGYS; SEQ ID NO: 3), HCDR2 (YIHYSGYTHYNPSLKS, SEQ ID NO: 4; or IHYSGY, SEQ ID NO: 5) and HCDR3 (SDYSANEGAMAY, SEQ ID NO: 6; or AGSDYSANEGAMAY, SEQ ID NO: 7); and/or wherein the light chain variable region comprises LCDR1 (RASESVDSFGNSFMH , SEQ ID NO: 8; or ESVDSFGNSF, SEQ ID NO: 9), LCDR2 (RTSNLQS, SEQ ID NO: 10; or R) and LCDR3 (QQSNEDPWT, SEQ ID NO: 1 1 ); ii.) the heavy chain variable region comprises HCDR1 (DEYWS, SEQ ID NO: 12; GGSFND, SEQ ID NO: 13; or GGSFNDEY; SEQ ID NO: 14), HCDR2 (EIHESGKTNYNPSLKP, SEQ ID NO: 15; or IHESGKT, SEQ ID NO: 16) and HCDR3 (LAYRDRWYGAFDV, SEQ ID NO: 17; or ARLAYRDRWYGAFDV, SEQ ID NO: 18); and/or wherein the light chain variable region comprises LCDR1 (QASHDITNYIN, SEQ ID NO: 19; or HDITNY, SEQ ID NO: 20), LCDR2 (HHTSKLQT, SEQ ID NO: 21 ; or H) and LCDR3 (QQFDNLLLT, SEQ ID NO: 22); iii.) the heavy chain variable region comprises HCDR1 (GYYMH, SEQ ID NO: 23; GYTFTG, SEQ ID NO: 24; or GYTFTGYY; SEQ ID NO: 25), HCDR2 (WINPNSGGTNYAQ, SEQ ID NO: 26; or INPNSGG, SEQ ID NO: 27) and HCDR3 (GAGVAAYDY, SEQ ID NO: 28; or ARGAGVAAYDY, SEQ ID NO: 29); and/or wherein the light chain variable region comprises LCDR1 (RASQSISSYLN, SEQ ID NO: 30; or QSISSY, SEQ ID NO: 31 ), LCDR2 (AASSLQS, SEQ ID NO: 32; or A) and LCDR3 (QQSYSTPVT, SEQ ID NO: 33); iv.) the heavy chain variable region comprises HCDR1 (DYAMS, SEQ ID NO: 34; GFTFSD, SEQ ID NO: 35; or GFTFSDYA; SEQ ID NO: 36), HCDR2 (TISGSGGDTFYADSVKG, SEQ ID NO: 37; or ISGSGGDT, SEQ ID NO: 38) and HCDR3 (DKNRGAYADAFD, SEQ ID NO: 39; or AKDKNRGAYADAFD, SEQ ID NO: 40); and/or wherein the light chain variable region comprises LCDR1 (TLRSDINVGTYRIY, SEQ ID NO: 41 ; or SDINVGTYR, SEQ ID NO: 42), LCDR2 (YKSDTDK, SEQ ID NO: 43; or YKSDTD, SEQ ID NO: 44) and LCDR3 (MVWHKSAPI, SEQ ID NO: 45); or v.) the heavy chain variable region comprises HCDR1 (DYAMS, SEQ ID NO: 34; GFTFSD, SEQ ID NO: 35; or GFTFSDYA; SEQ ID NO: 36), HCDR2 (TISGSGGDTFYADSVKG, SEQ ID NO: 37; or ISGSGGD, SEQ ID NO: 46) and HCDR3 (DKNRGAYADAFDI, SEQ ID NO: 47; or AKDKNRGAYADAFDI, SEQ ID NO: 48); and/or wherein the light chain variable region comprises LCDR1 (TLRSDINVATYRIY, SEQ ID NO: 49; or SDINVATYR, SEQ ID NO: 50), LCDR2 (YKSDSDK, SEQ ID NO: 51 ; or YKSDSD, SEQ ID NO: 52) and LCDR3 (MVWHKSAPI, SEQ ID NO: 45).

[00121] The present disclosure also provides an isolated delta-like ligand 4 (DLL4) antibody including, an antibody fragment (e.g., a Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2 or SCA (single chain antibody)), that binds to human DLL4 (SEQ ID NO: 63) and competes with the binding of an antibody comprising: i.) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 53 and a light chain variable region comprising the amino acid sequence of SEQ I D NO: 58, ii.) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 54 and a light chain variable region comprising the amino acid sequence of SEQ I D NO: 59, iii.) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 55 and a light chain variable region comprising the amino acid sequence of SEQ I D NO: 60, iv.) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 56 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 61 , or v.) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 57 and a light chain variable region comprising the amino acid sequence SEQ I D NO: 62.

[00122] In another aspect, the DLL4 antibodies disclosed herein find utility as reagents for detection and/or isolation of DLL4, such as detection of DLL4 in a sample, including a biological sample such as various tissues and cell types.

[00123] In another aspect, the DLL4 antibodies disclosed herein find utility in diagnosis, prognosis, and/or treatment of an individual, including as related to a disease or disorder.

[00124] The disclosure further provides methods of making DLL4 antibodies, and polynucleotides encoding DLL4 antibodies.

Definitions

[00125] An "isolated" biological molecule, such as the various polypeptides, polynucleotides, and antibodies disclosed herein, refers to a biological molecule that has been identified and separated and/or recovered from at least one component of its natural environment.

[00126] "Antagonist" refers to any molecule that partially or fully blocks, inhibits, or neutralizes an activity (e.g., biological activity) of a polypeptide, such as a DLL4 polypeptide. When the polypeptide is a DLL4 polypeptide, the antagonist is a DLL4 antagonist. Also encompassed by "antagonist" are molecules that fully or partially inhibit the transcription or translation of mRNA encoding the polypeptide. Suitable antagonist molecules include, e.g., antagonist antibodies or antibody fragments; fragments or amino acid sequence variants of a native polypeptide; peptides; antisense oligonucleotides; small organic molecules; and nucleic acids that encode polypeptide antagonists or antagonist antibodies. Reference to "an" antagonist encompasses a single antagonist or a combination of two or more different antagonists. [00127] "Agonist" refers to any molecule that partially or fully mimics a biological activity of a polypeptide, such as a DLL4 polypeptide. Also encompassed by "agonist" are molecules that stimulate the transcription or translation of mRNA encoding the polypeptide. Suitable agonist molecules include, e.g., agonist antibodies or antibody fragments; a native polypeptide; fragments or amino acid sequence variants of a native polypeptide; peptides; antisense oligonucleotides; small organic molecules; and nucleic acids that encode polypeptides agonists or antibodies. Reference to "an" agonist encompasses a single agonist or a combination of two or more different agonists.

[00128] An "isolated" antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody will be purified (1 ) to greater than 95% by weight of antibody {e.g., as determined by the Lowry method), and preferably to more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence {e.g., by use of a spinning cup sequenator), or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions {e.g., using Coomassie™ blue or, preferably, silver stain). Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Similarly, isolated antibody includes the antibody in medium around recombinant cells. An isolated antibody may be prepared by at least one purification step.

[00129] An "isolated" nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the antibody nucleic acid. An isolated nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from the nucleic acid molecule as it exists in natural cells. However, an isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that express an antibody where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.

[00130] Variable domain residue numbering as in Kabat or amino acid position numbering as in Kabat, and variations thereof, refers to the numbering system used for heavy chain variable domains or light chain variable domains of the compilation of antibodies in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991 ). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert {e.g., residue 52a according to Kabat) after residue 52 of H2 and inserted residues {e.g., residues 82a, 82b, and 82c, etc according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a "standard" Kabat numbered sequence.

[00131] "Substantially similar," or "substantially the same", refers to a sufficiently high degree of similarity between two numeric values (generally one associated with an antibody disclosed herein and the other associated with a reference/comparator antibody) such that one of skill in the art would consider the difference between the two values to be of little or no biological and/or statistical significance within the context of the biological characteristic measured by said values {e.g., Kd values). The difference between said two values is preferably less than about 50%, preferably less than about 40%, preferably less than about 30%, preferably less than about 20%, preferably less than about 10% as a function of the value for the reference/comparator antibody.

[00132] "Binding affinity" generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule {e.g., an antibody) and its binding partner {e.g., an antigen). Unless indicated otherwise, "binding affinity" refers to intrinsic binding affinity which reflects a 1 :1 interaction between members of a binding pair {e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant. Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high- affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure.

[00133] An "on-rate" or "rate of association" or "association rate" or "kon" can be determined with a surface plasmon resonance technique such as Biacore {e.g., Biacore A100, Biacore™-2000, Biacore™-3000, Biacore, Inc., Piscataway, N.J.) carboxymethylated dextran biosensor chips (CM5, Biacore Inc.) and according to the supplier's instructions.

[00134] "Vector" refers 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 phage vector. 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, "recombinant vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. Accordingly, "plasmid" and "vector" may, at times, be used interchangeably as the plasmid is a commonly used form of vector.

[00135] "Gene" refers to a nucleic acid {e.g., DNA) sequence that comprises coding sequences necessary for the production of a polypeptide, precursor, or RNA {e.g., rRNA, tRNA). The polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or functional properties {e.g., enzymatic activity, ligand binding, signal transduction, immunogenicity, etc.) of the full- length or fragment are retained. The term also encompasses the coding region of a structural gene and the sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of about 1 kb or more on either end such that the gene corresponds to the length of the full-length mRNA. Sequences located 5' of the coding region and present on the mRNA are referred to as 5' non-translated sequences. Sequences located 3' or downstream of the coding region and present on the mRNA are referred to as 3' non- translated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene. A genomic form or clone of a gene contains the coding region interrupted with non- coding sequences termed "introns" or "intervening regions" or "intervening sequences." Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA); introns can contain regulatory elements such as enhancers. Introns are removed or "spliced out" from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide. In addition to containing introns, genomic forms of a gene can also include sequences located on both the 5' and 3' end of the sequences that are present on the RNA transcript. These sequences are referred to as "flanking" sequences or regions (these flanking sequences are located 5' or 3' to the non-translated sequences present on the mRNA transcript). The 5' flanking region can contain regulatory sequences such as promoters and enhancers that control or influence the transcription of the gene. The 3' flanking region can contain sequences that direct the termination of transcription, post transcriptional cleavage and polyadenylation.

[00136] "Polynucleotide," or "nucleic acid," as used interchangeably herein, refers to polymers of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase, or by a synthetic reaction. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after synthesis, such as by conjugation with a label. Other types of modifications include, for example, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages {e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages {e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example, proteins {e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators {e.g., acridine, psoralen, etc.), those containing chelators {e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages {e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide(s). Further, any of the hydroxyl groups ordinarily present in the sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid or semi-solid supports. The 5' and 3' terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups. Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2'-0-methyl-, 2'-0-allyl, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, alpha-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and a basic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(0)S("thioate"), P(S)S ("dithioate"), "(0)NR2 ("amidate"), P(0)R, P(0)OR', CO or CH2 ("formacetal"), in which each R or R' is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether (-0-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.

[00137] "Oligonucleotide" refers to short, generally single stranded, generally synthetic polynucleotides that are generally, but not necessarily, less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The description above for polynucleotides is equally and fully applicable to oligonucleotides.

[00138] "Stringent hybridization conditions" refer to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acids, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology— Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993). Generally, stringent conditions are selected to be about 5-10°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 times background hybridization. Exemplary stringent hybridization conditions can be as following: 50% formamide, 5xSSC, and 1 % SDS, incubating at 42° C, or, 5xSSC, 1 % SDS, incubating at 65° C, with wash in 0.2xSSC, and 0.1 % SDS at 65° C.

[00139] "Recombinant" when used with reference to a cell, nucleic acid, protein or vector indicates that the cell, nucleic acid, protein or vector has been modified by the introduction of a heterologous nucleic acid or protein, the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. For example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are overexpressed or otherwise abnormally expressed such as, for example, expressed as non-naturally occurring fragments or splice variants. By the term "recombinant nucleic acid" herein is meant nucleic acid, originally formed in vitro, in general, by the manipulation of nucleic acid, e.g., using polymerases and endonucleases, in a form not normally found in nature. In this manner, operably linkage of different sequences is achieved. Thus an isolated nucleic acid, in a linear form, or an expression vector formed in vitro by ligating DNA molecules that are not normally joined, are both considered recombinant for the purposes of this disclosure. It is understood that once a recombinant nucleic acid is made and introduced into a host cell or organism, it will replicate non-recombinantly, e.g., using the in vivo cellular machinery of the host cell rather than in vitro manipulations; however, such nucleic acids, once produced recombinantly, although subsequently replicated non-recombinantly, are still considered recombinant for the purposes disclosed herein. Similarly, a "recombinant protein" is a protein made using recombinant techniques, e.g., through the expression of a recombinant nucleic acid as depicted above.

[00140] "Percent (%) amino acid sequence identity" with respect to a peptide or polypeptide sequence refers to the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MegAlign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[00141] "Polypeptide," "peptide," "protein," and "protein fragment" may be used interchangeably to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[00142] "Amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, gamma- carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, e.g., an alpha carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs can have modified R groups {e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions similarly to a naturally occurring amino acid.

[00143] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. "Amino acid variants" refers to amino acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical or associated {e.g., naturally contiguous) sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode most proteins. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to another of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes silent variations of the nucleic acid. One of skill will recognize that in certain contexts each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, silent variations of a nucleic acid which encodes a polypeptide is implicit in a described sequence with respect to the expression product, but not with respect to actual probe sequences. As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" including where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles disclosed herein. Typically conservative substitutions include: 1 ) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).

[00144] "DLL4" (interchangeably termed "delta-like ligand 4") refers, unless specifically or contextually indicated otherwise, to any native or variant (whether native or synthetic) DLL4 polypeptide, including a human DLL4 sequence (see, e.g., SEQ ID NO: 63). The term "native sequence" specifically encompasses naturally occurring truncated or secreted forms {e.g., an extracellular domain sequence), naturally occurring variant forms {e.g., alternatively spliced forms) and naturally-occurring allelic variants. The term "wild type DLL4" generally refers to a polypeptide comprising the amino acid sequence of a naturally occurring DLL4 protein. The term "wild type DLL4 sequence" generally refers to an amino acid sequence found in a naturally occurring DLL4.

[00145] A "DLL4 antibody" or "anti-DLL4 antibody" or "antibody to DLL4", used interchangeably herein, refers to an antibody that binds (e.g., selectively binds or specifically binds) any native or variant (whether native or synthetic) DLL4 polypeptide, for example, a human DLL4 sequence {see, e.g., SEQ ID NO: 63).

[00146] "Notch receptor" (interchangeably termed "Notch") refers, unless specifically or contextually indicated otherwise, to any native or variant (whether native or synthetic) Notch receptor polypeptide. Humans have four Notch receptors (Notchl , Notch 2, Notch3, and Notch4). The term Notch receptor may include any one of or all four human Notch receptors. The term "native sequence" specifically encompasses naturally occurring truncated or secreted forms {e.g., an extracellular domain sequence), naturally occurring variant forms {e.g., alternatively spliced forms) and naturally-occurring allelic variants. The term "wild type Notch receptor" generally refers to a polypeptide comprising the amino acid sequence of a naturally occurring Notch receptor protein. The term "wild type Notch receptor sequence" generally refers to an amino acid sequence found in a naturally occurring Notch receptor.

[00147] "Antibodies" (Abs) and "immunoglobulins" (Igs) are glycoproteins having similar structural characteristics. While antibodies may exhibit binding specificity to a specific antigen, immunoglobulins may include both antibodies and other antibody-like molecules which generally lack antigen specificity. Polypeptides of the latter kind are, for example, produced at low levels by the lymph system and at increased levels by myelomas.

[00148] "Antibody" and "immunoglobulin" are used interchangeably in the broadest sense and include monoclonal antibodies {e.g., full length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies {e.g., bispecific antibodies so long as they exhibit the desired biological activity) and may also include certain antibody fragments (as described in greater detail herein). An antibody can be human, humanized and/or affinity matured.

[00149] "Variable" refers to the fact that certain portions of the variable domains (also referred to as variable regions) differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions (HVRs) both in the light-chain and the heavy- chain variable domains. CDRs include those specified as Kabat, Chothia, and IMGT as shown herein within the variable region sequences. The more highly conserved portions of variable domains are called the framework (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a β-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the β-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991 )). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody- dependent cellular toxicity.

[00150] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-combining sites and is still capable of cross- linking antigen.

[00151] "Fv" refers to an antibody fragment which contains an antigen-recognition and antigen-binding site. In a two-chain Fv species, this region consists of a dimer of one heavy and one light chain variable domain in tight, non-covalent association. In a single chain Fv (scFv) species, one heavy chain and one light chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a "dimeric" structure analogous to that in a two-chain Fv (scFv) species. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[00152] The Fab fragment also contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[00153] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

[00154] Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., Igd, lgG2, lgG3, lgG4, IgA^ and lgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[00155] "Antibody fragments" comprise only a portion of an intact antibody, wherein the portion preferably retains at least one, preferably most or all, of the functions normally associated with that portion when present in an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. In one embodiment, an antibody fragment comprises an antigen binding site of the intact antibody and thus retains the ability to bind antigen. In another embodiment, an antibody fragment, for example one that comprises the Fc region, retains at least one of the biological functions normally associated with the Fc region when present in an intact antibody, such as FcRn binding, antibody half life modulation, ADCC function and complement binding. In one embodiment, an antibody fragment is a monovalent antibody that has an in vivo half life substantially similar to an intact antibody. For example, such an antibody fragment may comprise on antigen binding arm linked to an Fc sequence capable of conferring in vivo stability to the fragment.

[00156] "Hypervariable region", "HVR", or "HV", as well as "complementary determing region" or "CDR", may refer to the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops. Generally, antibodies comprise six hypervariable or CDR regions; three in the VH (H1 , H2, H3), and three in the VL (L1 , L2, L3). A number of hypervariable region or CDR delineations are in use and are encompassed herein. The Kabat Complementarity Determining Regions (Kabat CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991 )). Chothia refers instead to the location of the structural loops (Chothia and Lesk, J. Mol. Biol. 196:901 -917 (1987)). The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, (Chothia "CDRs") and are used by Oxford Molecular's AbM antibody modeling software. The "contact" hypervariable regions are based on an analysis of the available complex crystal structures. The residues from each of these hypervariable regions are noted below. (See also, for example, Figure 1 and bold, italicized text for Kabat CDRs and underlined text for Chothia CDRs for 12.3 ICI antibody).

Loop Kabat AbM Chothia Contact

L1 L24-L34 L24-L34 L26-L32 L30-L36

L2 L50-L56 L50-L56 L50-L52 L46-L55

L3 L89-L97 L89-L97 L91-L96 L89-L96

H1 H31-H35B H26-H35B H26-H32 H30-H35B

(Kabat Numbering)

H1 H31-H35 H26-H35 H26-H32 H30-H35

(Chothia Numbering)

H2 H50-H65 H50-H58 H53-H55 H47-H58

H3 H95-H102 H95-H102 H96-H101 H93-H101 IMGT referes to the international ImMunoGeneTics Information System, as described by Lefrace et al., Nucl. Acids, Res. 37; D1006-D1012 (2009), including for example, IMGT designated CDRs for antibodies (see also, for example, Figure 1 and bracketed text for 12.3 1 C1 antibody).

[00157] Hypervariable regions may comprise "extended hypervariable regions" as follows: 24-36 or 24-34 (L1 ), 46-56 or 50-56 (L2) and 89-97 (L3) in the VL and 26-35 (H 1 ), 50-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3) in the VH. The variable domain residues are numbered according to Kabat et al., Supra for each of these definitions.

[00158] "Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.

[00159] "Monoclonal antibody" refers to an antibody from a population of substantially homogeneous antibodies, that is, for example, the individual antibodies comprising the population are identical and/or bind the same epitope(s), except for possible variants that may arise during production of the monoclonal antibody, such variants generally being present in minor amounts. Such monoclonal antibody typically includes an antibody comprising a polypeptide sequence that binds a target, wherein the target-binding polypeptide sequence was obtained by a process that includes the selection of a single target binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones or recombinant DNA clones. It should be understood that the selected target binding sequence can be further altered, for example, to improve affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence is also a monoclonal antibody of this disclosure. In contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants {e.g., epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. In addition to their specificity, the monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present disclosure may be made by a variety of techniques, including, for example, the hybridoma method {e.g., Kohler et al., Nature, 256:495 (1975); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 , (Elsevier, N.Y., 1981 )), recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567), phage display technologies (see, e.g., Clackson et al., Nature, 352:624-628 (1991 ); Marks et al., J. Mol. Biol., 222:581-597 (1991 ); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Nat. Acad. Sci. USA 101 (34):12467-12472 (2004); and Lee et al. J. Immunol. Methods 284(1 -2):1 19-132 (2004), and technologies for producing human or human-like antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741 ; Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in Immune, 7:33 (1993); U.S. Patent Nos. 5,545,806; 5,569,825; 5,591 ,669; 5,545,807; WO 1997/17852; U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661 ,016; Marks et al., Bio/Technology, 10: 779-783 (1992); Lonberg et al., Nature, 368: 856-859 (1994); Morrison, Nature, 368: 812-813 (1994); Fishwild et al., Nature Biotechnology, 14: 845-851 (1996); Neuberger, Nature Biotechnology, 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol., 13: 65-93 (1995)).

[00160] "Humanized" or "Human engineered" forms of non-human {e.g., murine) antibodies are chimeric antibodies that contain amino acids represented in human immunoglobulin sequences, including, for example, wherein minimal sequence is derived from non-human immunoglobulin. For example, humanized antibodies may be human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in non-human {e.g., rodent) antibodies. Alternatively, humanized or human engineered antibodies may be non-human {e.g., rodent) antibodies in which some residues are substituted by residues from analogious sites in human antibodies (see, e.g., U.S. Patent No. 5,766,886). Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications may be made to further refine antibody performance. Humanized antibodies include human engineered antibodies, for example, as described by U.S. Patent No. 5,766,886, including methods for preparing modified antibody variable domains. A humanized antibody may comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. A humanized antibody optionally may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321 :522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the following review articles and references cited therein: Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1 : 105- 1 15 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994).

[00161] "Hybrid antibodies" are immunoglobulin molecules in which pairs of heavy and light chains from antibodies with different antigenic determinant regions are assembled together so that two different epitopes or two different antigens can be recognized and bound by the resulting tetramer.

[00162] "Chimeric" antibodies (immunoglobulins) have a portion of the heavy and/or light chain identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see e.g., Morrison et al., Proc. Natl. Acad. Sci. USA 81 :6851 - 6855 (1984)). Humanized antibody refers to a subset of chimeric antibodies.

[00163] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 1 13, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[00164] An "antigen" is a predetermined antigen to which an antibody can selectively bind. The target antigen may be polypeptide, carbohydrate, nucleic acid, lipid, hapten or other naturally occurring or synthetic compound. Preferably, the target antigen is a polypeptide.

[00165] "Epitope" or "antigenic determinant", used interchangeably herein, refer to that portion of an antigen capable of being recognized and specifically bound by a particular antibody. When the antigen is a polypeptide, epitopes can be formed both from contiguous amino acids and noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon protein denaturing, whereas epitopes formed by tertiary folding are typically lost upon protein denaturing. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Antibodies may bind to the same or a different epitope on an antigen, such as DLL4. Antibodies may be characterized in different epitope bins. Whether an antibody binds to the same or different epitope as another antibody {e.g., a reference antibody or benchmark antibody) may be determined by competition between antibodies in assays {e.g., competitive binding assays).

[00166] Competition between antibodies may be determined by an assay in which the immunoglobulin under test inhibits specific binding of a reference antibody to a common antigen. Numerous types of competitive binding assays are known, for example: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay or enzyme-linked immunosorbent assay (EIA or ELISA), sandwich competition assay including an ELISA assay (see Stahli et al., Methods in Enzymology 9:242-253 (1983)); solid phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614-3619 (1986)); solid phase direct labeled assay, solid phase direct labeled sandwich assay (see Harlow and Lane, "Antibodies, A Laboratory Manual," Cold Spring Harbor Press (1988)); solid phase direct label RIA using 1-125 label (see Morel et al., Molec. Immunol. 25(1 ):7-15 (1988)); solid phase direct biotin-avidin EIA (Cheung et al., Virology 176:546-552 (1990)); and direct labeled RIA (Moldenhauer et al., Scand. J. Immunol., 32:77-82 (1990)). Competition binding assays may be performed using Surface Plasmon Resonance (SPR), for example, with a Biacore® instrument for kinetic analysis of binding interactions. In such an assay, a DLL4 antibody of unknown epitope specificity may be evaluated for its ability to compete for binding against a comparator antibody {e.g., a BA1 or BA2 antibody as described herein). An assay may involve the use of purified antigen bound to a solid surface or cells bearing either of these, an unlabeled test immunoglobulin and a labeled reference immunoglobulin. Competitive inhibition may be measured by determining the amount of label bound to the solid surface or cells in the presence of the test immunoglobulin. Usually the test immunoglobulin is present in excess. An assay (competing antibodies) may include antibodies binding to the same epitope as the reference antibody and antibodies binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference antibody for steric hindrance to occur. Usually, when a competing antibody is present in excess, it will inhibit specific binding of a reference antibody to a common antigen by at least 50%, or at least about 70%, or at least about 80%, or least about 90%, or at least about 95%, or at least about 99% or about 100% for a competitor antibody.

[00167] That an antibody "selectively binds" or "specifically binds" means that the antibody reacts or associates more frequently, more rapidly, with greater duration, with greater affinity, or with some combination of the above to an antigen or an epitope than with alternative substances, including unrelated proteins. "Selectively binds" or "specifically binds" may mean, for example, that an antibody binds to a protein with a KD of at least about 0.1 mM, or at least about 1 μΜ or at least about 0.1 μΜ or better, or at least about 0.01 μΜ or better. Because of the sequence identity between homologous proteins in different species, specific binding can include an antibody that recognizes a given antigen in more than one species.

[00168] "Non-specific binding" and "background binding" when used in reference to the interaction of an antibody and a protein or peptide refer to an interaction that is not dependent on the presence of a particular structure {e.g., the antibody is binding to proteins in general rather that a particular structure such as an epitope).

[00169] "Diabodies" refer to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light- chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen- binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/1 1 161 ; and Hollinger et. al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[00170] A "human antibody" refers to one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non- human antigen-binding residues.

[00171] An "affinity matured" antibody refers to one with one or more alterations in one or more CDRs thereof which result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess those alteration(s). Preferred affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies are produced by procedures known in the art. Marks et al., Bio/Technology 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):3310-9 (1995); and Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

[00172] Antibody "effector functions" refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity; Fc receptor binding; antibody- dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation.

[00173] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs) present on certain cytotoxic cells {e.g., Natural Killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. The antibodies "arm" the cytotoxic cells and are absolutely required for such killing. The primary cells for mediating ADCC, NK cells, express FCYRI I I only, whereas monocytes express FcyRI, FcyRII and FCYRI I I . FCR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991 ). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al. Proc. Natl. Acad. Sci. USA 95:652-656 (1998).

[00174] "Human effector cells" are leukocytes which express one or more FcRs and perform effector functions. Preferably, the cells express at least FcyRIII and perform ADCC effector function. Examples of human leukocytes which mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK cells being preferred. The effector cells may be isolated from a native source, e.g., from blood.

[00175] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. The preferred FcR is a native sequence human FcR. Moreover, a preferred FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the FcyRI, FcyRII, and FcyRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcyRII receptors include FcyRIIA (an "activating receptor") and FcyRI IB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcyRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain, (see review M. in Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991 ); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term "FcR" herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 1 17:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and regulates homeostasis of immunoglobulins. For example, antibody variants with improved or diminished binding to FcRs have been described (see, e.g., Shields et al. J. Biol. Chem. 9(2): 6591-6604 (2001 )).

[00176] Methods of measuring binding to FcRn are known (see, e.g., Ghetie 1997, Hinton 2004). Binding to human FcRn in vivo and serum half life of human FcRn high affinity binding polypeptides can be assayed, e.g., in transgenic mice or transfected human cell lines expressing human FcRn, or in primates administered with the Fc variant polypeptides.

[00177] "Complement dependent cytotoxicity" or "CDC" refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (Clq) to antibodies (of the appropriate subclass) which are bound to their cognate antigen. To assess complement activation, a CDC assay, for example, as described in Gazzano- Santoro et al., J. Immunol. Methods 202:163 (1996), may be performed.

[00178] Polypeptide variants with altered Fc region amino acid sequences and increased or decreased Clq binding capability have been described {e.g., see, also, Idusogie et al. J. Immunol. 164: 4178-4184 (2000)).

[00179] "Fc region-comprising polypeptide" refers to a polypeptide, such as an antibody or immunoadhesin (see definitions below), which comprises an Fc region. The C- terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during purification of the polypeptide or by recombinant engineering the nucleic acid encoding the polypeptide.

[00180] "Blocking" antibody or an "antagonist" antibody refers to one which inhibits or reduces biological activity of the antigen it binds. Preferred blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.

[00181] "Agonist" antibody refers to an antibody which mimics {e.g., partially or fully) at least one of the functional activities of a polypeptide of interest.

[00182] "Acceptor human framework" refers to a framework comprising the amino acid sequence of a VL or VH framework derived from a human immunoglobulin framework, or from a human consensus framework. An acceptor human framework "derived from" a human immunoglobulin framework or human consensus framework may comprise the same amino acid sequence thereof, or may contain pre-existing amino acid sequence changes. Where pre-existing amino acid changes are present, preferably no more than 5 and preferably 4 or less, or 3 or less, pre-existing amino acid changes are present.

[00183] "Human consensus framework" refers to a framework which represents the most commonly occurring amino acid residue in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., supra.

[00184] "Disorder" or "disease" refers to any condition that would benefit from treatment with a substance/molecule (e.g., a DLL4 antibody including, for example, any DLL4 antibody as dislosed herein) or method disclosed herein. This includes chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question. For example, a disorder or disease may be a DLL4- associated disorder or disease. Non-limiting examples of disorders or diseases include cancers (e.g., malignant and benign tumors such as carcinoma, blastoma, and sarcoma) and autoimmune disorders or diseases. [00185] "Cell proliferative disorder" and "proliferative disorder" refer to disorders that are associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.

[00186] "Tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer", "cancerous", "cell proliferative disorder", "proliferative disorder" and "tumor" are not mutually exclusive as referred to herein.

[00187] "Cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation. Examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, gastric cancer, melanoma, and various types of head and neck cancer. Dysregulation of angiogenesis can lead to many disorders that can be treated by compositions and methods disclosed herein. These disorders include both non-neoplastic and neoplastic conditions. Neoplastics include but are not limited those described above. Non-neoplastic disorders include but are not limited to undesired or aberrant hypertrophy, arthritis, rheumatoid arthritis (RA), psoriasis, psoriatic plaques, sarcoidosis, atherosclerosis, atherosclerotic plaques, diabetic and other proliferative retinopathies including retinopathy of prematurity, retrolental fibroplasia, neovascular glaucoma, age-related macular degeneration, diabetic macular edema, corneal neovascularization, corneal graft neovascularization, corneal graft rejection, retinal/choroidal neovascularization, neovascularization of the angle (rubeosis), ocular neovascular disease, vascular restenosis, arteriovenous malformations (AVM), meningioma, hemangioma, angiofibroma, thyroid hyperplasias (including Grave's disease), corneal and other tissue transplantation, chronic inflammation, lung inflammation, acute lung injury/ARDS, sepsis, primary pulmonary hypertension, malignant pulmonary effusions, cerebral edema {e.g., associated with acute stroke/closed head injury/trauma), synovial inflammation, pannus formation in RA, myositis ossificans, hypertropic bone formation, osteoarthritis (OA), refractory ascites, polycystic ovarian disease, endometriosis, 3rd spacing of fluid diseases (pancreatitis, compartment syndrome, burns, bowel disease), uterine fibroids, premature labor, chronic inflammation such as Inflammatory Bowel Disease (IBD) {e.g., Crohn's disease and ulcerative colitis), renal allograft rejection, nephrotic syndrome, undesired or aberrant tissue mass growth (non-cancer), hemophilic joints, hypertrophic scars, inhibition of hair growth, Osier-Weber syndrome, pyogenic granuloma retrolental fibroplasias, scleroderma, trachoma, vascular adhesions, synovitis, dermatitis, preeclampsia, ascites, pericardial effusion (such as that associated with pericarditis), and pleural effusion.

[00188] "Metastasis" refers to the process by which a cancer spreads or transfers from the site of origin to other regions of the body with the development of a similar cancerous lesion at the new location. A "metastatic" or "metastasizing" cell is one that loses adhesive contacts with neighboring cells and migrates via the bloodstream or lymph from the primary site of disease to invade neighboring body structures.

[00189] "Treatment" refers to clinical intervention in an attempt to alter the natural course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, antibodies disclosed herein are used to delay development of a disease or disorder.

[00190] "Individual" {e.g., a "subject") refers to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, farm animals (such as cows), sport animals, pets (such as cats, dogs and horses), primates, mice and rats.

[00191] "Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, rodents {e.g., mice and rats), and monkeys; domestic and farm animals; and zoo, sports, laboratory, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. In some embodiments, the mammal is selected from a human, rodent, or monkey.

[00192] "Effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[00193] "Therapeutically effective amount" of a substance/molecule disclosed herein, or agonist or antagonist, including an antibody, may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance/molecule, agonist or antagonist to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the substance/molecule, agonist or antagonist 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 but not necessarily, 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.

[00194] "Pharmaceutically acceptable" refers to approved or approvable by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans.

[00195] "Pharmaceutically acceptable salt" refers to a salt of a compound that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound.

[00196] "Carriers" refer to pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

[00197] "Pharmaceutically acceptable excipient, carrier or adjuvant" refers to an excipient, carrier or adjuvant that can be administered to a subject, together with at least one antibody of the present disclosure, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the compound.

[00198] "Pharmaceutically acceptable vehicle" refers to a diluent, adjuvant, excipient, or carrier with which at least one antibody of the present disclosure is administered.

[00199] "Providing a diagnosis" or "diagnostic information" refers to any information, including for example the presence of human Th17 cells, that is useful in determining whether a patient has a disease or condition and/or in classifying the disease or condition into a phenotypic category or any category having significance with regards to the prognosis of or likely response to treatment (either treatment in general or any particular treatment) of the disease or condition. Similarly, diagnosis refers to providing any type of diagnostic information, including, but not limited to, whether a subject is likely to have a condition (such as a human Th17 cell-mediated disease or disorder), information related to the nature or classification of the condition, information related to prognosis and/or information useful in selecting an appropriate treatment. Selection of treatment can include the choice of a particular agent (such as an antibody to DLL4) or other treatment modality or a choice about whether to withhold or deliver therapy.

[00200] "Providing a prognosis", "prognostic information", or "predictive information" refer to providing information, including for example the presence of human Th17 cells, regarding the impact of the presence of the human Th17 cells {e.g., as determined by the diagnostic methods of the present disclosure) on an individual's future health.

[00201] "Treating" or "treatment" or "to treat" or "alleviating" or "to alleviate" refer to both 1 ) therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder and 2) prophylactic or preventative measures that prevent and/or slow the development of a targeted pathologic condition or disorder. Thus those in need of treatment include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented.

[00202] "Cytotoxic agent" refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes {e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32 and radioactive isotopes of Lu), chemotherapeutic agents e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and fragments thereof such as nucleolytic enzymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof, and the various antitumor or anticancer agents disclosed below. Other cytotoxic agents are described below. A tumoricidal agent causes destruction of tumor cells.

[00203] "Chemotherapeutic agent" refers to a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan (HYCAMTIN®), CPT-1 1 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1 -TM1 ); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics {e.g., calicheamicin, especially calicheamicin gammal l and calicheamicin omegaH (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxoids, e.g., TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™. Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, III.), and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine (VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); oxaliplatin; leucovovin; vinorelbine (NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine (XELODA®); pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU and leucovovin.

[00204] Also included in this definition are anti-hormonal agents that act to regulate, reduce, block, or inhibit the effects of hormones that can promote the growth of cancer, and are often in the form of systemic, or whole-body treatment. They may be hormones themselves. Examples include anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX® tamoxifen), EVISTA® raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY 1 17018, onapristone, and FARESTON® toremifene; anti-progesterones; estrogen receptor down-regulators (ERDs); agents that function to suppress or shut down the ovaries, for example, leutinizing hormone-releasing hormone (LHRH) agonists such as LUPRON® and ELIGARD® leuprolide acetate, goserelin acetate, buserelin acetate and tripterelin; other anti-androgens such as flutamide, nilutamide and bicalutamide; and aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole, RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole. In addition, such definition of chemotherapeutic agents includes bisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®), DIDROCAL® etidronate, NE-58095, ZOMETAP zoledronic acid/zoledronate, FOSAMAX® alendronate, AREDIA® pamidronate, SKELID® tiludronate, or ACTONEL® risedronate; as well as troxacitabine (a 1 ,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit expression of genes in signaling pathways implicated in abherant cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE vaccine and gene therapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; LURTOTECAN® topoisomerase 1 inhibitor; ABARELIX® rmRH; lapatinib ditosylate (an ErbB-2 and EGFR dual tyrosine kinase small-molecule inhibitor also known as GW572016); and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[00205] "Growth inhibitory agent" refers to a compound or composition which inhibits growth of a cell either in vitro or in vivo. Thus, the growth inhibitory agent may be one which significantly reduces the percentage of cells in S phase. Examples of growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase), such as agents that induce G1 arrest and M-phase arrest. Classical M-phase blockers include the vincas (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Those agents that arrest G1 also spill over into S-phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found in The Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1 , entitled "Cell cycle regulation, oncogenes, and antineoplastic drugs" by Murakami et al. (WB Saunders: Philadelphia, 1995), especially p. 13. The taxanes (paclitaxel and docetaxel) are anticancer drugs both derived from the yew tree. Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the European yew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, which results in the inhibition of mitosis in cells.

[00206] "Doxorubicin" refers to an anthracycline antibiotic. The full chemical name of doxorubicin is (8S-cis)-10-[(3-amino-2,3,6-trideoxy-a-L-lyxo-hexapyranosyl)oxy]-7,- 8,9,10-tetrahydro-6,8, 1-trihydroxy-8-(hydroxyacetyl)-1 -methoxy-5, 12-naphth- acenedione.

[00207] "Anti-neoplastic composition" refers to a composition useful in treating cancer comprising at least one active therapeutic agent, e.g., "anti-cancer agent". Examples of therapeutic agents (anti-cancer agents, also termed "anti-neoplastic agent" herein) include, but are limited to, e.g., chemotherapeutic agents, growth inhibitory agents, cytotoxic agents, agents used in radiation therapy, anti-angiogenesis agents, apoptotic agents, anti-tubulin agents, toxins, and other-agents to treat cancer, e.g., anti-VEGF neutralizing antibody, VEGF antagonist, anti-HER-2, anti-CD20, an epidermal growth factor receptor (EGFR) antagonist {e.g., a tyrosine kinase inhibitor), HER1/EGFR inhibitor, erlotinib, a COX-2 inhibitor {e.g., celecoxib), interferons, cytokines, antagonists {e.g., neutralizing antibodies) that bind to one or more of the ErbB2, ErbB3, ErbB4, or VEGF receptor(s), inhibitors for receptor tyrosine kinases for platet-derived growth factor (PDGF) and/or stem cell factor (SCF) {e.g., imatinib mesylate (Gleevec® Novartis)), TRAIL/Apo2L, and other bioactive and organic chemical agents, etc.

[00208] "Prodrug" refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active parent form. See, e.g., Wilman, "Prodrugs in Cancer Chemotherapy" Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) and Stella et al., "Prodrugs: A Chemical Approach to Targeted Drug Delivery," Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267, Humana Press (1985). The prodrugs of this disclosure include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, beta-lactam-containing prodrugs, optionally substituted phenoxyacetamide- containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5- fluorocytosine and other 5-fluorouridine prodrugs which can be converted into the more active cytotoxic free drug. Examples of cytotoxic drugs that can be derivatized into a prodrug form include, but are not limited to, those chemotherapeutic agents described above.

[00209] "Anti-angiogenesis agent" or "angiogenesis inhibitor" refers to a small molecular weight substance, a polynucleotide (including, e.g., an inhibitory RNA (RNAi or siRNA)), a polypeptide, an isolated protein, a recombinant protein, an antibody {e.g., DLL4 antibody), or conjugates or fusion proteins thereof, that inhibits angiogenesis, vasculogenesis, or undesirable vascular permeability, either directly or indirectly. For example, an anti-angiogenesis agent is an antibody or other antagonist to an angiogenic agent as defined above, e.g., antibodies to VEGF, antibodies to VEGF receptors, soluble VEGF receptor fragments or small molecules that block VEGF receptor signaling {e.g., PTK787/ZK2284, SU6668, SUTENT®/SU1 1248 (sunitinib malate), AMG706, or those described in, e.g., international patent application WO 2004/1 13304). Anti-angiogensis agents also include native angiogenesis inhibitors, e.g., angiostatin, endostatin, etc. See, e.g., Klagsbrun and D'Amore, Annu. Rev. Physiol., 53:217-39 (1991 ); Streit and Detmar, Oncogene, 22:3172-3179 (2003) {e.g., Table 3 listing anti-angiogenic therapy in malignant melanoma); Ferrara & Alitalo, Nature Medicine 5(12): 1359-1364 (1999); Tonini et al., Oncogene, 22:6549-6556 (2003) {e.g., Table 2 listing antiangiogenic factors); and, Sato Int. J Clin. Oncol., 8:200-206 (2003) {e.g., Table 1 lists Anti-angiogenic agents used in clinical trials). [00210] "Autoimmune disease" or "autoimmune disorder" or "autoimmunity" refers to any condition in which a humoral or cell-mediated immune response is mounted against a body's own tissue.

[0021 1] "Th17 cell-mediated disease or disorder" refers to any disease or disorder that is caused by, maintained, or exacerbated by human Th17 cells.

[00212] "Inflammation" refers to the accumulation of leukocytes and the dilation of blood vessels at a site of injury or infection, typically causing pain, swelling, and redness.

[00213] "Chronic inflammation" refers to inflammation in which the cause of the inflammation persists and is difficult or impossible to remove.

[00214] "Autoimmune inflammation" refers to inflammation associated with an autoimmune disorder.

[00215] "Lupus" refers to an autoimmune disease and/or an inflammatory condition associated with an autoimmune disease or condition that manifests as a disease of the skin, heart, lungs, kidney, joints and/or nervous system.

[00216] "Cytokine" refers to proteins released by one cell population which act on another cell population as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-a and -β; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-β; platelet-growth factor; transforming growth factors (TGFs) such as TGF-a and TGF-β; insulin-like growth factor-l and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-a, -β, and -γ; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1 , IL-1 a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-1 1 , IL-12, IL-17, IL-22; a tumor necrosis factor such as TNF-a or TNF-β; and other polypeptide factors including LIF and kit ligand (KL). The term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.

[00217] "Inflammatory cells" refers to cells that enhance the inflammatory response such as mononuclear cells, eosinophils, macrophages, and polymorphonuclear neutrophils (PMN). Compositions Disclosed Herein and Methods of Making Same

[00218] This disclosure encompasses compositions, including pharmaceutical compositions, comprising an anti-DLL4 antibody; and polynucleotides comprising sequences encoding a DLL4 antibody. As used herein, compositions comprise one or more antibodies that bind to DLL4, and/or one or more polynucleotides comprising sequences encoding one or more antibodies that bind to DLL4. These compositions may further comprise suitable carriers, such as pharmaceutically acceptable excipients including buffers, which are well known in the art.

[00219] The disclosure also encompasses isolated antibody and polynucleotide embodiments. The disclosure also encompasses substantially pure antibody and polynucleotide embodiments.

[00220] The DLL4 antibodies disclosed herein are preferably monoclonal. Also encompassed within the scope of the disclosure are Fab, Fab', Fab'-SH and F(ab')2 fragments of the DLL4 antibodies provided herein. These antibody fragments can be created by traditional means, such as enzymatic digestion, or may be generated by recombinant techniques. Such antibody fragments may be chimeric or humanized. These fragments are useful for the diagnostic and therapeutic purposes set forth below.

[00221] Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies.

[00222] The DLL4 monoclonal antibodies disclosed herein can be made using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods.

[00223] In the hybridoma method, a mouse or other appropriate host animal, is immunized to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Antibodies to DLL4 generally are raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of DLL4 and an adjuvant or cells expressing recombinant DLL4. DLL4 may be prepared using methods well-known in the art, some of which are further described herein. For example, animals may be immunized with a derivative of DLL4 that contains the extracellular domain (ECD) of DLL4 fused to the Fc portion of an immunoglobulin heavy chain {e.g., an DLL4- lgG1 fusion protein). Animals may be boosted by subsequent immunizations, their serum may be assayed for anti-DLL4 titer, and animals may be further boosted until titer plateaus. [00224] Lymphocytes from immunized animals (or alternatively lymphocytes immunized in vitro) then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

[00225] The hybridoma cells may be seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.

[00226] Preferred myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, myeloma cell lines may be murine myeloma lines, such as those derived from MOPC-21 and MPC-1 1 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et at., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[00227] Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against DLL4. For example, the binding specificity of monoclonal antibodies produced by hybridoma cells may be determined by immunoprecipitation or by an in vitro binding assay, such as an enzyme-linked immunoadsorbent assay (ELISA).

[00228] The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[00229] After hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal.

[00230] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[00231] The DLL4 antibodies disclosed herein may be made by using combinatorial libraries to screen for synthetic antibody clones with the desired activity or activities. For example, synthetic antibody clones are selected by screening phage libraries containing phage that display various fragments of antibody variable regions {e.g., scFv or Fab) fused to phage coat protein. Such phage libraries may be panned, for example, by affinity chromatography against the desired antigen. Clones expressing antibody fragments capable of binding to the desired antigen may be adsorbed to the antigen and thus separated from the non-binding clones in the library. The binding clones may then be eluted from the antigen, and can be further enriched by additional cycles of antigen adsorption/elution. Any of the DLL4 antibodies disclosed herein may be obtained by designing a suitable antigen screening procedure to select for the phage clone of interest followed by construction of a full length DLL4 antibody clone using the VH and VL {e.g., from scFv or Fab) sequences from the phage clone of interest and suitable constant region (Fc) sequences described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91 -3242, Bethesda Md. (1991 ), vols. 1-3.

[00232] The antigen-binding domain of an antibody is formed from two variable (V) regions of about 1 10 amino acids, one each from the light (VL) and heavy (VH) chains, that both present three hypervariable loops or complementarity-determining regions (CDRs). Variable domains may be displayed functionally on phage, either as single-chain Fv (scFv, also referred to as single-chain antibody (SCA)) fragments, in which VH and VL are covalently linked through a short, flexible peptide, or as Fab fragments, in which they are each fused to a constant domain and interact non-covalently, as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). scFv or SCA encoding phage clones and Fab encoding phage clones may be separately or collectively referred to as "Fv phage clones" or "Fv clones".

[00233] Repertoires of VH and VL genes may be separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be searched for antigen-binding clones as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire may be cloned to provide a single source of human antibodies to a wide range of non-self and also self antigens without any immunization as described by Griffiths et al., EMBO J. 12: 725-734 (1993). Finally, naive libraries can also be made synthetically by cloning the unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro as described by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).

[00234] Filamentous phage is used to display antibody fragments by fusion to the minor coat protein pill. Protein pill may include truncated forms of pill. The antibody fragments can be displayed as single chain Fv fragments, in which VH and VL domains are connected on the same polypeptide chain by a flexible polypeptide spacer, {e.g., as described by Marks et al., J. Mol. Biol., 222: 581-597 (1991 )), or as Fab fragments, in which one chain is fused to pill {e.g., a truncated pill) and the other is secreted into the bacterial host cell periplasm where assembly of a Fab-coat protein structure which becomes displayed on the phage surface by displacing some of the wild type coat proteins, (e.g., as described in Hoogenboom et al., Nucl. Acids Res., 19: 4133-4137 (1991 )).

[00235] Nucleic acids encoding antibody gene fragments may be obtained from immune cells harvested from humans or animals. If a library biased in favor of anti-DLL4 clones is desired, the subject is immunized with DLL4 to generate an antibody response, and spleen cells and/or circulating B cells or other peripheral blood lymphocytes (PBLs) are recovered for library construction. For example, a human antibody gene fragment library biased in favor of anti-DLL4 clones is obtained by generating a DLL4 antibody response in transgenic mice carrying a functional human immunoglobulin gene array (and lacking a functional endogenous antibody production system) such that DLL4 immunization gives rise to B cells producing human antibodies against DLL4.

[00236] Additional enrichment for anti-DLL4 reactive cell populations can be obtained by using a suitable screening procedure to isolate B cells expressing DLL4- specific membrane bound antibody, for example by cell separation with DLL4 affinity chromatography or adsorption of cells to fluorochrome-labeled DLL4 followed by flow- activated cell sorting (FACS).

[00237] Alternatively, the use of spleen cells and/or B cells or other PBLs from an unimmunized donor may provide a better representation of the possible antibody repertoire, and also permits the construction of an antibody library using any animal (human or non-human) species in which DLL4 is not antigenic. For libraries incorporating in vitro antibody gene construction, stem cells are harvested from the subject to provide nucleic acids encoding unrearranged antibody gene segments. The immune cells of interest can be obtained from a variety of animal species, such as human, mouse, rat, lagomorpha, luprine, canine, feline, porcine, bovine, equine, and avian species, etc.

[00238] Nucleic acid encoding antibody variable gene segments (including VH and

VL segments) are recovered from the cells of interest and and may be amplified or copies made by recombinant DNA techniques {e.g., Kunkel mutagenesis). For example, in the case of rearranged VH and VL gene libraries, the desired DNA may be obtained by isolating genomic DNA or mRNA from lymphocytes followed by polymerase chain reaction (PCR) with primers matching the 5' and 3' ends of rearranged VH and VL genes as described in Orlandi et al., Proc. Natl. Acad. Sci. (USA), 86: 3833-3837 (1989), thereby making diverse V gene repertoires for expression. The V genes may be amplified from cDNA and genomic DNA, with back primers at the 5' end of the exon encoding the mature V-domain and forward primers based within the J-segment as described in Orlandi et al. (1989) and in Ward et al., Nature, 341 : 544-546 (1989). For amplifying from cDNA, back primers can also be based in the leader exon as described in Jones et al., Biotechnol., 9: 88-89 (1991 ), and forward primers within the constant region as described in Sastry et al., Proc. Natl. Acad. Sci. (USA), 86: 5728-5732 (1989). To enhance or maximize complementarity, degeneracy may be incorporated in the primers as described in Orlandi et al. (1989) or Sastry et al. (1989). Library diversity may be enhanced or maximized by using PCR primers targeted to each V-gene family in order to amplify available VH and VL arrangements present in the immune cell nucleic acid sample, for example, as described in the method of Marks et al., J. Mol. Biol., 222: 581 -597 (1991 ) or as described in the method of Orum et al., Nucleic Acids Res., 21 : 4491 -4498 (1993). For cloning of the amplified DNA into expression vectors, rare restriction may can be introduced within the PCR primer as a tag at one end as described in Orlandi et al. (1989), or by further PCR amplification with a tagged primer as described in Clackson et al., Nature, 352: 624-628 (1991 ).

[00239] Repertoires of synthetically rearranged V genes may be derived in vitro from V gene segments. Most of the human VH-gene segments have been cloned and sequenced {e.g., reported in Tomlinson et al., J. Mol. Biol., 227: 776-798 (1992)), and mapped {e.g., reported in Matsuda et al., Nature Genet., 3: 88-94 (1993); these cloned segments (including all the major conformations of the H 1 and H2 loop) may be used to generate diverse VH gene repertoires with PCR primers encoding H3 loops of diverse sequence and length as described in Hoogenboom and Winter, J. Mol. Biol., 227: 381 -388 (1992). VH repertoires may also be made with all the sequence diversity focused in a long H3 loop of a single length as described in Barbas et al., Proc. Natl. Acad. Sci. USA, 89: 4457-4461 (1992). Human VK and VA. segments have been cloned and sequenced (reported in Williams and Winter, Eur. J. Immunol., 23: 1456-1461 (1993)) and can be used to make synthetic light chain repertoires. Synthetic V gene repertoires, based on a range of VH and VL folds, and L3 and H3 lengths, will encode antibodies of considerable structural diversity. Following amplification of V-gene encoding DNAs, germline V-gene segments can be rearranged in vitro according to the methods of Hoogenboom and Winter, J. Mol. Biol., 227: 381 -388 (1992).

[00240] Repertoires of antibody fragments may be constructed by combining VH and VL gene repertoires together in several ways. Each repertoire may be created in different vectors, and the vectors recombined in vitro, for example, as described in Hogrefe et al., Gene, 128: 1 19-126 (1993), or in vivo by combinatorial infection, for example, the loxP system described in Waterhouse et al., Nucl. Acids Res., 21 : 2265-2266 (1993). The in vivo recombination approach exploits the two-chain nature of Fab fragments to overcome the limit on library size imposed by E. coli transformation efficiency. Naive VH and VL repertoires are cloned separately, one into a phagemid and the other into a phage vector. The two libraries are then combined by phage infection of phagemid-containing bacteria so that each cell contains a different combination and the library size is limited only by the number of cells present (about 1012 clones). Both vectors contain in vivo recombination signals so that the VH and VL genes are recombined onto a single replicon and are co-packaged into phage virions. These large libraries may provide large numbers of diverse antibodies of good affinity (Kd "1 of about 10"8 M).

[00241] Alternatively, the repertoires may be cloned sequentially into the same vector, for example, as described in Barbas et al., Proc. Natl. Acad. Sci. USA, 88: 7978- 7982 (1991 ), or assembled together by PCR and then cloned, for example, as described in Clackson et al., Nature, 352: 624-628 (1991 ). PCR assembly may also be used to join VH and VL DNAs with DNA encoding a flexible peptide spacer to form single chain Fv (scFv) repertoires. In yet another technique, "in cell PCR assembly" may be used to combine VH and VL genes within lymphocytes by PCR and then clone repertoires of linked genes as described in Embleton et al., Nucl. Acids Res., 20: 3831 -3837 (1992).

[00242] The antibodies produced by naive libraries (either natural or synthetic) can be of moderate affinity (Kd "1 of about 106 to 107M"1), but affinity maturation may also be mimicked in vitro by constructing and reselecting from secondary libraries as described in Winter et al. (1994), supra. For example, mutation can be introduced at random in vitro by using error-prone polymerase (reported in Leung et al., Technique, 1 : 1 1 -15 (1989)) in the method of Hawkins et al., J. Mol. Biol., 226: 889-896 (1992) or in the method of Gram et al., Proc. Natl. Acad. Sci. USA, 89: 3576-3580 (1992). Additionally, affinity maturation may be performed by randomly mutating one or more CDRs, for example, using PCR with primers carrying random sequence spanning the CDR of interest, in selected individual Fv clones and screening for higher affinity clones. WO 9607754 described a method for inducing mutagenesis in a complementarity determining region of an immunoglobulin light chain to create a library of light chain genes. Another effective approach is to recombine the VH or VL domains selected by phage display with repertoires of naturally occurring V domain variants obtained from unimmunized donors and screen for higher affinity in several rounds of chain reshuffling as described in Marks et al., Biotechnol., 10: 779-783 (1992). This technique allows the production of antibodies and antibody fragments with affinities in the 10"9 M range.

[00243] Human and murine DLL4 nucleic acid and amino acid sequences are known in the art. An exemplary human DLL4 cDNA sequence and DLL4 protein sequence are represented by GenBank Accession Nos: NM_019074 and NP_061947.1 (SEQ ID NO: 63), respectively. An exemplary murine DLL4 cDNA sequence and DLL4 protein sequence are represented by GenBank Accession Nos: NM_019454 and NP_062327.2, respectively. DLL4 is a transmembrane protein and comprises an extracellular region that includes 8 EGF-like repeats, and a DSL domain that is conserved among all Notch ligands which is believed to be necessary for receptor binding. Human DLL4 (see, e.g., SEQ ID NO: 63) is 685 amino acid in length and contains the following regions: signal peptide (amino acids 1 - 26); DSL domain (amino acids 173-217); EGF-Like 1 domain (amino acids 218-251 ); EGF- Like 2 domain (amino acids 252-282); EGF-Like 3 domain (amino acids 284-322); EGF- Like 4 domain (amino acids 324-360); EGF-Like 5 domain (amino acids 362-400); EGF- Like 6 domain (amino acids 402-438); EGF-Like 7 domain (amino acids 440-476); EGF- Like 8 domain (amino acids 480-518); transmembrane domain (amino acids 530-550); and cytoplasmic domain (amino acids 551-685).

[00244] DNAs encoding DLL4 can be prepared by a variety of methods known in the art. These methods include, but are not limited to, chemical synthesis by any of the methods described in Engels et al., Agnew. Chem. Int Ed. Engl., 28: 716-734 (1989), such as the triester, phosphite, phosphoramidite and H-phosphonate methods. In one embodiment, codons preferred by the expression host cell are used in the design of the DLL4 encoding DNA. Alternatively, DNA encoding the DLL4 can be isolated from a genomic or cDNA library.

[00245] Following construction of the DNA molecule encoding the DLL4, the DNA molecule is operably linked to an expression control sequence in an expression vector, such as a plasmid, wherein the control sequence is recognized by a host cell transformed with the vector. In general, plasmid vectors contain replication and control sequences which are derived from species compatible with the host cell. The vector ordinarily carries a replication site, as well as sequences which encode proteins that are capable of providing phenotypic selection in transformed cells. Suitable vectors for expression in prokaryotic and eukaryotic host cells are known in the art and some are further described herein. Eukaryotic organisms, such as yeasts, or cells derived from multicellular organisms, such as mammals, may be used.

[00246] Optionally, the DNA encoding the DLL4 is operably linked to a secretory leader sequence resulting in secretion of the expression product by the host cell into the culture medium. Examples of secretory leader sequences include stll, ecotin, lamB, herpes GD, Ipp, alkaline phosphatase, invertase, and alpha factor. Also suitable for use herein is the 36 amino acid leader sequence of protein A (see, e.g., Abrahmsen et al., EMBO J., 4: 3901 (1985)).

[00247] Host cells are transfected and preferably transformed with the above- described expression or cloning vectors of this disclosure and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences, including, for example, human DLL4. A DLL-expressing cell line may be obtained and is useful, including for assays with antibodies with antibodies that bind to DLL4, as described herein. Alternatively, DLL4 protein may be purchased from commercial sources {e.g., R&D Systems, Minneapolis, MN).

[00248] Transfection refers to the taking up of an expression vector by a host cell whether or not any coding sequences are in fact expressed. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, CaP04 precipitation and electroporation. Successful transfection is generally recognized when any indication of the operation of this vector occurs within the host cell. Methods for transfection are well known in the art, and some are further described herein.

[00249] Transformation means introducing DNA into an organism so that the DNA is replicable, either as an extrachromosomal element or by chromosomal integrant. Depending on the host cell used, transformation is done using standard techniques appropriate to such cells. Methods for transformation are well known in the art, and some are further described herein.

[00250] Prokaryotic host cells used to produce the DLL4 may be cultured as described generally in Sambrook et al., supra.

[00251] The mammalian host cells used to produce the DLL4 may be cultured in a variety of media, which is well known in the art and some of which is described herein.

[00252] The host cells referred to in this disclosure encompass cells in in vitro culture as well as cells that are within a host animal.

[00253] Purification of DLL4 may be accomplished using art-recognized methods, some of which are described herein. Alternatively, purified DLL4 may be purchased from commercial sources, such as R&D Systems (Minneapolis, MN). [00254] The purified DLL4 may be attached to a suitable matrix such as agarose beads, acrylamide beads, glass beads, cellulose, various acrylic copolymers, hydroxyl methacrylate gels, polyacrylic and polymethacrylic copolymers, nylon, neutral and ionic carriers, and the like, for use in the affinity chromatographic separation of phage display clones. Attachment of the DLL4 protein to the matrix may be accomplished by the methods described in Methods in Enzymology, vol. 44 (1976). A commonly employed technique for attaching protein ligands to polysaccharide matrices, {e.g., agarose, dextran or cellulose), involves activation of the carrier with cyanogen halides and subsequent coupling of the peptide ligand's primary aliphatic or aromatic amines to the activated matrix.

[00255] Alternatively, DLL4 may be used to coat the wells of adsorption plates, expressed on host cells affixed to adsorption plates or used in cell sorting, or conjugated to biotin for capture with streptavidin-coated beads, or used in any other art-known method for panning phage display libraries.

[00256] The phage library samples are contacted with immobilized DLL4 under conditions suitable for binding of at least a portion of the phage particles with the adsorbent. Normally, the conditions, including pH, ionic strength, temperature and the like are selected to mimic physiological conditions. The phages bound to the solid phase are washed and then eluted by acid, e.g., as described in Barbas et al., Proc. Natl. Acad. Sci. USA, 88: 7978-7982 (1991 ), or by alkali, (e.g., as described in Marks et al., J. Mol. Biol., 222: 581-597 (1991 )), or by DLL4 antigen competition, (e.g., in a procedure similar to the antigen competition method of Clackson et al., Nature, 352: 624-628 (1991 )). Phages may be enriched 20-1 ,000-fold in a single round of selection. Moreover, the enriched phages may be grown in bacterial culture and subjected to further rounds of selection.

[00257] The efficiency of selection depends on many factors, including the kinetics of dissociation during washing, and whether multiple antibody fragments on a single phage can simultaneously engage with antigen. Antibodies with fast dissociation kinetics (and weak binding affinities) may be retained by use of short washes, multivalent phage display and high coating density of antigen in solid phase. The high density not only stabilizes the phage through multivalent interactions, but favors rebinding of phage that has dissociated. The selection of antibodies with slow dissociation kinetics (and good binding affinities) may be promoted by use of long washes and monovalent phage display as described in Bass et al., Proteins, 8: 309-314 (1990) and in WO 92/09690, and a low coating density of antigen as described in Marks et al., Biotechnol., 10: 779-783 (1992).

[00258] It is possible to select between phage antibodies of different affinities, even with affinities that differ slightly, for DLL4. However, random mutation of a selected antibody {e.g., as performed in some of the affinity maturation techniques described above) is likely to give rise to many mutants, most binding to antigen, and a few with higher affinity. With limiting DLL4, rare high affinity phage may be competed out. To retain all the higher affinity mutants, phages may be incubated with excess, labeled {e.g., biotinylated) DLL4, but with the labeled {e.g., biotinylated) DLL4 at a concentration of lower molarity than the target molar affinity constant for DLL4. The high affinity-binding phages may then be captured by streptavidin-coated paramagnetic beads. Such "equilibrium capture" allows the antibodies to be selected according to their affinities of binding, with sensitivity that permits isolation of mutant clones with as little as two-fold higher affinity from a great excess of phages with lower affinity. Conditions used in washing phages bound to a solid phase may also be manipulated to discriminate on the basis of dissociation kinetics.

[00259] Anti-DLL4 clones may be activity selected. In one embodiment, the disclosure provides DLL4 antibodies that block the binding between a Notch receptor (such as Notchl , Notch2, Notch3 and/or Notch4) and DLL4, but do not block the binding between a Notch receptor and a second protein. Fv clones corresponding to such DLL4 antibodies can be selected by (1 ) isolating anti-DLL4 clones from a phage library as described above, and optionally amplifying the isolated population of phage clones by growing up the population in a suitable bacterial host; (2) selecting DLL4 and a second protein against which blocking and non-blocking activity, respectively, is desired; (3) adsorbing the anti- DLL4 phage clones to immobilized DLL4; (4) using an excess of the second protein to elute any undesired clones that recognize DLL4-binding determinants which overlap or are shared with the binding determinants of the second protein; and (5) eluting the clones which remain adsorbed following step (4). Optionally, clones with the desired blocking/non- blocking properties may be further enriched by repeating the selection procedures described herein one or more times.

[00260] DNA encoding the hybridoma-derived monoclonal antibodies or phage display Fv clones disclosed herein is readily isolated and sequenced using conventional procedures {e.g., by using oligonucleotide primers designed to specifically amplify the heavy and light chain coding regions of interest from hybridoma or phage DNA template). Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of the desired monoclonal antibodies in the recombinant host cells. Recombinant expression in bacteria of antibody-encoding DNA has been described by Better et al., U.S. Patent No. 6,204,023 (see also, e.g., Skerra et al., Curr. Opinion in Immunol., 5: 256 (1993) and Pluckthun, Immunol. Revs, 130: 151 (1992)). [00261] DNA encoding Fv clones as disclosed herein may be combined with known DNA sequences encoding heavy chain and/or light chain constant regions {e.g., the appropriate DNA sequences can be obtained from Kabat et al., supra) to form clones encoding full or partial length heavy and/or light chains. It will be appreciated that constant regions of any isotype can be used for this purpose, including IgG, IgM, IgA, IgD, and IgE constant regions, and that such constant regions may be obtained from any human or animal species. A Fv clone derived from the variable domain DNA of one animal (such as human) species and then fused to constant region DNA of another animal species to form coding sequence(s) for "hybrid", full length heavy chain and/or light chain is included in the definition of "chimeric" and "hybrid" antibody as used herein. In a preferred Fv clone embodiment, aFv clone derived from human variable DNA is fused to human constant region DNA to form coding sequence(s) for all human, full or partial length heavy and/or light chains.

[00262] DNA encoding DLL4 antibody derived from a hybridoma disclosed herein may also be modified, for example, by substituting the coding sequence for human heavy- and light-chain constant domains in place of homologous murine sequences derived from the hybridoma clone {e.g., as in the method of Morrison et al., Proc. Natl. Acad. Sci. USA, 81 : 6851-6855 (1984)). DNA encoding a hybridoma or Fv clone-derived antibody or fragment can be further modified by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. In this manner, "chimeric" or "hybrid" antibodies are prepared that have the binding specificity of the Fv clone or hybridoma clone-derived antibodies disclosed herein.

Antibody Fragments

[00263] The present disclosure encompasses antibody fragments. In certain circumstances there are advantages of using antibody fragments, rather than whole antibodies. The smaller size of the fragments allows for rapid clearance, and may lead to improved access to solid tumors.

[00264] Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-1 17 (1992); and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv and ScFv antibody fragments can all be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of these fragments (see, e.g., U.S. Patent No. 6,204,023). Antibody fragments can be isolated from antibody phage libraries as discussed above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (see, e.g., Carter et al., Bio/Technology 10: 163-167 (1992)). According to another approach, F(ab')2 fragments can be isolated directly from recombinant host cell culture. Fab and F(ab')2 fragment with increased in vivo half-life comprising a salvage receptor binding epitope residues (see, e.g., in U.S. Patent No. 5,869,046). Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv or single chain antibody (SCA)). See WO 93/16185; U.S. Patent Nos. 5,571 ,894; and 5,587,458. Fv and sFv are the only species with intact combining sites that are devoid of constant regions; thus, they are suitable for reduced nonspecific binding during in vivo use. sFv fusion proteins may be constructed to yield fusion of an effector protein at either the amino or the carboxy terminus of an sFv. See Antibody Engineering, ed. Borrebaeck, Supra. The antibody fragment may also be a "linear antibody", for example, as described in U.S. Patent No. 5,641 ,870. Such linear antibody fragments may be monospecific or bispecific.

Humanized Antibodies

[00265] The present disclosure encompasses humanized antibodies. Humanized antibodies include human engineered antibodies (see, e.g., U.S. Patent No. 5,766,886). Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody can have one or more amino acid residues introduced into it from a source which is human or non-human. Humanization may be performed following the method of Studnicka (U.S. Patent No. 5,766,886), including the preparation of modified antibody variable domains. Humanization may alternatively be performed following the method of Winter and co-workers (Jones et al. (1986) Nature 321 :522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536), by substituting hypervariable region sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" or "human engineered" antibodies are chimeric antibodies, including wherein substantially less than an intact human variable domain has been substituted by or incorporated into the corresponding sequence from a non-human species. For example, humanized antibodies may be human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. Alternatively, humanized or human engineered antibodies may be non-human (e.g, rodent) antibodies in which some residues are substituted by residues from analogious sites in human antibodies (see, e.g., U.S. Patent No. 5,766,886). [00266] The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is important to reduce antigenicity. For example, to the so-called "best-fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences. The human sequence which is closest to that of the rodent is then accepted as the human framework for the humanized antibody (Sims et al. (1993) J. Immunol. 151 :2296; Chothia et al. (1987) J. Mol. Biol. 196:901 ). Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; Presta et al. (1993) J. Immunol., 151 :2623).

[00267] It is further important that antibodies be humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, according to one method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three- dimensional models of the parental and humanized sequences. 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, e.g., 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 recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.

Human Antibodies

[00268] Human DLL4 antibodies disclosed herein may be constructed by combining Fv clone variable domain sequence(s) selected from human-derived phage display libraries with known human constant domain sequences(s) as described above. Alternatively, human monoclonal DLL4 antibodies disclosed herein may be made by the hybridoma method. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described, for example, by Kozbor J. Immuunol., 133: 3001 (1984); Brodeur et al, Monoclonal Antibody Production Techniques and Applications, pp. 51 -63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al, J. Immunol., 147: 86 (1991 ).

[00269] It is possible to produce transgenic animals {e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al, Nature, 362: 255 (1993); Bruggermann et al, Year in Immunol., 7: 33 (1993).

[00270] Gene shuffling can also be used to derive human antibodies from non- human, {e.g., rodent), antibodies, where the human antibody has similar affinities and specificities to the starting non-human antibody. According to this method, which is also called "epitope imprinting", either the heavy or light chain variable region of a non-human antibody fragment obtained by phage display techniques as described above is replaced with a repertoire of human V domain genes, creating a population of non-human chain/human chain scFv or Fab chimeras. Selection with antigen results in isolation of a non-human chain/human chain chimeric scFv or Fab wherein the human chain restores the antigen binding site destroyed upon removal of the corresponding non-human chain in the primary phage display clone. The epitope governs (imprints) the choice of the human chain partner. When the process is repeated in order to replace the remaining non-human chain, a human antibody is obtained (see, e.g., WO93/06213). Unlike traditional humanization of non-human antibodies by CDR grafting, this technique provides completely human antibodies, which have no FR or CDR residues of non-human origin.

Bispecific Antibodies

[00271] Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. For example, one of the binding specificities may be for DLL4 and the other may be for any other antigen. Exemplary bispecific antibodies may bind to two different epitopes of the DLL4 protein. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express DLL4. These antibodies possess a DLL4-binding arm and an arm which binds the cytotoxic agent {e.g., saporin, anti-interferon-a, vinca alkaloid, ricin A chain, methotrexate or radioactive isotope hapten). Bispecific antibodies may be prepared as full length antibodies or antibody fragments {e.g., F(ab')2 bispecific antibodies). [00272] Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co- expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305: 537 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93/08829, and in Traunecker et al., EMBO J., 10: 3655 (1991 ).

[00273] According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1 ), containing the site necessary for light chain binding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co- transfected into a suitable host organism. This provides for flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yields. It is, however, possible to insert the coding sequences for two or all three polypeptide chains in one expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are not of particular significance.

[00274] In a preferred embodiment of this approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure may facilitate the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in WO 94/04690. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121 :210 (1986).

[00275] According to another approach, the interface between a pair of antibody molecules may can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains {e.g., tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones {e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.

[00276] Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate may be coupled to avidin, the other to biotin. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Patent No. 4,676,980), and for treatment of HIV infection (WO 91/00360, WO 92/00373, and EP 03089). Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Patent No. 4,676,980, along with a number of cross-linking techniques.

[00277] Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies may be prepared using chemical linkage. Brennan et al., Science, 229: 81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced may be used as agents for the selective immobilization of enzymes.

[00278] Recent progress has facilitated the direct recovery of Fab'-SH fragments from E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab')2 molecule. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the HER2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

[00279] Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. See, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re- oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy- chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen- binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See, Gruber et al., J. Immunol., 152:5368 (1994).

[00280] Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. See, e.g., Tutt et al. J. Immunol. 147: 60 (1991 ).

Multivalent Antibodies

[00281] A multivalent antibody may be internalized (and/or catabolized) faster than a bivalent antibody by a cell expressing an antigen to which the antibodies bind. The antibodies of the present disclosure may be multivalent antibodies (which are other than of the IgM class) with three or more antigen binding sites (e.g., tetravalent antibodies), which may be produced by recombinant expression of nucleic acid encoding the polypeptide chains of the antibody. The multivalent antibody may comprise a dimerization domain and three or more antigen binding sites. A preferred dimerization domain may comprise (or consist of) an Fc region or a hinge region. In this scenario, the antibody will comprise an Fc region and three or more antigen binding sites amino-terminal to the Fe region. A preferred multivalent antibody may comprise (or consist of) three to about eight, but preferably four, antigen binding sites. The multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain(s) comprise two or more variable domains. For instance, the polypeptide chain(s) may comprise VD1-(X1 )n- VD2-(X2)n-Fc, wherein VD1 is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of an Fc region, X1 and X2 represent an amino acid or polypeptide, and n is 0 or 1 . For instance, the polypeptide chain(s) may comprise: VH-CH1 - flexible linker-VH-CH1-Fc region chain; or VH-CH1-VH-CH1-Fc region chain. A multivalent antibody may preferably further comprises at least two (and preferably four) light chain variable domain polypeptides. A multivalent antibody may, for instance, comprise from about two to about eight light chain variable domain polypeptides. The light chain variable domain polypeptides may comprise a light chain variable domain and, optionally, further comprise a CL domain.

Antibody Variants

[00282] In some embodiments, amino acid sequence modification(s) of the antibodies to DLL4 described herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of the antibody are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics. The amino acid alterations may be introduced in the subject antibody amino acid sequence at the time that sequence is made.

[00283] A useful method for identification of certain residues or regions of the antibody that are preferred locations for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science, 244:1081-1085. Here, a residue or group of target residues are identified {e.g., charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acids with antigen. Those amino acid locations demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at, or for, the sites of substitution. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to analyze the performance of a mutation at a given site, ala scanning or random mutagenesis is conducted at the target codon or region and the expressed immunoglobulins are screened for the desired activity.

[00284] Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue or the antibody fused to a cytotoxic polypeptide. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme {e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody.

[00285] Glycosylation of polypeptides is typically either N-linked or O-linked. relinked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X- threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N- aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[00286] Addition of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).

[00287] Where the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. For example, antibodies with a mature carbohydrate structure that lacks fucose attached to an Fc region of the antibody have been described (see, e.g., US 2003/0157108, US 2004/0093621. Antibodies with a bisecting N-acetylglucosamine (GlcNAc) in the carbohydrate attached to an Fc region of the antibody have been described (see, e.g., WO 2003/01 1878, and U.S. Patent No. 6,602,684). Antibodies with at least one galactose residue in the oligosaccharide attached to an Fc region of the antibody WO 1997/30087; see, also, WO 1998/58964 and WO 1999/22764 concerning antibodies with altered carbohydrate attached to the Fc region thereof). Antigen-binding molecules with modified glycosylation have been described (see, e.g., WO 99/54342, U.S. Patent Nos. 6,602,684 and 7,517,670, and US 2004/0072290; see also, e.g., U.S. Patent Nos. 7,214,775 and 7,682,610).

[00288] The preferred glycosylation variant herein comprises an Fc region, wherein a carbohydrate structure attached to the Fc region lacks fucose. Such variants have improved ADCC function. Optionally, the Fc region further comprises one or more amino acid substitutions therein which further improve ADCC, for example, substitutions at positions 298, 333, and/or 334 of the Fc region (Eu numbering of residues). Examples of publications related to "defucosylated" or "fucose-deficient" antibodies include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/01 15614 (now U.S. Patent No.6,946,292) US 2002/0164328 (now U.S. Patent No. 7,064, 191 ); US 2004/0093621 ; US 2004/0132140; US 2004/01 10704; US 2004/01 10282 (now U.S. Patent No 7,749,753); US 2004/0109865; WO 2003/0851 19; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of cell lines producing defucosylated antibodies include Led 3 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US 2003/0157108 A1 , Presta, L; and WO 2004/056312 A1 , Adams et al., especially at Example 1 1 ), and knockout cell lines, such as alpha-1 ,6-fucosyltransferase gene, FUT8, knockout CHO cells (Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004)).

[00289] Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue in the antibody molecule replaced by a different residue. The sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but FR alterations are also contemplated. Conservative substitutions are shown in Table 2 under the heading of "preferred substitutions". If such substitutions result in a change in biological activity, then more substantial changes, denominated "exemplary substitutions", or as further described below in reference to amino acid classes, may be introduced and the products screened.

Original Residue Exemplary Substitutions Preferred Substitutions

Ala (A) Val; Leu; lie Val

Arg (R) Lys; Gin; Asn Lys

Asn (N) Gin; His; Asp, Lys; Arg Gin

Asp (D) Glu; Asn Glu

Cys (C) Ser; Ala Ser

Gin (Q) Asn; Glu Asn

Glu (E) Asp; Gin Asp

Gly (G) Ala Ala

His (H) Asn; Gin; Lys; Arg Arg

lie (I) Leu; Val; Met; Ala; Phe; Norleucine Leu

Leu (L) Norleucine; lie; Val; Met; Ala; Phe lie

Lys (K) Arg; Gin; Asn Arg

Met (M) Leu; Phe; lie Leu

Phe (F) Trp; Leu; Val; lie; Ala; Tyr Tyr

Pro (P) Ala Ala

Ser (S) Thr Thr

Thr (T) Val; Ser Ser

Trp (W) Tyr; Phe Tyr

Tyr (Y) Trp; Phe; Thr; Ser Phe

Val (V) lie; Leu; Met; Phe; Ala; Norleucine Leu

[00290] Substantial modifications in the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side-chain properties: (1 ) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: asp, glu; (4) basic: his, lys, arg; (5) residues that influence chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe.

[00291] Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

[00292] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody {e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated. A convenient way for generating such substitutional variants involves affinity maturation using phage display. Briefly, several hypervariable region sites {e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibodies thus generated are displayed from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity {e.g., binding affinity) as herein disclosed. In order to identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues contributing significantly to antigen binding. Alternatively, or additionally, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues are candidates for substitution according to the techniques elaborated herein. Once such variants are generated, the panel of variants is subjected to screening as described herein and antibodies with superior properties in one or more relevant assays may be selected for further development.

[00293] Nucleic acid molecules encoding amino acid sequence variants of the antibody are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of the antibody.

[00294] It may be desirable to introduce one or more amino acid modifications in an Fc region of the immunoglobulin polypeptides disclosed herein, thereby generating a Fc region variant. The Fc region variant may comprise a human Fc region sequence {e.g., a human lgG1 , lgG2, lgG3 or lgG4 Fc region) comprising an amino acid modification {e.g., a substitution) at one or more amino acid positions including that of a hinge cysteine. In accordance with this description and the teachings of the art, it is contemplated that in some embodiments, an antibody used in methods disclosed herein may comprise one or more alterations as compared to the wild type counterpart antibody, e.g,. in the Fc region. These antibodies would nonetheless retain substantially the same characteristics required for therapeutic utility as compared to their wild type counterpart. For example, it is thought that certain alterations can be made in the Fc region that would result in altered {e.g., either improved or diminished) Clq binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in W099/51642. See also Duncan & Winter Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821 ; and W094/29351 concerning other examples of Fc region variants. WO00/42072 and WO 2004/056312 describe antibody variants with improved or diminished binding to FcRs. See, also, Shields et al. J. Biol. Chem. 9(2): 6591 -6604 (2001 ). Antibodies with increased half lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 1 17:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in US2005/0014934 (Hinton et al.). These antibodies comprise an Fc reg on with one or more substitutions therein which improve binding of the Fc region to FcRn. Polypeptide variants with altered Fc region amino acid sequences and increased or decreased Clq binding capability are described in U.S. Patent No. 6, 194,551 , W099/51642. See, also, Idusogie et al. J. Immunol. 164:4178-4184 (2000).

Antibody Derivatives

[00295] The antibodies of the present disclosure may be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available. Preferably, the moieties suitable for derivatization of the antibody are water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1 ,3- dioxolane, poly-1 ,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols {e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymers are attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc. Antibody Characterization

[00296] The antibodies of the present disclosure can be characterized for their physical/chemical properties and biological functions by various assays known in the art. In some embodiments, antibodies are characterized for any one or more of binding to DLL4; and/or disruption or blocking of Notch receptor binding to DLL4; and/or reduction or blocking of Notch receptor activation; and/or reduction or blocking of Notch receptor downstream molecular signaling; and/or promotion of endothelial cell proliferation; and/or inhibition of endothelial cell differentiation; and/or inhibition of arterial differentiation; and/or inhibition of tumor vascular perfusion; and/or treatment and/or prevention of a tumor, cell proliferative disorder or a cancer; and/or treatment or prevention of a disorder associated with DLL4 expression and/or activity; and/or treatment or prevention of a disorder associated with Notch receptor expression and/or activity.

[00297] The purified antibodies can be further characterized by a series of assays including, but not limited to, N-terminal sequencing, amino acid analysis, non-denaturing size exclusion high pressure liquid chromatography (HPLC), mass spectrometry, ion exchange chromatography and papain digestion.

[00298] In certain embodiments disclosed herein, the antibodies produced herein are analyzed for their biological activity. In some embodiments, the antibodies of the present disclosure are tested for their antigen binding activity. The antigen binding assays that are known in the art and can be used herein include without limitation any direct or competitive binding assays using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, fluorescent immunoassays, and protein A immunoassays. Illustrative antigen binding assays include those described herein, including in the Examples section.

[00299] In certain embodiments disclosed herein, the antibodies to DLL4 recognize unique or novel epitopes of DLL4. For example, DLL4 monoclonal antibodies may recognize a DLL4 epitope that is different from or only partially overlaps with a DLL4 epitope recognized by referenced antibody such as BA1 and/or BA2 as described herein (see, e.g., Examples 2 and 3). Such antibodies can be obtained by screening anti-DLL4 hybridoma supernatants for binding to immobilized DLL4 in competition with a labeled reference antibody such as a BA1 and/or BA2 antibody. A hybridoma supernatant containing antibodies with partially overlapping epitope binding regions may reduce the amount of bound, labeled antibody detected in the subject competition binding mixture as compared to the amount of bound, labeled antibody detected in a control binding mixture containing irrelevant (or no) antibody. Any of the competition binding assays described herein are suitable for use in the foregoing procedure.

[00300] In certain embodiments disclosed herein, DLL4 antibodies are provided that comprise one or more (such as 2, 3, 4, 5, and/or 6) CDRs of a 12.3 1 C1 , 12.73, 12.08, 12.14, or 12.55 antibody. A DLL4 monoclonal antibody that comprises one or more CDRs of a 12.3 1 C1 , 12.73, 12.08, 12.14, or 12.55 antibody can be constructed by grafting one or more CDRs of a 12.3 1 C1 , 12.73, 12.08, 12.14, or 12.55 antibody onto a template antibody sequence, e.g., a human antibody sequence which shares some homology to or identity with {e.g., is closest in sequence to) the corresponding murine sequence of the parental antibody, or a consensus sequence of human antibodies in the particular subgroup of the parental antibody light or heavy chain, and expressing the resulting chimeric light and/or heavy chain variable region sequence(s), with or without accompanying constant region sequence(s), in recombinant host cells as described herein.

[00301] DLL4 antibodies disclosed herein possessing the unique properties described herein can be obtained by screening anti-DLL4 hybridoma clones for the desired properties by any convenient method. For example, if a DLL4 monoclonal antibody that blocks or does not block the binding of Notch receptors to DLL4 is desired, the candidate antibody can be tested in a binding competition assay, such as a competitive binding ELISA, wherein plate wells are coated with DLL4, and a solution of antibody in an excess of the Notch receptor of interest is layered onto the coated plates, and bound antibody is detected. Such detection may be by any means including enzymatically, for example, by contacting the bound antibody with a labeled anti-lg antibody {e.g., with HRP-conjugated anti-lg antibody or biotinylated anti-lg antibody and developing the HRP color reaction, for example, by developing plates with streptavidin-HRP and/or hydrogen peroxide and detecting the HRP color reaction by spectrophotometry at 490 nm with an ELISA plate reader).

[00302] In an embodiment, the present disclosure contemplates an altered antibody that possesses some but not all effector functions, which make it a desired candidate for many applications in which the half life of the antibody in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In certain embodiments, the Fc activities of the produced immunoglobulin are measured to ensure that only the desired properties are maintained. In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcyR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes express FcyRI, FcyRII and FCYRI I I . FCR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991 ). An example of an in vitro assay to assess ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 or 5,821 ,337. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, {e.g., in a animal model such as that disclosed in Clynes et al. Proc. Natl. Acad. Sci. A 95:652-656 (1998)). Clq binding assays may also be carried out to confirm that the antibody is unable to bind Clq and hence lacks CDC activity. To assess complement activation, a CDC assay, e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be performed. FcRn binding and in vivo clearance/half life determinations can also be performed using methods known in the art.

Vectors, Host Cells and Recombinant Methods

[00303] For recombinant production of an antibody disclosed herein, the nucleic acid encoding it is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. DNA encoding the antibody is readily isolated and sequenced using conventional procedures {e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). Many vectors are available. The choice of vector depends in part on the host cell to be used. Generally, preferred host cells are of either prokaryotic or eukaryotic (generally mammalian) origin. It will be appreciated that constant regions of any isotype can be used for this purpose, including IgG, IgM, IgA, IgD, and IgE constant regions, and that such constant regions can be obtained from any human or animal species.

a. Generating Antibodies Using Prokaryotic Host Cells:

i. Vector Construction

[00304] Polynucleotide sequences encoding polypeptide components of the antibodies disclosed herein can be obtained using standard recombinant techniques. Desired polynucleotide sequences may be isolated and sequenced from antibody producing cells such as hybridoma cells. Alternatively, polynucleotides can be synthesized using nucleotide synthesizer or PCR techniques. Once obtained, sequences encoding the polypeptides are inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in prokaryotic hosts. Many vectors that are available and known in the art can be used for the purpose of the present disclosure. Selection of an appropriate vector will depend mainly on the size of the nucleic acids to be inserted into the vector and the particular host cell to be transformed with the vector. Each vector contains various components, depending on its function (amplification or expression of heterologous polynucleotide, or both) and its compatibility with the particular host cell in which it resides. The vector components generally include, but are not limited to: an origin of replication, a selection marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, the heterologous nucleic acid insert and a transcription termination sequence.

[00305] In general, plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts. The vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells. For example, E. coli is typically transformed using pBR322, a plasmid derived from an E. coli species. pBR322 contains genes encoding ampicillin (Amp) and tetracycline (Tet) resistance and thus provides easy means for identifying transformed cells. pBR322, its derivatives, or other microbial plasmids or bacteriophage may also contain, or be modified to contain, promoters which can be used by the microbial organism for expression of endogenous proteins. Examples of pBR322 derivatives used for expression of particular antibodies have been described (see, e.g., U.S. Patent No. 5,648,237).

[00306] In addition, phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as transforming vectors in connection with these hosts. For example, bacteriophage such as AGEM™-1 1 may be utilized in making a recombinant vector which can be used to transform susceptible host cells such as E. coli LE392.

[00307] The expression vectors disclosed herein may comprise two or more promoter-cistron pairs, encoding each of the polypeptide components. A promoter is an untranslated regulatory sequence located upstream (5') to a cistron that modulates its expression. Prokaryotic promoters typically fall into two classes, inducible and constitutive. Inducible promoter is a promoter that initiates increased levels of transcription of the cistron under its control in response to changes in the culture condition, e.g., the presence or absence of a nutrient or a change in temperature.

[00308] A large number of promoters recognized by a variety of potential host cells are well known. The selected promoter can be operably linked to cistron DNA encoding the light or heavy chain by removing the promoter from the source DNA via restriction enzyme digestion and inserting the isolated promoter sequence into the vector disclosed herein. Both the native promoter sequence and many heterologous promoters may be used to direct amplification and/or expression of the target genes. In some embodiments, heterologous promoters are utilized, as they generally permit greater transcription and higher yields of expressed target gene as compared to the native target polypeptide promoter.

[00309] Promoters suitable for use with prokaryotic hosts include: an ara B promoter, a PhoA promoter, β-galactamase and lactose promoter systems, a tryptophan (trp) promoter system and hybrid promoters such as the tac or the trc promoter. However, other promoters that are functional in bacteria (such as other known bacterial or phage promoters) are suitable as well. Their nucleotide sequences have been published, thereby enabling a skilled worker operably to ligate them to cistrons encoding the target light and heavy chains {e.g., Siebenlist et al. (1980) Cell 20: 269) using linkers or adaptors to supply any required restriction sites.

[00310] In one aspect disclosed herein, each cistron within the recombinant vector comprises a secretion signal sequence component that directs translocation of the expressed polypeptides across a membrane. In general, the signal sequence may be a component of the vector, or it may be a part of the target polypeptide DNA that is inserted into the vector. The signal sequence should be one that is recognized and processed (e.g., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the signal sequences native to the heterologous polypeptides, the signal sequence is substituted by a prokaryotic signal sequence selected, for example PelB, OmpA, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II (STII) leaders, LamB, PhoE, and MBP. In one embodiment disclosed herein, the signal sequences used in both cistrons of the expression system are STII signal sequences or variants thereof.

[0031 1] In another aspect, the production of the immunoglobulins according to the disclosure can occur in the cytoplasm of the host cell, and therefore does not require the presence of secretion signal sequences within each cistron. In that regard, immunoglobulin light and heavy chains are expressed, folded and assembled to form functional immunoglobulins within the cytoplasm. Certain host strains {e.g., the E. coli trxB-strains) provide cytoplasm conditions that are favorable for disulfide bond formation, thereby permitting proper folding and assembly of expressed protein subunits (see e.g., Proba and Pluckthun Gene, 159:203 (1995)).

[00312] Prokaryotic host cells suitable for expressing antibodies disclosed herein include Archaebacteria and Eubacteria, such as Gram-negative or Gram-positive organisms. Examples of useful bacteria include Escherichia {e.g., E. coli), Bacilli {e.g., B. subtilis), Enterobacteria, Pseudomonas species {e.g., P. aeruginosa), Salmonella typhimurium, Serratia marcescans, Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, or Paracoccus. In one embodiment, gram-negative cells are used. In one embodiment, E. coli cells are used as hosts for the disclosure. Examples of E. coli strains include strain W31 10 (Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, D.C.: American Society for Microbiology, 1987), pp. 1 190-1219; ATCC® Deposit No. 27,325) and derivatives thereof, including strain 33D3 having genotype W31 10 AfhuA. (AtonA) ptr3 lac Iq lacL8 AompTA (nmpc-fepE) degP41 kanR (U.S. Patent No. 5,639,635). Other strains and derivatives thereof, such as E. coli 294 (ATCC 31 ,446), E. coli B, E. coli λ 1776 (ATCC 31 ,537) and E. coli RV308 (ATCC 31 ,608) are also suitable. These examples are illustrative rather than limiting. Methods for constructing derivatives of any of the above-mentioned bacteria having defined genotypes are known in the art and described in, for example, Bass et al., Proteins, 8:309-314 (1990). It is generally necessary to select the appropriate bacteria taking into consideration replicability of the replicon in the cells of a bacterium. For example, E. coli, Serratia, or Salmonella species can be suitably used as the host when well known plasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to supply the replicon. Typically the host cell should secrete minimal amounts of proteolytic enzymes, and additional protease inhibitors may desirably be incorporated in the cell culture.

/'/'. Antibody Production

[00313] Host cells are transformed with the above-described expression vectors and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

[00314] Transformation means introducing DNA into the prokaryotic host so that the DNA is replicable, either as an extrachromosomal element or by chromosomal integrant. Depending on the host cell used, transformation is done using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride is generally used for bacterial cells that contain substantial cell-wall barriers. Another method for transformation employs polyethylene glycol/DMSO. Yet another technique used is electroporation.

[00315] Prokaryotic cells used to produce the polypeptides disclosed herein are grown in media known in the art and suitable for culture of the selected host cells. Examples of suitable media include luria broth (LB) plus necessary nutrient supplements. In some embodiments, the media also contains a selection agent, chosen based on the construction of the expression vector, to selectively permit growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to media for growth of cells expressing ampicillin resistant gene. [00316] Any necessary supplements besides carbon, nitrogen, and inorganic phosphate sources may also be included at appropriate concentrations introduced alone or as a mixture with another supplement or medium such as a complex nitrogen source. Optionally the culture medium may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycollate, dithioerythritol and dithiothreitol.

[00317] The prokaryotic host cells are cultured at suitable temperatures. For E. coli growth, for example, the preferred temperature ranges from about 20° C. to about 39° C, more preferably from about 25° C. to about 37° C, even more preferably at about 30° C. The pH of the medium may be any pH ranging from about 5 to about 9, depending mainly on the host organism. For E. coli, the pH is preferably from about 6.8 to about 7.4, and more preferably about 7.0.

[00318] If an inducible promoter is used in the expression vector disclosed herein, protein expression is induced under conditions suitable for the activation of the promoter. For example, an ara B or phoA promoter may be used for controlling transcription of the polypeptides. A variety of inducers may be used, according to the vector construct employed, as is known in the art.

[00319] The expressed polypeptides of the present disclosure are secreted into and recovered from the periplasm of the host cells or transported into the culture media. Protein recovery from the periplasm typically involves disrupting the microorganism, generally by such means as osmotic shock, sonication or lysis. Once cells are disrupted, cell debris or whole cells may be removed by centrifugation or filtration. The proteins may be further purified, for example, by affinity resin chromatography. Alternatively, proteins that are transported into the culture media may be isolated therein. Cells may be removed from the culture and the culture supernatant being filtered and concentrated for further purification of the proteins produced. The expressed polypeptides can be further isolated and identified using commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assay.

[00320] Antibody production may be conducted in large quantity by a fermentation process. Various large-scale fed-batch fermentation procedures are available for production of recombinant proteins. Large-scale fermentations have at least 1000 liters of capacity, preferably about 1 ,000 to 100,000 liters of capacity. These fermentors use agitator impellers to distribute oxygen and nutrients, especially glucose (a preferred carbon/energy source). Small scale fermentation refers generally to fermentation in a fermentor that is no more than approximately 100 liters in volumetric capacity, and can range from about 1 liter to about 100 liters. [00321] In a fermentation process, induction of protein expression is typically initiated after the cells have been grown under suitable conditions to a desired density, e.g., an OD550 of about 180-220, at which stage the cells are in the early stationary phase. A variety of inducers may be used, according to the vector construct employed, as is known in the art and described above. Cells may be grown for shorter periods prior to induction. Cells are usually induced for about 12-50 hours, although longer or shorter induction time may be used.

[00322] To improve the production yield and quality of the polypeptides disclosed herein, various fermentation conditions can be modified. For example, to improve the proper assembly and folding of the secreted antibody polypeptides, additional vectors overexpressing chaperone proteins, such as Dsb proteins (DsbA, DsbB, DsbC, DsbD and or DsbG) or FkpA (a peptidylprolyl cis,trans-isomerase with chaperone activity) may be used to co-transform the host prokaryotic cells. The chaperone proteins have been demonstrated to facilitate the proper folding and solubility of heterologous proteins produced in bacterial host cells, (see e.g., Chen et al. (1999) J Bio Chem 274:19601 - 19605; U.S. Patent No. 6,083,715; U.S. Patent No. 6,027,888; Bothmann and Pluckthun (2000) J. Biol. Chem. 275:17100-17105; Ramm and Pluckthun (2000) J. Biol. Chem. 275:17106-171 13; Arie et al. (2001 ) Mol. Microbiol. 39:199-210).

[00323] To minimize proteolysis of expressed heterologous proteins (especially those that are proteolytically sensitive), certain host strains deficient for proteolytic enzymes can be used for the present disclosure. For example, host cell strains may be modified to effect genetic mutation(s) in the genes encoding known bacterial proteases such as Protease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V, Protease VI and combinations thereof. Some E. coli protease-deficient strains are available (see, e.g., My et al. (1998), supra; U.S. Patent No. 5,264,365; U.S. Patent No. 5,508, 192; Hara et al., Microbial Drug Resistance, 2:63-72 (1996)).

[00324] E. coli strains deficient for proteolytic enzymes and transformed with plasmids overexpressing one or more chaperone proteins may be used as host cells in the expression systems disclosed herein.

/'/'/'. Antibody Purification

[00325] Standard protein purification methods known in the art can be employed. The following procedures are exemplary of suitable purification procedures: fractionation on immunoaffinity or ion-exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica or on a cation-exchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gel filtration using, for example, Sephadex G-75. [00326] In one aspect, Protein A immobilized on a solid phase is used for immunoaffinity purification of the full length antibody products disclosed herein. Protein A is a 41 kD cell wall protein from Staphylococcus aureas which binds with a high affinity to the Fc region of antibodies (see, e.g., Lindmark et al (1983) J. Immunol. Meth. 62:1 -13). The solid phase to which Protein A is immobilized is preferably a column comprising a glass or silica surface, more preferably a controlled pore glass column or a silicic acid column. In some applications, the column has been coated with a reagent, such as glycerol, in an attempt to prevent nonspecific adherence of contaminants.

[00327] As the first step of purification, the preparation derived from the cell culture as described above is applied onto the Protein A immobilized solid phase to allow specific binding of the antibody of interest to Protein A. The solid phase is then washed to remove contaminants non-specifically bound to the solid phase. Finally the antibody of interest is recovered from the solid phase by elution.

b. Generating Antibodies Using Eukaryotic Host Cells:

[00328] The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

(i) Signal Sequence Component

[00329] A vector for use in a eukaryotic host cell may also contain a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide of interest. The heterologous signal sequence selected preferably is one that is recognized and processed {e.g., cleaved by a signal peptidase) by the host cell. In mammalian cell expression, mammalian signal sequences as well as viral secretory leaders, for example, the herpes simplex gD signal, are available.

[00330] The DNA for such precursor region is ligated in reading frame to DNA encoding the antibody.

(ii) Origin of Replication

[00331] Generally, an origin of replication component is not needed for mammalian expression vectors. For example, the SV40 origin may be used only because it contains the early promoter.

(Hi) Selection Gene Component

[00332] Expression and cloning vectors may contain a selection gene, also termed a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, where relevant, or (c) supply critical nutrients not available from complex media. [00333] One example of a selection scheme utilizes a drug to arrest growth of a host cell. Those cells that are successfully transformed with a heterologous gene produce a protein conferring drug resistance and thus survive the selection regimen. Examples of such dominant selection use the drugs neomycin, mycophenolic acid and hygromycin.

[00334] Another example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the antibody nucleic acid, such as DHFR, thymidine kinase, metallothionein-l and -II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc.

[00335] For example, cells transformed with the DHFR selection gene are first identified by culturing all of the transformants in a culture medium that contains methotrexate (Mtx), a competitive antagonist of DHFR. An appropriate host cell when wild- type DHFR is employed is the Chinese hamster ovary (CHO) cell line deficient in DHFR activity {e.g., ATCC CRL-9096).

[00336] Alternatively, host cells (particularly wild-type hosts that contain endogenous DHFR) transformed or co-transformed with DNA sequences encoding an antibody, wild-type DHFR protein, and another selectable marker such as aminoglycoside 3'-phosphotransferase (APH) can be selected by cell growth in medium containing a selection agent for the selectable marker such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See U.S. Patent No. 4,965, 199.

(iv) Promoter Component

[00337] Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the antibody polypeptide nucleic acid. Promoter sequences are known for eukaryotes. Virtually alleukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CNCAAT region where N may be any nucleotide. At the 3' end of most eukaryotic genes is an AATAAA sequence that may be the signal for addition of the poly A tail to the 3' end of the coding sequence. All of these sequences are suitably inserted into eukaryotic expression vectors.

[00338] Antibody polypeptide transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, from heat-shock promoters, provided such promoters are compatible with the host cell systems. [00339] The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication. The immediate early promoter of the human cytomegalovirus is conveniently obtained as a Hindi 11 E restriction fragment. A system for expressing DNA in mammalian hosts using the bovine papilloma virus as a vector is disclosed in U.S. Patent No. 4,419,446. A modification of this system is described in U.S. Patent No. 4,601 ,978. Alternatively, the Rous Sarcoma Virus long terminal repeat can be used as the promoter.

(v) Enhancer Element Component

[00340] Transcription of DNA encoding the antibody polypeptide of this disclosure by higher eukaryotes is often increased by inserting an enhancer sequence into the vector. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, a-fetoprotein, and insulin). An enhancer from a eukaryotic cell virus may also be used. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18 (1982) on enhancing elements for activation of eukaryotic promoters. The enhancer may be spliced into the vector at a position 5' or 3' to the antibody polypeptide-encoding sequence, but is preferably located at a site 5' from the promoter.

(vi) Transcription Termination Component

[00341] Expression vectors used in eukaryotic host cells will typically also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding an antibody. One useful transcription termination component is the bovine growth hormone polyadenylation region. See W094/1 1026 and the expression vector disclosed therein.

(vii) Selection and Transformation of Host Cells

[00342] Suitable host cells for cloning or expressing the DNA in the vectors herein include higher eukaryote cells described herein, including vertebrate host cells. Propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651 ); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51 ); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).

[00343] Host cells are transformed with the above-described expression or cloning vectors for antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

(viii) Culturing the Host Cells

[00344] The host cells used to produce an antibody of this disclosure may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Patent Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5, 122,469; WO 90/03430; WO 87/00195; or U.S. Patent Reissue 30,985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN™ drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

(ix) Purification of Antibody

[00345] When using recombinant techniques, the antibody can be produced intracellularly, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, are removed, for example, by centrifugation or ultrafiltration. Where the antibody is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon® ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.

[00346] The antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human γ1 , γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1 -13 (1983)). Protein G is recommended for all mouse isotypes and for human γ3 (Guss et al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH3 domain, the Bakerbond ABX™ resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™ chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.

[00347] Soluble forms of antibody or fragment present either in the cytoplasm or released from the periplasmic space may be further purified using methods known in the art, for example Fab fragments are released from the bacterial periplasmic space by osmotic shock techniques.

[00348] If inclusion bodies comprising an antibody or fragment have formed, they can often bind to the inner and/or outer cellular membranes and thus will be found primarily in the pellet material after centrifugation. The pellet material can then be treated at pH extremes or with chaotropic agent such as a detergent, guanidine, guanidine derivatives, urea, or urea derivatives in the presence of a reducing agent such as dithiothreitol at alkaline pH or tris carboxyethyl phosphine at acid pH to release, break apart, and solubilize the inclusion bodies. The soluble antibody or fragment can then be analyzed using gel electrophoresis, immunoprecipitation or the like. If it is desired to isolate a solublized antibody or antigen binding fragment isolation may be accomplished using standard methods such as those set forth below and in Marston et al. (Meth. Enz., 182:264-275 (1990)). [00349] Following any preliminary purification step(s), the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations {e.g., from about 0-0.25 M salt).

[00350] In some cases, an antibody or fragment may not be biologically active upon isolation. Various methods for "refolding" or converting a polypeptide to its tertiary structure and generating disulfide linkages, can be used to restore biological activity. Such methods include exposing the solubilized polypeptide to a pH usually above 7 and in the presence of a particular concentration of a chaotrope. The selection of chaotrope is very similar to the choices used for inclusion body solubilization, but usually the chaotrope is used at a lower concentration and is not necessarily the same as chaotropes used for the solubilization. In most cases the refolding/oxidation solution will also contain a reducing agent or the reducing agent plus its oxidized form in a specific ratio to generate a particular redox potential allowing for disulfide shuffling to occur in the formation of the protein's cysteine bridge(s). Some of the commonly used redox couples include cysteine/cystamine, glutathione (GSH)/dithiobis GSH, cupric chloride, dithiothreitol(DTT)/dithiane DTT, and 2- mercaptoethanol(bME)/di-thio-b(ME). In many instances, a cosolvent may be used to increase the efficiency of the refolding, and common reagents used for this purpose include glycerol, polyethylene glycol of various molecular weights, arginine and the like.

Immunoconjugates

[00351] The disclosure also provides immunoconjugates (interchangeably termed "antibody-drug conjugates" or "ADC"), comprising any of the DLL4 antibodies described herein conjugated to a cytotoxic agent such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin {e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope {e.g., a radioconjugate).

[00352] The use of antibody-drug conjugates for the local delivery of cytotoxic or cytostatic agents. For example, drugs to kill or inhibit tumor cells in the treatment of cancer (Syrigos and Epenetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drg Del. Rev. 26:151-172; U.S. Patent No. 4,975,278) allows targeted delivery of the drug moiety to tumors, and intracellular accumulation therein, where systemic administration of these unconjugated drug agents may result in unacceptable levels of toxicity to normal cells as well as the tumor cells sought to be eliminated (Baldwin et at., (1986) Lancet pp. (Mar. 15, 1986):603-05; Thorpe, (1985) "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical Applications, A. Pinchera et al. (ed.s), pp. 475-506). Maximal efficacy with minimal toxicity is sought thereby. Both polyclonal antibodies and monoclonal antibodies have been reported as useful in these strategies (Rowland et al., (1986) Cancer Immunol. Immunother., 21 :183-87). Drugs used in these methods include daunomycin, doxorubicin, methotrexate, and vindesine (Rowland et al., (1986) Supra). Toxins used in antibody-toxin conjugates include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins such as geldanamycin (Mandler et al (2000) Jour, of the Nat. Cancer Inst. 92(19):1573-1581 ; Mandler et al (2000) Bioorganic & Med. Chem. Letters 10: 1025-1028; Mandler et al (2002) Bioconjugate Chem. 13:786-791 ), maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al (1998) Cancer Res. 58:2928; Hinman et al (1993) Cancer Res. 53:3336-3342). The toxins may effect their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition. Some cytotoxic drugs tend to be inactive or less active when conjugated to large antibodies or protein receptor ligands.

[00353] ZEVALIN® (ibritumomab tiuxetan, Biogen/ldec) is an antibody-radioisotope conjugate composed of a murine lgG1 kappa monoclonal antibody directed against the CD20 antigen found on the surface of normal and malignant B lymphocytes and 111ln or 90Y radioisotope bound by a thiourea linker-chelator (Wiseman et al (2000) Eur. Jour. Nucl. Med. 27(7):766-77; Wiseman et al (2002) Blood 99(12):4336-42; Witzig et al (2002) J. Clin. Oncol. 20(10):2453-63; Witzig et al (2002) J. Clin. Oncol. 20(15):3262-69). Although ZEVALIN has activity against B-cell non-Hodgkin's Lymphoma (NHL), administration results in severe and prolonged cytopenias in most patients. MYLOTARG™ (gemtuzumab ozogamicin, Wyeth Pharmaceuticals), an antibody drug conjugate composed of a hu CD33 antibody linked to calicheamicin, was approved in 2000 for the treatment of acute myeloid leukemia by injection (Drugs of the Future (2000) 25(7):686; U.S. Patent Nos. 4,970, 198; 5,079,233; 5,585,089; 5,606,040; 5,693,762; 5,739,1 16; 5,767,285; 5,773,001 ). Cantuzumab mertansine (Immunogen, Inc.), an antibody drug conjugate composed of the huC242 antibody linked via the disulfide linker SPP to the maytansinoid drug moiety, DM1 , is advancing into Phase II trials for the treatment of cancers that express CanAg, such as colon, pancreatic, gastric, and others. MLN-2704 (Millennium Pharm., BZL Biologies, Immunogen Inc.), an antibody drug conjugate composed of the anti-prostate specific membrane antigen (PSMA) monoclonal antibody linked to the maytansinoid drug moiety, DM1 , is under development for the potential treatment of prostate tumors. The auristatin peptides, auristatin E (AE) and monomethylauristatin (MMAE), synthetic analogs of dolastatin, were conjugated to chimeric monoclonal antibodies cBR96 (specific to Lewis Y on carcinomas) and cAC10 (specific to CD30 on hematological malignancies) (Doronina et al (2003) Nature Biotechnology 21 (7):778-784) and are under therapeutic development.

[00354] Chemotherapeutic agents useful in the generation of immunoconjugates are described herein. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. See, e.g., WO 93/21232 published Oct. 28, 1993. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 212Bi, 1311, 131 In, 90Y, and 186Re. Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCI), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)- ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1 ,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin may be prepared as described in Vitetta et al., Science, 238: 1098 (1987). Carbon-14- labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/1 1026.

[00355] Conjugates of an antibody and one or more small molecule toxins, such as a calicheamicin, maytansinoids, dolastatins, aurostatins, a trichothecene, and CC1065, and the derivatives of these toxins that have toxin activity, are also contemplated herein.

a. Maytansine and Maytansinoids

[00356] In some embodiments, the immunoconjugate comprises a DLL4 antibody (full length or fragments) disclosed herein conjugated to one or more maytansinoid molecules.

[00357] Maytansinoids are mitototic inhibitors which act by inhibiting tubulin polymerization. Maytansine was first isolated from the east African shrub Maytenus serrata (U.S. Patent No. 3,896, 1 1 1 ). Subsequently, it was discovered that certain microbes also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Patent No. 4, 151 ,042). Synthetic maytansinol and derivatives and analogues thereof are disclosed, for example, in U.S. Patent Nos. 4, 137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821 ; 4,322,348; 4,331 ,598; 4,361 ,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371 ,533.

[00358] Maytansinoid drug moieties are attractive drug moieties in antibody drug conjugates because they are: (i) relatively accessible to prepare by fermentation or chemical modification, derivatization of fermentation products, (ii) amenable to derivatization with functional groups suitable for conjugation through the non-disulfide linkers to antibodies, (iii) stable in plasma, and (iv) effective against a variety of tumor cell lines.

[00359] Immunoconjugates containing maytansinoids, methods of making same, and their therapeutic use are disclosed, for example, in U.S. Patent Nos. 5,208,020, 5,416,064 and EP 0 425 235. Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) described immunoconjugates comprising a maytansinoid designated DM1 linked to the monoclonal antibody C242 directed against human colorectal cancer. The conjugate was found to be highly cytotoxic towards cultured colon cancer cells, and showed antitumor activity in an in vivo tumor growth assay. Chari et al., Cancer Research 52:127-131 (1992) describe immunoconjugates in which a maytansinoid was conjugated via a disulfide linker to the murine antibody A7 binding to an antigen on human colon cancer cell lines, or to another murine monoclonal antibody TA.1 that binds the HER-2/neu oncogene. The cytotoxicity of the TA.1 -maytansinoid conjugate was tested in vitro on the human breast cancer cell line SK-BR-3, which expresses 3x105 HER-2 surface antigens per cell. The drug conjugate achieved a degree of cytotoxicity similar to the free maytansinoid drug, which could be increased by increasing the number of maytansinoid molecules per antibody molecule. The A7-maytansinoid conjugate showed low systemic cytotoxicity in mice.

[00360] Antibody-maytansinoid conjugates are prepared by chemically linking an antibody to a maytansinoid molecule without significantly diminishing the biological activity of either the antibody or the maytansinoid molecule. See, e.g., U.S. Patent No. 5,208,020. An average of 3-4 maytansinoid molecules conjugated per antibody molecule has shown efficacy in enhancing cytotoxicity of target cells without negatively affecting the function or solubility of the antibody, although even one molecule of toxin/antibody would be expected to enhance cytotoxicity over the use of naked antibody. Maytansinoids are well known in the art and can be synthesized by known techniques or isolated from natural sources. Suitable maytansinoids are disclosed, for example, in U.S. Patent No. 5,208,020 and in the other patents and nonpatent publications referred to hereinabove. Preferred maytansinoids are maytansinol and maytansinol analogues modified in the aromatic ring or at other positions of the maytansinol molecule, such as various maytansinol esters.

[00361] There are many linking groups known in the art for making antibody- maytansinoid conjugates, including, for example, those disclosed in U.S. Patent Nos. 5,208,020, 6,441 , 163, or EP Patent 0 425 235, Chari et al., Cancer Research 52:127-131 (1992). Antibody-maytansinoid conjugates comprising the linker component SMCC may be prepared. The linking groups include disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups, or esterase labile groups, as disclosed in the above-identified patents, disulfide and thioether groups being preferred. Additional linking groups are described and exemplified herein.

[00362] Conjugates of the antibody and maytansinoid may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1 -carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCI), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis- diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1 ,5- difluoro-2,4-dinitrobenzene). Particularly preferred coupling agents include N-succinimidyl- 3-(2-pyridyldithio) propionate (SPDP) (Carlsson et al., Biochem. J. 173:723-737 (1978)) and N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for a disulfide linkage.

[00363] The linker may be attached to the maytansinoid molecule at various positions, depending on the type of the link. For example, an ester linkage may be formed by reaction with a hydroxyl group using conventional coupling techniques. The reaction may occur at the C-3 position having a hydroxyl group, the C-14 position modified with hydroxymethyl, the C-15 position modified with a hydroxyl group, and the C-20 position having a hydroxyl group. In a preferred embodiment, the linkage is formed at the C-3 position of maytansinol or a maytansinol analogue.

b. Auristatins and Dolastatins

[00364] In some embodiments, the immunoconjugate comprises an antibody disclosed herein conjugated to dolastatins or dolostatin peptidic analogs and derivatives, the auristatins (U.S. Patent Nos. 5,635,483; 5,780,588). Dolastatins and auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cellular division (Woyke et al (2001 ) Antimicrob. Agents and Chemother. 45(12):3580- 3584) and have anticancer (U.S. Patent No. 5,663, 149) and antifungal activity (Pettit et al (1998) Antimicrob. Agents Chemother. 42:2961 -2965). The dolastatin or auristatin drug moiety may be attached to the antibody through the N (amino) terminus or the C (carboxyl) terminus of the peptidic drug moiety (WO 02/088172).

[00365] Exemplary auristatin embodiments include the N-terminus linked monomethylauristatin drug moieties DE and DF, (see, e.g., U.S. Patent No. 7,498,298).

[00366] Typically, peptide-based drug moieties can be prepared by forming a peptide bond between two or more amino acids and/or peptide fragments. Such peptide bonds can be prepared, for example, according to the liquid phase synthesis method (see, e.g., E. Schroder and K. Lubke, "The Peptides", volume 1 , pp 76-136, 1965, Academic Press) that is well known in the field of peptide chemistry. The auristatin/dolastatin drug moieties may be prepared according to the methods of: U.S. Patent No. 5,635,483; U.S. Patent No. 5,780,588; Pettit et al (1989) J. Am. Chem. Soc. 1 1 1 :5463-5465; Pettit et al. (1998) Anti-Cancer Drug Design 13:243-277; Pettit, G. R., et al. Synthesis, 1996, 719-725; and Pettit et al. (1996) J. Chem. Soc. Perkin Trans. 1 5:859-863. See also Doronina (2003) Nat Biotechnol 21 (7):778-784; U.S. Patent No. 7,498,289, (disclosing, linkers and methods of preparing monomethylvaline compounds such as MMAE and MMAF conjugated to linkers).

c. Calicheamicin

[00367] In other embodiments, the immunoconjugate comprises an antibody disclosed herein conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics are capable of producing double-stranded DNA breaks at sub- picomolar concentrations. For the preparation of conjugates of the calicheamicin family, see U.S. Patent Nos. 5,712,374, 5,714,586, 5,739, 1 16, 5,767,285, 5,770,701 , 5,770,710, 5,773,001 , 5,877,296. Structural analogues of calicheamicin which may be used include, but are not limited to, γ , α2', α3', N-acetyl-y , PSAG and θΊ (see, e.g., Hinman et al., Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928 (1998) and the aforementioned U.S. patents). Another anti-tumor drug that the antibody can be conjugated is QFA which is an antifolate. Both calicheamicin and QFA have intracellular sites of action and do not readily cross the plasma membrane. Therefore, cellular uptake of these agents through antibody mediated internalization greatly enhances their cytotoxic effects.

d. Other Cytotoxic Agents

[00368] Other antitumor agents that can be conjugated to the antibodies disclosed herein include BCNU, streptozoicin, vincristine and 5-fluorouracil, the family of agents known collectively LL-E33288 complex described in U.S. Patent Nos. 5,053,394, 5,770,710, as well as esperamicins (U.S. Patent No. 5,877,296). [00369] Enzymatically active toxins and fragments thereof which can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. See, for example, WO 93/21232 published Oct. 28, 1993.

[00370] The present disclosure further contemplates an immunoconjugate formed between an antibody and a compound with nucleolytic activity {e.g., a ribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).

[00371] For selective destruction of the tumor, the antibody may comprise a highly radioactive atom. A variety of radioactive isotopes are available for the production of radioconjugated antibodies. Examples include At 211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and radioactive isotopes of Lu. When the conjugate is used for detection, it may comprise a radioactive atom for scintigraphic studies, for example tc99m or I123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123 again, iodine-131 , indium-1 1 1 , fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

[00372] The radiolabels or other labels may be incorporated in the conjugate in known ways. For example, the peptide may be biosynthesized or may be synthesized by chemical amino acid synthesis using suitable amino acid precursors involving, for example, fluorine-19 in place of hydrogen. Labels such as tc99m or I123, Re186, Re188 and In111 can be attached via a cysteine residue in the peptide. Yttrium-90 can be attached via a lysine residue. The IODOGEN method (Fraker et al (1978) Biochem. Biophys. Res. Commun. 80: 49-57) can be used to incorporate iodine-123. "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989) describes other methods.

[00373] Conjugates of the antibody and cytotoxic agent may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1 -carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCI), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis- diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1 ,5- difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled 1- isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/1 1026. The linker may be a "cleavable linker" facilitating release of the cytotoxic drug in the cell. For example, an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide-containing linker (Chari et al., Cancer Research 52:127- 131 (1992); U.S. Patent No. 5,208,020) may be used.

[00374] The compounds disclosed herein expressly contemplate, but are not limited to, ADC prepared with cross-linker reagents: BMPS, EMCS, GMBS, HBVS, LC- SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) which are commercially available {e.g., from Pierce Biotechnology, Inc., Rockford, III., U.S.A). See pages 467-498, 2003-2004 Applications Handbook and Catalog.

e. Preparation of Antibody Drug Conjugates

[00375] In the antibody drug conjugates (ADC) disclosed herein, an antibody (Ab) is conjugated to one or more drug moieties (D), e.g,. about 1 to about 20 drug moieties per antibody, through a linker (L). An ADC of Formula I [Ab-(L-D)p] may be prepared by several routes, employing organic chemistry reactions, conditions, and reagents known to those skilled in the art, including: (1 ) reaction of a nucleophilic group of an antibody with a bivalent linker reagent, to form Ab-L, via a covalent bond, followed by reaction with a drug moiety D; and (2) reaction of a nucleophilic group of a drug moiety with a bivalent linker reagent, to form D-L, via a covalent bond, followed by reaction with the nucleophilic group of an antibody. Additional methods for preparing ADC are described herein.

[00376] The linker may be composed of one or more linker components. Exemplary linker components include 6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP"), valine-citrulline ("val-cit"), alanine-phenylalanine ("ala-phe"), p- aminobenzyloxycarbonyl ("PAB"), N-Succinimidyl 4-(2-pyridylthio) pentanoate ("SPP"), N- Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate ("SMCC"), and N- Succinimidyl (4-iodo-acetyl)aminobenzoate ("SIAB"). Additional linker components are known in the art and some are disclosed herein (see, e.g., U.S. Patent No. 7,498,298).

[00377] In some embodiments, the linker may comprise amino acid residues. Exemplary amino acid linker components include a dipeptide, a tripeptide, a tetrapeptide or a pentapeptide. Exemplary dipeptides include: valine-citrulline (vc or val-cit), alanine- phenylalanine (af or ala-phe). Exemplary tripeptides include: glycine-valine-citrulline (gly- val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acid residues which comprise an amino acid linker component include those occurring naturally, as well as minor amino acids and non-naturally occurring amino acid analogs, such as citrulline. Amino acid linker components can be designed and optimized in their selectivity for enzymatic cleavage by a particular enzymes, for example, a tumor-associated protease, cathepsin B, C and D, or a plasmin protease.

[00378] Nucleophilic groups on antibodies include, but are not limited to: (i) N- terminal amine groups, (ii) side chain amine groups, e.g., lysine, (iii) side chain thiol groups, e.g., cysteine, and (iv) sugar hydroxyl or amino groups where the antibody is glycosylated. Amine, thiol, and hydroxyl groups are nucleophilic and capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups. Certain antibodies have reducible interchain disulfides, e.g., cysteine bridges. Antibodies may be made reactive for conjugation with linker reagents by treatment with a reducing agent such as DTT (dithiothreitol). Each cysteine bridge will thus form, theoretically, two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into antibodies through the reaction of lysines with 2- iminothiolane (Traut's reagent) resulting in conversion of an amine into a thiol. Reactive thiol groups may be introduced into the antibody (or fragment thereof) by introducing one, two, three, four, or more cysteine residues {e.g., preparing mutant antibodies comprising one or more non-native cysteine amino acid residues).

[00379] Antibody drug conjugates disclosed herein may also be produced by modification of the antibody to introduce electrophilic moieties, which can react with nucleophilic substituents on the linker reagent or drug. The sugars of glycosylated antibodies may be oxidized, e.g., with periodate oxidizing reagents, to form aldehyde or ketone groups which may react with the amine group of linker reagents or drug moieties. The resulting imine Schiff base groups may form a stable linkage, or may be reduced, e.g., by borohydride reagents to form stable amine linkages. In one embodiment, reaction of the carbohydrate portion of a glycosylated antibody with either glactose oxidase or sodium meta-periodate may yield carbonyl (aldehyde and ketone) groups in the protein that can react with appropriate groups on the drug (Hermanson, Bioconjugate Techniques). In another embodiment, proteins containing N-terminal serine or threonine residues can react with sodium meta-periodate, resulting in production of an aldehyde in place of the first amino acid (Geoghegan & Stroh, (1992) Bioconjugate Chem. 3:138-146; U.S. Patent No. 5,362,852). Such aldehyde can be reacted with a drug moiety or linker nucleophile.

[00380] Likewise, nucleophilic groups on a drug moiety include, but are not limited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups.

[00381] Alternatively, a fusion protein comprising the antibody and cytotoxic agent may be made, e.g., by recombinant techniques or peptide synthesis. The length of DNA may comprise respective regions encoding the two portions of the conjugate either adjacent one another or separated by a region encoding a linker peptide which does not destroy the desired properties of the conjugate.

[00382] In yet another embodiment, the antibody may be conjugated to a

"receptor" (such streptavidin) for utilization in tumor pre-targeting wherein the antibody- receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand" {e.g., avidin) which is conjugated to a cytotoxic agent {e.g., a radionucleotide).

Pharmaceutical Formulations

[00383] DLL4 binding antibodies, antibody fragments, nucleic acids, or vectors disclosed herein can be formulated in compositions, especially pharmaceutical compositions. Such compositions with antibodies to DLL4 comprise a therapeutically or prophylactically effective amount of a DLL4 binding antibody, antibody fragment, nucleic acid, or vector disclosed herein in admixture with a suitable carrier, e.g., a pharmaceutically acceptable agent. Typically, DLL4 binding antibodies, antibody fragments, nucleic acids, or vectors disclosed herein are sufficiently purified for administration before formulation in a pharmaceutical composition.

[00384] Pharmaceutically acceptable agents for use in the present pharmaceutical compositions include carriers, excipients, diluents, antioxidants, preservatives, coloring, flavoring and diluting agents, emulsifying agents, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, tonicity agents, cosolvents, wetting agents, complexing agents, buffering agents, antimicrobials, and surfactants.

[00385] Neutral buffered saline or saline mixed with serum albumin are exemplary appropriate carriers. The pharmaceutical compositions may include antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as Tween, pluronics, or polyethylene glycol (PEG). Also by way of example, suitable tonicity enhancing agents include alkali metal halides (preferably sodium or potassium chloride), mannitol, sorbitol, and the like. Suitable preservatives include benzalkonium chloride, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid and the like. Hydrogen peroxide also may be used as preservative. Suitable cosolvents include glycerin, propylene glycol, and PEG. Suitable complexing agents include caffeine, polyvinylpyrrolidone, beta- cyclodextrin or hydroxy-propyl-beta-cyclodextrin. Suitable surfactants or wetting agents include sorbitan esters, polysorbates such as polysorbate 80, tromethamine, lecithin, cholesterol, tyloxapal, and the like. The buffers may be conventional buffers such as acetate, borate, citrate, phosphate, bicarbonate, or Tris-HCI. Acetate buffer may be about pH 4-5.5, and Tris buffer can be about pH 7-8.5. Additional pharmaceutical agents are set forth in Remington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, ed., Mack Publishing Company, 1990.

[00386] The composition may be in liquid form or in a lyophilized or freeze-dried form and may include one or more lyoprotectants, excipients, surfactants, high molecular weight structural additives and/or bulking agents (see, for example, U.S. Patent Nos. 6,685,940, 6,566,329, and 6,372,716). In one embodiment, a lyoprotectant is included, which is a non-reducing sugar such as sucrose, lactose or trehalose. The amount of lyoprotectant generally included is such that, upon reconstitution, the resulting formulation will be isotonic, although hypertonic or slightly hypotonic formulations also may be suitable. In addition, the amount of lyoprotectant should be sufficient to prevent an unacceptable amount of degradation and/or aggregation of the protein upon lyophilization. Exemplary lyoprotectant concentrations for sugars {e.g., sucrose, lactose, trehalose) in the pre- lyophilized formulation are from about 10 mM to about 400 mM. In another embodiment, a surfactant is included, such as for example, nonionic surfactants and ionic surfactants such as polysorbates {e.g., polysorbate 20, polysorbate 80); poloxamers {e.g., poloxamer 188); poly(ethylene glycol) phenyl ethers {e.g., Triton); sodium dodecyl sulfate (SDS); sodium laurel sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl, myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine {e.g., lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodium methyl ofeyl-ta urate; and the MONAQUAT™. series (Mona Industries, Inc., Paterson, N.J.), polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol {e.g., Pluronics, PF68 etc). Exemplary amounts of surfactant that may be present in the pre-lyophilized formulation are from about 0.001-0.5%. High molecular weight structural additives {e.g., fillers, binders) may include for example, acacia, albumin, alginic acid, calcium phosphate (dibasic), cellulose, carboxymethylcellulose, carboxymethylcellulose sodium, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, dextran, dextrin, dextrates, sucrose, tylose, pregelatinized starch, calcium sulfate, amylose, glycine, bentonite, maltose, sorbitol, ethylcellulose, disodium hydrogen phosphate, disodium phosphate, disodium pyrosulfite, polyvinyl alcohol, gelatin, glucose, guar gum, liquid glucose, compressible sugar, magnesium aluminum silicate, maltodextrin, polyethylene oxide, polymethacrylates, povidone, sodium alginate, tragacanth microcrystalline cellulose, starch, and zein. Exemplary concentrations of high molecular weight structural additives are from 0.1 % to 10% by weight. In other embodiments, a bulking agent {e.g., mannitol, glycine) may be included.

[00387] Compositions may be suitable for parenteral administration. Exemplary compositions are suitable for injection or infusion into an animal by any route available to the skilled worker, such as intraarticular, subcutaneous, intravenous, intramuscular, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, or intralesional routes. A parenteral formulation typically will be a sterile, pyrogen-free, isotonic aqueous solution, optionally containing pharmaceutically acceptable preservatives.

[00388] Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringers' dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, anti-microbials, anti-oxidants, chelating agents, inert gases and the like. See generally, Remington's Pharmaceutical Science, 16th Ed., Mack Eds., 1980.

[00389] Pharmaceutical compositions described herein may be formulated for controlled or sustained delivery in a manner that provides local concentration of the product {e.g., bolus, depot effect) and/or increased stability or half-life in a particular local environment. The compositions can include the formulation of DLL4 binding antibodies, antibody fragments, nucleic acids, or vectors disclosed herein with particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc., as well as agents such as a biodegradable matrix, injectable microspheres, microcapsular particles, microcapsules, bioerodible particles beads, liposomes, and implantable delivery devices that provide for the controlled or sustained release of the active agent which then can be delivered as a depot injection. Techniques for formulating such sustained- or controlled- delivery means are known and a variety of polymers have been developed and used for the controlled release and delivery of drugs. Such polymers are typically biodegradable and biocompatible. Polymer hydrogels, including those formed by complexation of enantiomeric polymer or polypeptide segments, and hydrogels with temperature or pH sensitive properties, may be desirable for providing drug depot effect because of the mild and aqueous conditions involved in trapping bioactive protein agents {e.g., antibodies to DLL4). See, for example, the description of controlled release porous polymeric microparticles for the delivery of pharmaceutical compositions in WO 93/15722.

[00390] Suitable materials for this purpose include polylactides (see, e.g., U.S. Patent No. 3,773,919), polymers of poly-(a-hydroxycarboxylic acids), such as poly-D-(-)-3- hydroxybutyric acid (EP 133,988A), copolymers of L-glutamic acid and gamma ethyl-L- glutamate (Sidman et al., Biopolymers, 22: 547-556 (1983)), poly(2-hydroxyethyl- methacrylate) (Langer et al., J. Biomed. Mater. Res., 15: 167-277 (1981 ), and Langer, Chem. Tech., 12: 98-105 (1982)), ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyric acid. Other biodegradable polymers include poly(lactones), poly(acetals), poly(orthoesters), and poly(orthocarbonates). Sustained-release compositions also may include liposomes, which can be prepared by any of several methods known in the art (see, e.g., Eppstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688-92 (1985)). The carrier itself, or its degradation products, should be nontoxic in the target tissue and should not further aggravate the condition. This can be determined by routine screening in animal models of the target disorder or, if such models are unavailable, in normal animals.

[00391] Microencapsulation of recombinant proteins for sustained release has been performed successfully with human growth hormone (rhGH), interferon-(rhlFN-), interleukin-2, and MN rgp120. Johnson et al., Nat. Med., 2:795-799 (1996); Yasuda, Biomed. Ther., 27:1221-1223 (1993); Hora et al., Bio/Technology. 8:755-758 (1990); Cleland, "Design and Production of Single Immunization Vaccines Using Polylactide Polyglycolide Microsphere Systems," in Vaccine Design: The Subunit and Adjuvant Approach, Powell and Newman, eds, (Plenum Press: New York, 1995), pp. 439-462; WO 97/03692, WO 96/40072, WO 96/07399; and U.S. Patent No. 5,654,010. The sustained- release formulations of these proteins were developed using poly-lactic-coglycolic acid (PLGA) polymer due to its biocompatibility and wide range of biodegradable properties. The degradation products of PLGA, lactic and glycolic acids can be cleared quickly within the human body. Moreover, the degradability of this polymer can be depending on its molecular weight and composition. Lewis, "Controlled release of bioactive agents from lactide/glycolide polymer," in: M. Chasin and R. Langer (Eds.), Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: New York, 1990), pp. 1 -41 . Additional examples of sustained release compositions include, for example, EP 58,481A, U.S. Patent No. 3,887,699, EP 158,277A, Canadian Patent No. 1 176565, U. Sidman et al., Biopolymers 22, 547 [1983], R. Langer et al., Chem. Tech. 12, 98 [1982], Sinha et al., J. Control. Release 90, 261 [2003], Zhu et al., Nat. Biotechnol. 18, 24 [2000], and Dai et al., Colloids Surf B Biointerfaces 41 , 1 17 [2005].

[00392] Bioadhesive polymers are also contemplated for use in or with compositions of the present disclosure. Bioadhesives are synthetic and naturally occurring materials able to adhere to biological substrates for extended time periods. For example, Carbopol and polycarbophil are both synthetic cross-linked derivatives of poly(acrylic acid). Bioadhesive delivery systems based on naturally occurring substances include for example hyaluronic acid, also known as hyaluronan. Hyaluronic acid is a naturally occurring mucopolysaccharide consisting of residues of D-glucuronic and N-acetyl-D-glucosamine. Hyaluronic acid is found in the extracellular tissue matrix of vertebrates, including in connective tissues, as well as in synovial fluid and in the vitreous and aqueous humor of the eye. Esterified derivatives of hyaluronic acid have been used to produce microspheres for use in delivery that are biocompatible and biodegradable (see, for example, Cortivo et al., Biomaterials (1991 ) 12:727-730; EP 517,565; WO 96/29998; Ilium et al., J. Controlled Rel. (1994) 29:133-141 ). Exemplary hyaluronic acid containing compositions of the present disclosure comprise a hyaluronic acid ester polymer in an amount of approximately 0.1 % to about 40% (w/w) of an DLL4 binding antibody or fragment to hyaluronic acid polymer.

[00393] Both biodegradable and non-biodegradable polymeric matrices may be used to deliver compositions of the present disclosure, and such polymeric matrices may comprise natural or synthetic polymers. Biodegradable matrices are preferred. The period of time over which release occurs is based on selection of the polymer. Typically, release over a period ranging from between a few hours and three to twelve months is most desirable. Exemplary synthetic polymers which may be used to form the biodegradable delivery system include: polymers of lactic acid and glycolic acid, polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, poly-vinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyanhydrides, polyurethanes and co- polymers thereof, poly(butic acid), poly(valeric acid), alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose, cellulose triacetate, cellulose sulphate sodium salt, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene, polypropylene, poly(ethylene glycol), poly(ethylene oxide), poly(ethylene terephthalate), polyvinyl alcohols), polyvinyl acetate, poly vinyl chloride, polystyrene and polyvinylpyrrolidone. Exemplary natural polymers include alginate and other polysaccharides including dextran and cellulose, collagen, chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins, zein and other prolamines and hydrophobic proteins, copolymers and mixtures thereof. In general, these materials degrade either by enzymatic hydrolysis or exposure to water in vivo, by surface or bulk erosion. The polymer optionally is in the form of a hydrogel (see, for example, WO 04/009664, WO 05/087201 , Sawhney, et al., Macromolecules, 1993, 26, 581 -587) that can absorb up to about 90% of its weight in water and further, optionally is cross-linked with multi-valent ions or other polymers.

[00394] Delivery systems also include non-polymer systems that are lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-di- and tri-glycerides; hydrogel release systems; silastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like. Specific examples include, but are not limited to: (a) erosional systems in which the product is contained in a form within a matrix such as those described in U.S. Patent Nos. 4,452,775, 4,675,189 and 5,736, 152 and (b) diffusional systems in which a product permeates at a controlled rate from a polymer such as described in U.S. Patent Nos. 3,854,480, 5, 133,974 and 5,407,686. Liposomes containing the product may be prepared by methods known methods, such as for example (DE 3,218,121 ; Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77: 4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641 ; JP 83-1 18008; U.S. Patent Nos. 4,485,045 and 4,544,545; and EP 102,324).

[00395] Alternatively or additionally, the compositions may be administered locally via implantation into the affected area of a membrane, sponge, or other appropriate material on to which an DLL4 binding antibody, antibody fragment, nucleic acid, or vector disclosed herein has been absorbed or encapsulated. Where an implantation device is used, the device may be implanted into any suitable tissue or organ, and delivery of an DLL4 binding antibody, antibody fragment, nucleic acid, or vector disclosed herein can be directly through the device via bolus, or via continuous administration, or via catheter using continuous infusion.

[00396] A pharmaceutical composition comprising an DLL4 binding antibody, antibody fragment, nucleic acid, or vector disclosed herein may be formulated for inhalation, such as for example, as a dry powder. Inhalation solutions also may be formulated in a liquefied propellant for aerosol delivery. In yet another formulation, solutions may be nebulized. Additional pharmaceutical composition for pulmonary administration include, those described, for example, in WO 94/20069, which discloses pulmonary delivery of chemically modified proteins. For pulmonary delivery, the particle size should be suitable for delivery to the distal lung. For example, the particle size may be from 1 μηη to 5 μηη; however, larger particles may be used, for example, if each particle is fairly porous.

[00397] Certain formulations containing DLL4 binding antibodies, antibody fragments, nucleic acids, or vectors disclosed herein may be administered orally. Formulations administered in this fashion may be formulated with or without those carriers customarily used in the compounding of solid dosage forms such as tablets and capsules. For example, a capsule can be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. Additional agents may be included to facilitate absorption of a selective binding agent. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders also can be employed.

[00398] Another preparation may involve an effective quantity of a DLL4 binding antibody, antibody fragment, nucleic acid, or vector disclosed herein in a mixture with nontoxic excipients which are suitable for the manufacture of tablets. By dissolving the tablets in sterile water, or another appropriate vehicle, solutions may be prepared in unit dose form. Suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.

[00399] Suitable and/or preferred pharmaceutical formulations may be determined in view of the present disclosure and general knowledge of formulation technology, depending upon the intended route of administration, delivery format, and desired dosage. Regardless of the manner of administration, an effective dose may be calculated according to patient body weight, body surface area, or organ size. Further refinement of the calculations for determining the appropriate dosage for treatment involving each of the formulations described herein are routinely made in the art and is within the ambit of tasks routinely performed in the art. Appropriate dosages may be ascertained through use of appropriate dose-response data.

[00400] In some embodiments, antibodies to DLL4 or fragments thereof are provided with a modified Fc region where a naturally-occurring Fc region is modified to increase the half-life of the antibody or fragment in a biological environment, for example, the serum half-life or a half-life measured by an in vitro assay. Methods for altering the original form of a Fc region of an IgG also are described in U.S. Patent No. 6,998,253.

[00401] In certain embodiments, it may be desirable to modify the antibody or fragment in order to increase its serum half-life, for example, adding molecules such as PEG or other water soluble polymers, including polysaccharide polymers, to antibody fragments to increase the half-life. This may also be achieved, for example, by incorporation of a salvage receptor binding epitope into the antibody fragment {e.g., by mutation of the appropriate region in the antibody fragment or by incorporating the epitope into a peptide tag that is then fused to the antibody fragment at either end or in the middle, e.g., by DNA or peptide synthesis) (see, International Publication No. W096/32478). Salvage receptor binding epitope refers to an epitope of the Fc region of an IgG molecule {e.g., lgG1 , lgG2, lgG3, or lgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.

[00402] A salvage receptor binding epitope may include a region wherein any one or more amino acid residues from one or two loops of a Fc domain are transferred to an analogous position of the antibody fragment. Even more preferably, three or more residues from one or two loops of the Fc domain are transferred. Still more preferred, the epitope is taken from the CH2 domain of the Fc region {e.g., of an IgG) and transferred to the CH1 , CH3, or VH region, or more than one such region, of the antibody. Alternatively, the epitope is taken from the CH2 domain of the Fc region and transferred to the CL region or VL region, or both, of the antibody fragment. See also WO 97/34631 and WO 96/32478 which describe Fc variants and their interaction with the salvage receptor.

[00403] Mutation of residues within Fc receptor binding sites may result in altered effector function, such as altered ADCC or CDC activity, or altered half-life. Potential mutations include insertion, deletion or substitution of one or more residues, including substitution with alanine, a conservative substitution, a non-conservative substitution, or replacement with a corresponding amino acid residue at the same position from a different IgG subclass {e.g., replacing an lgG1 residue with a corresponding lgG2 residue at that position). For example, it has been reported that mutating the serine at amino acid position 241 in lgG4 to proline (found at that position in lgG1 and lgG2) led to the production of a homogeneous antibody, as well as extending serum half-life and improving tissue distribution compared to the original chimeric lgG4. (Angal et al., Mol. Immunol. 30:105-8, 1993).

Uses and Therapeutic Methods

[00404] An antibody to DLL4 of the present disclosure may be used in, for example, in vitro, ex vivo and in vivo methods, including, for example, therapeutic, diagnostic and/or prognostic methods.

[00405] In one aspect, the disclosure provides methods for treating or preventing a tumor, a cancer, and/or a cell proliferative disorder associated with increased expression and/or activity of DLL4, the methods comprising administering an effective amount of a

DLL4 antibody to a subject in need of such treatment.

[00406] In one aspect, the disclosure provides methods for reducing, inhibiting, blocking, or preventing growth of a tumor or cancer, the methods comprising administering an effective amount of a DLL4 antibody to a subject in need of such treatment.

[00407] In one aspect, the disclosure provides methods for treating a tumor, a cancer, and/or a cell proliferative disorder comprising administering an effective amount of a DLL4 antibody to a subject in need of such treatment.

[00408] In one aspect, the disclosure provides methods for inhibiting angiogenesis comprising administering an effective amount of a DLL4 antibody to a subject in need of such treatment.

[00409] In one aspect, the disclosure provides methods for treating a pathological condition associated with angiogenesis comprising administering an effective amount of a

DLL4 antibody to a subject in need of such treatment. In some embodiments, the pathological condition associated with angiogenesis is a tumor, a cancer, and/or a cell proliferative disorder. In some embodiments, the pathological condition associated with angiogenesis is an intraocular neovascular disease.

[00410] Moreover, at least some of the antibodies to DLL4 disclosed herein can bind to DLL4 antigen from other species. Accordingly, the antibodies disclosed herein may be used to bind specific antigen activity, e.g., in a cell culture containing the antigen, in human subjects or in other mammalian subjects having the antigen with which an antibody disclosed herein cross-reacts {e.g., chimpanzee, baboon, marmoset, cynomolgus and rhesus, pig or mouse). In one embodiment, the antibody disclosed herein can be used for inhibiting antigen activities by contacting the antibody with the antigen such that antigen activity is inhibited. Preferably, the antigen is a human protein molecule {e.g., a human DLL4 sequence).

[0041 1] In one embodiment, an antibody disclosed herein can be used in a method for binding an antigen in a subject suffering from a disorder associated with increased antigen expression and/or activity, comprising administering to the subject an antibody disclosed herein such that the antigen in the subject is bound. Preferably, the antigen is a human protein molecule and the subject is a human subject. Alternatively, the subject can be a mammal expressing the antigen with which an antibody disclosed herein binds. Still further, the subject can be a mammal into which the antigen has been introduced {e.g., by administration of the antigen or by expression of an antigen transgene). An antibody to DLL4 disclosed herein can be administered to a human subject for therapeutic purposes. Moreover, an antibody disclosed herein can be administered to a non-human mammal expressing an antigen with which the immunoglobulin cross-reacts {e.g., a primate, pig or mouse) 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 disclosed herein {e.g., testing of dosages and time courses of administration).

[00412] The antibodies to DLL4 disclosed herein can be used to treat, inhibit, delay progression of, prevent/delay recurrence of, ameliorate, or prevent diseases, disorders or conditions associated with expression and/or activity of one or more DLL4 antigen molecules.

[00413] Exemplary disorders include cancers such as carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer {e.g., epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, melanoma, multiple myeloma and B-cell lymphoma, brain, as well as head and neck cancer, and associated metastases. In some embodiments, the cancer is selected from the group consisting of small cell lung cancer, neuroblastomas, melanoma, breast carcinoma, gastric cancer, colorectal cancer (CRC), and hepatocellular carcinoma. In some embodiments, the cancer is selected from the group consisting of non-small cell lung cancer, colorectal cancer and breast carcinoma, including metastatic forms of those cancers.

[00414] In certain embodiments, an immunoconjugate comprising an antibody to DLL4 conjugated with one or more cytotoxic agent(s) is administered to the patient. In some embodiments, the immunoconjugate and/or antigen to which it is bound is/are internalized by the cell, resulting in increased therapeutic efficacy of the immunoconjugate in killing the target cell to which it binds. In one embodiment, the cytotoxic agent targets or interferes with nucleic acid in the target cell. In one embodiment, the cytotoxic agent targets or interferes with microtubule polymerization. Examples of such cytotoxic agents include any of the chemotherapeutic agents noted herein (such as a maytansinoid, auristatin, dolastatin, or a calicheamicin), a radioactive isotope, or a ribonuclease or a DNA endonuclease.

[00415] Antibodies disclosed herein can be used either alone or in combination with other compositions in a therapy. For instance, an antibody disclosed herein may be co-administered with another antibody, chemotherapeutic agent(s) (including cocktails of chemotherapeutic agents), other cytotoxic agent(s), anti-angiogenic agent(s), cytokines, and/or growth inhibitory agent(s). Where an antibody disclosed herein inhibits tumor growth, it may be particularly desirable to combine it with one or more other therapeutic agent(s) which also inhibits tumor growth, e.g., anti-VEGF agents including antibodies to VEGF. Alternatively, or additionally, the patient may receive combined radiation therapy (e.g., external beam irradiation or therapy with a radioactive labeled agent, such as an antibody). Such combined therapies noted above include combined administration (where the two or more agents are included in the same or separate formulations), and separate administration, in which case, administration of the antibody disclosed herein can occur prior to, and/or following, administration of the adjunct therapy or therapies.

[00416] DLL4 antibodies include those that can have one or more of the following effects, for example: interfere with the expression of a cancer cell marker; interfere with activation of a cancer cell signal transduction pathway by, for example, sterically inhibiting interactions between a cancer cell marker and its ligand, receptor or co-receptors; activate a cancer cell signal transduction pathway by, for example, acting as a ligand or promoting the binding of an endogenous ligand; or bind to a cancer cell marker and inhibit tumor cell proliferation. Cancer cells may include cancer stem cells.

[00417] In certain embodiments, antibodies against a cancer cell marker act extracellularly to modulate the function of a cancer cell marker protein. In some embodiments, extracellular binding of an antibody against a cancer cell marker can inhibit the signaling of a cancer cell marker protein by, for example, inhibiting intrinsic activation {e.g., kinase activity) of a cancer cell marker and/or by sterically inhibiting the interaction, for example, of a cancer cell marker with its ligand, with its receptor, with a co-receptor or, with the extracellular matrix. In some embodiments, extracellular binding of an antibody against a cancer cell marker can downregulate cell-surface expression of a cancer cell marker such as, for example, by internalization of a cancer cell marker protein or decreasing cell surface trafficking of a cancer cell marker. In some embodiments, extracellular binding of an antibody against a cancer cell marker can promote the signaling of a cancer cell marker protein by, for example, acting as a decoy ligand or increasing ligand binding.

[00418] In certain embodiments, antibodies against a cancer cell marker bind to a cancer cell marker protein and have one or more of the following effects: inhibit proliferation of tumor cells, trigger cell death of tumor cells, promote differentiation of tumor cells into a less tumorigenic cell type, or prevent metastasis of tumor cells. In certain embodiments, antibodies against a cancer cell marker trigger cell death via a conjugated toxin, chemotherapeutic agent, radioisotope, or other such agent. For example, an antibody against a cancer cell marker is conjugated to a toxin that is activated in tumor cells expressing the cancer cell marker by protein internalization.

[00419] In certain embodiments, antibodies against a cancer cell marker mediate cell death of a cell expressing the cancer cell marker protein via antibody-dependent cellular cytotoxicity (ADCC). ADCC involves cell lysis by effector cells that recognize the Fc portion of an antibody. Many lymphocytes, monocytes, tissue macrophages, granulocytes and eosinophiles, for example, have Fc receptors and can mediate cytolysis (Dillman, 1994, J. Clin. Oncol. 12:1497).

[00420] In certain embodiments, antibodies against a cancer cell marker trigger cell death of a cell expressing a cancer cell marker protein by activating complement- dependent cytotoxicity (CDC). CDC involves binding of serum complement to the Fc portion of an antibody and subsequent activation of the complement protein cascade, resulting in cell membrane damage and eventual cell death. Biological activity of antibodies is known to be determined, to a large extent, by the constant domains or Fc region of the antibody molecule (Uananue and Benacerraf, Textbook of Immunology, 2nd Edition, Williams & Wilkins, p. 218 (1984)). Antibodies of different classes and subclasses differ in this respect, as do antibodies of the same subclass but from different species. Of human antibodies, IgM is the most efficient class of antibodies to bind complement, followed by lgG1 , lgG3, and lgG2 whereas lgG4 appears quite deficient in activating the complement cascade (Dillman, 1994, J. Clin. Oncol. 12:1497; Jefferis et ai, 1998, Immunol. Rev. 163:59-76). Antibodies of those classes having the desired biological activity may be prepared.

[00421] The ability of any particular antibody against a cancer cell to mediate lysis of the target cell by complement activation and/or ADCC can be assayed. The cells of interest are grown and labeled in vitro; the antibody is added to the cell culture in combination with either serum complement or immune cells which can be activated by the antigen antibody complexes. Cytolysis of the target cells is detected, for example, by the release of label from the lysed cells. In fact, antibodies can be screened using the patient's own serum as a source of complement and/or immune cells. The antibody that is capable of activating complement or mediating ADCC in the in vitro test can then be used therapeutically in that particular patient.

[00422] In certain embodiments, antibodies against a cancer cell marker can trigger cell death by inhibiting angiogenesis. Angiogenesis is the process by which new blood vessels form from pre-existing vessels and is a fundamental process required for normal growth, for example, during embryonic development, wound healing, and in response to ovulation. Solid tumor growth larger than 1-2 mm2 also requires angiogenesis to supply nutrients and oxygen without which tumor cells die. In certain embodiments, an antibody against a cancer stem cell marker targets vascular cells that express the cancer stem cell marker including, for example, endothelial cells, smooth muscle cells, or components of the extracellular matrix required for vascular assembly. In certain embodiments, an antibody against a cancer stem cell marker inhibits growth factor signaling required by vascular cell recruitment, assembly, maintenance, or survival.

[00423] The antibodies against a cancer cell marker find use in the diagnostic and therapeutic methods described herein. In certain embodiments, the antibodies of the present disclosure are used to detect the expression of a cancer cell marker protein in biological samples such as, for example, a patient tissue biopsy, pleural effusion, or blood sample. Tissue biopsies can be sectioned and protein detected using, for example, immunofluorescence or immunohistochemistry. In addition, individual cells from a sample can be isolated, and protein expression detected on fixed or live cells by FACS analysis. In certain embodiments, antibodies can be used on protein arrays to detect expression of a cancer cell marker, for example, on tumor cells, in cell lysates, or in other protein samples. In certain embodiments, the antibodies of the present disclosure are used to inhibit the growth of tumor cells by contacting the antibodies with tumor cells in in vitro cell based assays, in vivo animal models, etc. In certain embodiments, the antibodies are used to treat cancer in a patient by administering a therapeutically effective amount of an antibody against a cancer cell marker, including, for example, an antibody to DLL4. [00424] Therapeutic methods are provided using any of the antibodies to DLL4 as described herein, including compositions or pharmaceutical formulations comprising such antibodies to DLL4. Methods include in vitro, ex vivo, and in vivo therapeutic methods, unless otherwise indicated. In various aspects, methods of modulating (e.g., stimulating or inhibiting) human T cell activation, cytokine production, and differentiation are provided. Methods of modulating {e.g., inhibiting) a human Th17 cell function are provided. Methods of treating human inflammatory and/or autoimmune disorders are also provided. Methods of treating a Th17 cell mediated disease or disorder in humans are further provided. These and other aspects of the methods are provided below.

[00425] In one aspect, a method of inhibiting a signaling pathway in a biological system involving human Th17 cells is provided, the method comprising providing an antibody to DLL4 to the biological system. Additionally or alternatively a DLL4 antagonist may be used. Biological systems include, e.g., human cells in an in vitro cell culture system or in a human in vivo.

[00426] In another aspect, a method of inhibiting a human Th17 cell function is provided, the method comprising exposing a human Th17 cell to an antibody to DLL4. Additionally or alternatively a DLL4 antagonist may be used. In one embodiment, the antibody is a neutralizing DLL4 antibody. Exemplary humanTh17 cell functions include, but are not limited to, stimulation of cell-mediated immunity (delayed-type hypersensitivity); recruitment of innate immune cells, such as myeloid cells (e.g., monocytes and neutrophils) to sites of inflammation; and stimulation of inflammatory cell infiltration into tissues.

[00427] In yet another aspect, a method of treating inflammation in a human is provided, the method comprising administering to a human in need of such treatment an effective amount of a pharmaceutical formulation comprising an antibody to DLL4. Additionally or alternatively a DLL4 antagonist may be used. In one embodiment, the antibody is a neutralizing DLL4 antibody. Inflammation includes, but is not limited to, an inflammatory disease or disorder, including, for example, autoimmune inflammation (inflammation associated with an autoimmune disorder), chronic inflammation, skin inflammation, arthritic inflammation (including inflammation associated with rheumatoid arthritis), and systemic inflammatory response. In one embodiment, the inflammation is mediated by a cytokine, including, for example, IL-17 and/or IL-22. Inflammatory diseases may include multiple sclerosis, psoriasis, psoriatic arthritis, rheumatoid arthritis, autoimmune ocular diseases, endotoxemia, IBS, colitis, asthma, allograft rejection, immune mediated renal diseases, hepatobiliary diseases, atherosclerosis, or degenerative joint disease atopic and contact dermatitis, colitis, endotoxemia, arthritis, rheumatoid arthritis, psoriatic arthritis, autoimmune ocular diseases (uveitis, scleritis), adult respiratory disease (ARD), demyelinating diseases, septic shock, multiple organ failure, inflammatory lung injury such as asthma, chronic obstructive pulmonary disease (COPD), airway hyper- responsiveness, chronic bronchitis, allergic asthma, psoriasis, eczema, IBS and inflammatory bowel disease (IBD) such as ulcerative colitis and Crohn's disease, diabetes, Helicobacter pylori infection, intraabdominal adhesions and/or abscesses as results of peritoneal inflammation (i.e. from infection, injury, etc.), systemic lupus erythematosus (SLE), multiple sclerosis, systemic sclerosis, nephrotic syndrome, organ allograft rejection, graft vs. host disease (GVHD), kidney, lung, heart, etc. transplant rejection, streptococcal cell wall (SCW)-induced arthritis, osteoarthritis, gingivitis/periodontitis, herpetic stromal keratitis, restenosis, and Kawasaki disease.

[00428] In yet another aspect, a method of treating an autoimmune disease or disorder in a human is provided, the method comprising administering to a human in need of such treatment an effective amount of a pharmaceutical formulation comprising an antibody to DLL4. Additionally or alternatively a DLL4 antagonist may be used. In one embodiment, the antibody is a neutralizing DLL4 antibody. Autoimmune diseases or disorders may include connective tissue disease, multiple sclerosis, systemic lupus erythematosus or lupus, inflammatory arthritis (e.g., rheumatoid arthritis), autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin- dependent diabetes mellitus, uveitis, myasthenia gravis, graft-versus-host disease, autoimmune inflammatory eye disease, psoriasis, arthritis associated with autoimmunity {e.g., rheumatoid arthritis), autoimmune inflammation of the brain, and inflammatory bowel disease.

[00429] In a particular aspect, methods for the treatment of lupus and/or disorders characterized by lupus-like symptoms are provided. Lupus may be characterized as a systemic autoimmune disease that can affect any part of the body. As occurs in other autoimmune diseases, the immune system attacks the body's cells and tissues, resulting in inflammation and tissue damage. Lupus may also be considered a Type III hypersensitivity reaction caused by antibody-immune complex formation. Lupus most often harms the heart, joints, skin, lungs, blood vessels, liver, kidneys and nervous system. The course of lupus is unpredictable, with periods of illness (called flares) alternating with remissions.

[00430] In one embodiment, a method of treating lupus in a human comprises administering to a patient an effective amount of a pharmaceutical formulation comprising an antibody to DLL4. Additionally or alternatively a DLL4 antagonist may be used. In one embodiment, the antibody is a neutralizing DLL4 antibody. In various embodiments, the method further comprises administering (either in the same pharmaceutical formulation or a separate pharmaceutical formulation) at least one additional therapeutic agent. In one such embodiment, the additional therapeutic agent is at least one antagonist of a cytokine. Any number of such antibodies may be selected in any combination. In another embodiment, the additional therapeutic agent is an agent used in the treatment of lupus. Such an agent may include: nonsteroidal anti-inflammatory drugs (NSAIDs), disease- modifying antirheumatic drugs (DMARDs) such as antimalarials, immunosuppressants, cyclophosphamide, corticosteroids, analgesics including opioids, and intravenous immunoglobulins (IVIGs).

[00431] Compositions of the present disclosure (e.g., polypeptides, antibodies, antagonists, agonists and pharmaceutical formulations comprising any of the foregoing), are administered to a human, in accord with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation (intranasal, intrapulmonary) routes. Intravenous or inhaled administration of polypeptides and antibodies is preferred.

[00432] In certain embodiments, administration of an anti-cancer agent may be combined with the administration of a composition of the present disclosure. For example, a patient to be treated with an antibody, a composition, or formulation as disclosed herein may also receive an anti-cancer agent {e.g., chemotherapeutic agent) or other therapy (e.g., radiation, chemotherapy, surgery). Preparation and dosing schedules for such other agents may be used according to manufacturers' instructions or as determined empirically by the skilled practitioner.

[00433] It may be desirable to also administer antibodies against other immune disease associated- or tumor associated-antigens. Alternatively, or in addition, two or more antibodies binding the same or two or more different antigens disclosed herein may be co- administered to the patient. In certain embodiments, it may be beneficial to also administer one or more cytokines to a patient. In certain embodiments, an antibody, a composition or for mutation as disclosed herein is co-administered with a growth inhibitory agent. For example, the growth inhibitory agent may be administered before, after, or contemporaneously with administration of the composition. Suitable dosages for the growth inhibitory agent are those presently used and may be lowered due to the combined action (synergy) of the growth inhibitory agent and the composition.

[00434] For the treatment or reduction in the severity of an immune disease or disorder (e.g., an autoimmune disease or disorder), the appropriate dosage of an antibody a composition or fumulation as disclosed herein will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the agent is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the compound, and the discretion of the attending physician. The compound is suitably administered to the patient at one time or over a series of treatments.

[00435] For example, depending on the type and severity of a disease, about 1 μg/kg to 15 mg/kg (e.g., 0.1 -20 mg/kg) of a polypeptide or antibody is an initial candidate dosage for administration to a patient, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

Combination Therapies

[00436] As indicated above, the disclosure provides combined therapies in which an DLL4 antibody is administered with another therapy. For example, DLL4 antibodies are used in combinations with anti-cancer therapeutics or anti-neovascularization therapeutics to treat various neoplastic or non-neoplastic conditions. In one embodiment, the neoplastic or non-neoplastic condition is characterized by pathological disorder associated with aberrant or undesired angiogenesis. The DLL4 antibody may be administered serially or in combination with another agent that is effective for those purposes, either in the same composition or as separate compositions. Alternatively, or additionally, multiple inhibitors of DLL4 {e.g., antigonists, whether small molecules, proteins or antibodies) may be administered.

[00437] The administration of the DLL4 antibody may be done simultaneously, e.g., as a single composition or as two or more distinct compositions using the same or different administration routes. Alternatively, or additionally, the administration may be done sequentially, in any order. In certain embodiments, intervals ranging from minutes to days, to weeks to months, can be present between the administrations of the two or more compositions. For example, the anti-cancer agent may be administered first, followed by the DLL4 inhibitor. However, simultaneous administration or administration of the DLL4 antibody first is also contemplated.

[00438] The effective amounts of therapeutic agents administered in combination with a DLL4 antibody will be at the physician's or veterinarian's discretion. Dosage administration and adjustment is done to achieve maximal management of the conditions to be treated. The dose will additionally depend on such factors as the type of therapeutic agent to be used and the specific patient being treated. Suitable dosages for the anti- cancer agent are those presently used and can be lowered due to the combined action (synergy) of the anti-cancer agent and the DLL4 antibody. In certain embodiments, the combination of the inhibitors potentiates the efficacy of a single inhibitor. The term "potentiate" refers to an improvement in the efficacy of a therapeutic agent at its common or approved dose. See also the section entitled Pharmaceutical Compositions herein.

[00439] Typically, the DLL4 antibodies and anti-cancer agents are suitable for the same or similar diseases to block or reduce a pathological disorder such as tumor growth or growth of a cancer cell. In one embodiment, the anti-cancer agent is an anti- angiogenesis agent.

[00440] Antiangiogenic therapy in relationship to cancer is a cancer treatment strategy aimed at inhibiting the development of tumor blood vessels required for providing nutrients to support tumor growth. Because angiogenesis is involved in both primary tumor growth and metastasis, the antiangiogenic treatment provided by the disclosure is capable of inhibiting the neoplastic growth of tumor at the primary site as well as preventing metastasis of tumors at the secondary sites, therefore allowing attack of the tumors by other therapeutics.

[00441] Many anti-angiogenic agents have been identified and are known in the arts, including those listed herein, e.g., listed under Definitions, and by, e.g., Carmeliet and Jain, Nature 407:249-257 (2000); Ferrara et al., Nature Reviews. Drug Discovery, 3:391 - 400 (2004); and Sato Int. J. Clin. Oncol., 8:200-206 (2003). See also, US 2003/0055006. In one embodiment, a DLL4 antibody is used in combination with an anti-VEGF neutralizing antibody (or fragment) and/or another VEGF antagonist or a VEGF receptor antagonist including, but not limited to, for example, soluble VEGF receptor {e.g., VEGFR-1 , VEGFR- 2, VEGFR-3, neuropillins {e.g., NRP1 , NRP2)) fragments, aptamers capable of blocking VEGF or VEGFR, neutralizing anti-VEGFR antibodies, low molecule weight inhibitors of VEGFR tyrosine kinases (RTK), antisense strategies for VEGF, ribozymes against VEGF or VEGF receptors, antagonist variants of VEGF; and any combinations thereof. Alternatively, or additionally, two or more angiogenesis inhibitors may optionally be coadministered to the patient in addition to VEGF antagonist and other agent. In certain embodiment, one or more additional therapeutic agents, e.g., anti-cancer agents, can be administered in combination with DLL4 antibody, the VEGF antagonist, and an anti- angiogenesis agent.

[00442] In certain aspects disclosed herein, other therapeutic agents useful for combination tumor therapy with a DLL4 antibody include other cancer therapies, {e.g., surgery, radiological treatments {e.g., involving irradiation or administration of radioactive substances), chemotherapy, treatment with anti-cancer agents listed herein and known in the art, or combinations thereof). Alternatively, or additionally, two or more antibodies binding the same or two or more different antigens disclosed herein can be coadministered to the patient. Sometimes, it may be beneficial to also administer one or more cytokines to the patient.

Chemotherapeutic Agents

[00443] Methods of blocking or reducing tumor growth or growth of a cancer cell, comprise administering effective amounts of an antagonist of DLL4 and/or an angiogenesis inhibitor(s) and one or more chemotherapeutic agents to a patient susceptible to, or diagnosed with, cancer. A variety of chemotherapeutic agents may be used in the combined treatment methods disclosed herein. An exemplary and non-limiting list of chemotherapeutic agents contemplated is provided herein under "Definitions."

[00444] As will be understood by those of ordinary skill in the art, the appropriate doses of chemotherapeutic agents will be generally around those already employed in clinical therapies wherein the chemotherapeutics are administered alone or in combination with other chemotherapeutics. Variation in dosage will likely occur depending on the condition being treated. The physician administering treatment will be able to determine the appropriate dose for the individual subject.

[00445] Methods and compositions for inhibiting or preventing relapse tumor growth or relapse cancer cell growth are disclosed. Relapse tumor growth or relapse cancer cell growth is used to describe a condition in which patients undergoing or treated with one or more currently available therapies {e.g., cancer therapies, such as chemotherapy, radiation therapy, surgery, hormonal therapy and/or biological therapy/immunotherapy, anti-VEGF antibody therapy, particularly a standard therapeutic regimen for the particular cancer) is not clinically adequate to treat the patients or the patients are no longer receiving any beneficial effect from the therapy such that these patients need additional effective therapy. As used herein, the phrase can also refer to a condition of the "non-responsive/refractory" patient, e.g., which describe patients who respond to therapy yet suffer from side effects, develop resistance, do not respond to the therapy, do not respond satisfactorily to the therapy, etc. In various embodiments, a cancer is relapse tumor growth or relapse cancer cell growth where the number of cancer cells has not been significantly reduced, or has increased, or tumor size has not been significantly reduced, or has increased, or fails any further reduction in size or in number of cancer cells. The determination of whether the cancer cells are relapse tumor growth or relapse cancer cell growth can be made either in vivo or in vitro by any method known in the art for assaying the effectiveness of treatment on cancer cells, using the art-accepted meanings of "relapse" or "refractory" or "non-responsive" in such a context. A tumor resistant to anti- VEGF treatment is an example of a relapse tumor growth.

[00446] Methods of blocking or reducing relapse tumor growth or relapse cancer cell growth in a subject comprise administering one or more DLL4 antibodies to block or reduce the relapse tumor growth or relapse cancer cell growth in subject. In certain embodiments, the antagonist may be administered subsequent to the cancer therapeutic. In certain embodiments, the DLL4 antibodies are administered simultaneously with cancer therapy. Alternatively, or additionally, the DLL4 antibody therapy alternates with another cancer therapy, which can be performed in any order. The disclosure also encompasses methods for administering one or more inhibitory antibodies to prevent the onset or recurrence of cancer in patients predisposed to having cancer. Generally, the subject was or is concurrently undergoing cancer therapy. In one embodiment, the cancer therapy is treatment with an anti-angiogenesis agent, e.g., a VEGF antagonist. The anti-angiogenesis agent includes those known in the art and those found under the Definitions herein. In one embodiment, the anti-angiogenesis agent is an anti-VEGF neutralizing antibody or fragment (e.g., AVASTIN® (bevacizumab) or LUCENTIS® (ranibizumab) Y0317, M4, G6, B20, 2C3, etc.). See, e.g., 6,582,959, 6,884,879, 6,703,020; W098/45332; WO 96/30046; WO94/10202; EP 0 666 868; US 2003/0206899, US 2003/0190317, US 2003/0203409, and US 2005/01 12126; Popkov et al., Journal of Immunological Methods 288:149-164 (2004); and, WO2005012359. Additional agents can be administered in combination with VEGF antagonist and a DLL4 antibody for blocking or reducing relapse tumor growth or relapse cancer cell growth, e.g., see section entitled Combination Therapies herein.

[00447] The antibodies to DLL4 as disclosed herein (and adjunct therapeutic agent) is/are administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional or intravitreal administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In addition, the antibody is suitably administered by pulse infusion, particularly with declining doses of the antibody. Dosing can be by any suitable route, e.g., by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.

[00448] The antibody compositions disclosed herein will be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The antibody need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antibodies disclosed herein present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as used hereinbefore or about from 1 to 99% of the heretofore employed dosages.

[00449] For the prevention or treatment of disease, the appropriate dosage of an antibody disclosed herein (when used alone or in combination with other agents such as chemotherapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μg/kg to 15 mg/kg {e.g., 0.1 mg/kg-10 mg/kg) of antibody is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. One typical daily dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. One exemplary dosage of the antibody would be in the range from about 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 3.0 mg/kg, 4.0 mg/kg, 5.0 mg/kg or 10 mg/kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, e.g., every week or every other week or every three weeks {e.g., such that the patient receives from about two to about twenty, e.g., about six doses of the antibody). An initial higher loading dose, followed by one or more lower doses may be administered. For example, a dosing regimen may comprise administering an initial loading dose of about 1-4 mg/kg, followed by a weekly maintenance dose of about 0.5-2 mg/kg of the antibody. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

Uses In Assays and Methods

[00450] The DLL4 antibodies disclosed herein are useful in assays detecting DLL4 expression (such as diagnostic or prognostic assays), for example, in a sample such as a biological sample {e.g., in specific cells or tissues) wherein the antibodies are labeled as described below and/or are immobilized on an insoluble matrix.

[00451] In another aspect, the disclosure provides methods for detection of DLL4, the methods comprising detecting DLL4-DLL4 antibody complex in a sample. The term "detection" as used herein includes qualitative and/or quantitative detection (measuring levels) with or without reference to a control.

[00452] In another aspect, the disclosure provides methods for diagnosing a disorder associated with DLL4 expression and/or activity, the methods comprising detecting DLL4-DLL4 antibody complex in a biological sample from a patient having or suspected of having the disorder. In some embodiments, the DLL4 expression is increased expression or abnormal (undesired) expression. In some embodiments, the disorder is a tumor, cancer, and/or a cell proliferative disorder.

[00453] In another aspect, the disclosure provides any of the DLL4 antibodies described herein, wherein the DLL4 antibody comprises a label such as a detectable label.

[00454] In another aspect, the disclosure provides a complex of any of the DLL4 antibodies described herein and DLL4. In some embodiments, the complex is in vivo or in vitro. In some embodiments, the complex comprises a cancer cell. In some embodiments, the DLL4 antibody is detectably labeled.

[00455] DLL4 antibodies can be used for the detection of DLL4 in any one of a number of well known detection assay methods. For example, a sample such as a biological sample may be assayed for DLL4 by obtaining the sample from a desired source, admixing the sample with DLL4 antibody to allow the antibody to form antibody/DLL4 complex with any DLL4 present in the mixture, and detecting any antibody/DLL4 complex present in the mixture. A sample such as a biological sample may be prepared for assay by methods known in the art which are suitable for the particular sample. The methods of admixing the sample with antibodies and the methods of detecting antibody/DLL4 complex are chosen according to the type of assay used. Such assays include immunohistochemistry, competitive and sandwich assays, and steric inhibition assays.

[00456] Analytical methods for DLL4 all use one or more of the following reagents: labeled DLL4 analogue, immobilized DLL4 analogue, labeled DLL4 antibody, immobilized DLL4 antibody and steric conjugates. The labeled reagents also are known as "tracers."

[00457] The label used is any detectable functionality that does not interfere with the binding of DLL4 and DLL4 antibody. Numerous labels are known for use in immunoassay, examples including moieties that may be detected directly, such as fluorochrome, chemiluminescent, and radioactive labels, as well as moieties, such as enzymes, that must be reacted or derivatized to be detected. The label used is any detectable functionality that does not interfere with the binding of DLL4 and DLL4 antibody. Numerous labels are known for use in immunoassay, examples including moieties that may be detected directly, such as fluorochrome, chemiluminescent, and radioactive labels, as well as moieties, such as enzymes, that must be reacted or derivatized to be detected. Examples of such labels include the radioisotopes 32P, 14C, 125l, 3H, and 1311, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β galactosidase, glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, coupled with an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase, or microperoxidase, biotin/avidin, spin labels, bacteriophage labels, stable free radicals, and the like.

[00458] Conventional methods are available to bind these labels covalently to proteins or polypeptides. For instance, coupling agents such as dialdehydes, carbodiimides, dimaleimides, bis-imidates, bis-diazotized benzidine, and the like may be used to tag the antibodies with the above-described fluorescent, chemiluminescent, and enzyme labels. See, for example, U.S. Patent Nos. 3,940,475 (fluorimetry) and 3,645,090 (enzymes); Hunter et al., Nature, 144: 945 (1962); David et al., Biochemistry, 13: 1014- 1021 (1974); Pain et al., J. Immunol. Methods, 40: 219-230 (1981 ); and Nygren, J. Histochem. and Cytochem., 30: 407-412 (1982). Preferred labels herein are enzymes such as horseradish peroxidase and alkaline phosphatase. The conjugation of such label, including the enzymes, to the antibody is a standard manipulative procedure for one of ordinary skill in immunoassay techniques. See, for example, O'Sullivan et al., "Methods for the Preparation of Enzyme-antibody Conjugates for Use in Enzyme Immunoassay," in Methods in Enzymology, ed. J. J. Langone and H. Van Vunakis, Vol. 73 (Academic Press, New York, N.Y., 1981 ), pp. 147-166.

[00459] Immobilization of reagents is required for certain assay methods.

Immobilization entails separating the DLL4 antibody from any DLL4 that remains free in solution. This conventionally is accomplished by either insolubilizing the DLL4 antibody or DLL4 analogue before the assay procedure, as by adsorption to a water-insoluble matrix or surface (Bennich et al., U.S. Patent No. 3,720,760), by covalent coupling (for example, using glutaraldehyde cross-linking), or by insolubilizing the DLL4 antibody or DLL4 analogue afterward, e.g., by immunoprecipitation. [00460] The expression of proteins in a sample may be examined using immunohistochemistry and staining protocols, including using antibodies to DLL4. Immunohistochemical staining of tissue sections has been shown to be a reliable method of assessing or detecting presence of proteins in a sample. Immunohistochemistry ("IHC") techniques utilize an antibody to probe and visualize cellular antigens in situ, generally by chromogenic or fluorescent methods. For sample preparation, a tissue or cell sample from a mammal (typically a human patient) may be used. Examples of samples include, but are not limited to, cancer cells such as colon, breast, prostate, ovary, lung, stomach, pancreas, lymphoma, and leukemia cancer cells. The sample can be obtained by a variety of procedures known in the art including, but not limited to surgical excision, aspiration or biopsy. The tissue may be fresh or frozen. In one embodiment, the sample is fixed and embedded in paraffin or the like. The tissue sample may be fixed {e.g., preserved) by conventional methodology. One of ordinary skill in the art will appreciate that the choice of a fixative is determined by the purpose for which the sample is to be histologically stained or otherwise analyzed. One of ordinary skill in the art will also appreciate that the length of fixation depends upon the size of the tissue sample and the fixative used.

[00461] IHC may be performed in combination with additional techniques such as morphological staining and/or fluorescence in-situ hybridization. Two general methods of IHC are available; direct and indirect assays. According to the first assay, binding of antibody to the target antigen {e.g., DLL4) is determined directly. This direct assay uses a labeled reagent, such as a fluorescent tag or an enzyme-labeled primary antibody, which can be visualized without further antibody interaction. In a typical indirect assay, unconjugated primary antibody binds to the antigen and then a labeled secondary antibody binds to the primary antibody. Where the secondary antibody is conjugated to an enzymatic label, a chromogenic or fluorogenic substrate is added to provide visualization of the antigen. Signal amplification occurs because several secondary antibodies may react with different epitopes on the primary antibody.

[00462] The primary and/or secondary antibody used for immunohistochemistry typically will be labeled with a detectable moiety. Numerous labels are available.

[00463] Aside from the sample preparation procedures discussed above, further treatment of the tissue section prior to, during or following IHC may be desired, For example, epitope retrieval methods, such as heating the tissue sample in citrate buffer may be carried out (see, e.g., Leong et al. Appl. Immunohistochem. 4(3):201 (1996)).

[00464] Following an optional blocking step, the tissue section is exposed to primary antibody for a sufficient period of time and under suitable conditions such that the primary antibody binds to the target protein antigen in the tissue sample. Appropriate conditions for achieving this can be determined by routine experimentation. The extent of binding of antibody to the sample is determined by using any one of the detectable labels discussed above. The label may be an enzymatic label (e.g., HRP) which catalyzes a chemical alteration of the chromogenic substrate such as 3,3'-diaminobenzidine chromogen. An enzymatic label may be conjugated to antibody which binds specifically to the primary antibody (e.g., the primary antibody is rabbit polyclonal antibody and secondary antibody is goat anti-rabbit antibody). Specimens thus prepared may be mounted and coverslipped. Slide evaluation may then be determined, e.g., using a microscope, and staining intensity criteria, routinely used in the art, may be employed. Staining intensity criteria may be evaluated as follows:

Staining Pattern Score

No staining is observed in cells 0

Faint/barely perceptible staining is detected in more than 1 +

10% of the cells.

Weak to moderate staining is observed in more than 2+

10% of the cells.

Moderate to strong staining is observed in more than 3+

10% of the cells.

[00465] A staining pattern score of about 2+ or higher in an IHC assay may be diagnostic and/or prognostic. In some embodiments, a staining pattern score of about 1 + or higher may be diagnostic and/or prognostic. In other embodiments, a staining pattern score of about 3 of higher may be diagnostic and/or prognostic. It is understood that when cells and/or tissue from a tumor or colon adenoma are examined using IHC, staining is generally determined or assessed in tumor cell and/or tissue (as opposed to stromal or surrounding tissue that may be present in the sample).

[00466] Other assay methods, known as competitive or sandwich assays, are well established and widely used in the commercial diagnostics industry.

[00467] Competitive assays may rely on the ability of a tracer DLL4 molecule (e.g., analogue) to compete with the test sample DLL4 for a limited number of DLL4 antibody antigen-binding sites. A DLL4 antibody may be insolubilized before or after the competition and then the tracer and DLL4 bound to the DLL4 antibody may be separated from the unbound tracer and DLL4. This separation may be accomplished by decanting (where the binding partner was preinsolubilized) or by centrifuging (where the binding partner was precipitated after the competitive reaction). The amount of test sample DLL4 is inversely proportional to the amount of bound tracer as measured by the amount of marker substance. Dose-response curves with known amounts of DLL4 are prepared and compared with the test results to quantitatively determine the amount of DLL4 present in the test sample. These assays are called ELISA {e.g., enzyme-linked immunosorbant assays) systems when enzymes are used as the detectable markers.

[00468] Another example of a competitive assay, called a "homogeneous" assay, does not require a phase separation. Here, a conjugate of an enzyme with the DLL4 may be prepared and used such that when DLL4 antibody binds to the DLL4 the presence of the DLL4 antibody modifies the enzyme activity. In this case, the DLL4 or its immunologically active fragments may be conjugated with a bifunctional organic bridge to an enzyme such as peroxidase. Conjugates may be selected for use with DLL4 antibody so that binding of the DLL4 antibody inhibits or potentiates the enzyme activity of the label. This method is practiced under the name of EMIT.

[00469] Steric conjugates may be used in steric hindrance methods for homogeneous assay. These conjugates may be synthesized by covalently linking a low- molecular-weight hapten to a small DLL4 fragment so that antibody to hapten is substantially unable to bind the conjugate at the same time as DLL4 antibody. Under this assay procedure, the DLL4 present in the test sample may bind DLL4 antibody, thereby allowing anti-hapten to bind the conjugate, resulting in a change in the character of the conjugate hapten, e.g., a change in fluorescence when the hapten is a fluorophore.

[00470] Sandwich assays are useful for the determination of DLL4 or DLL4 antibodies. In sequential sandwich assays, an immobilized DLL4 antibody is used to adsorb test sample DLL4, the test sample is removed as by washing, the bound DLL4 is used to adsorb a second, labeled DLL4 antibody and bound material is then separated from residual tracer. The amount of bound tracer is directly proportional to test sample DLL4. In "simultaneous" sandwich assays the test sample is not separated before adding the labeled anti-DLL4. A sequential sandwich assay using a DLL4 monoclonal antibody as one antibody and a polyclonal DLL4 antibody as the other is useful in testing samples for DLL4.

[00471] The foregoing are merely exemplary detection assays for DLL4. Other methods now or hereafter developed that use DLL4 antibody for the determination of DLL4 are included within the scope hereof, including the assays such as bioassays as described herein.

Articles of Manufacture

[00472] In another aspect disclosed herein, an article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the disorders described above is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition, for example, comprising an antibody to DLL4, which is by itself or when combined with another composition(s) effective for treating, preventing and/or diagnosing the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is a DLL4 antibody as disclosed herein. The label or package insert indicates that the composition is used for treating a condition of choice, such as cancer. Moreover, the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises an antibody disclosed herein; and (b) a second container with a composition contained therein, wherein the composition comprises a further therapeutic agent, including, e.g., a chemotherapeutic agent or an anti-angiogenesis agent, including, e.g., an anti-VEGF antibody {e.g., bevacizumab or ranibizumab). The article of manufacture in this embodiment disclosed herein may further comprise a package insert indicating that the first and second antibody compositions can be used to treat a particular condition, e.g., cancer. Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[00473] The following are examples of the methods and compositions of the disclosure. It is understood that various other embodiments may be practiced, given the general description provided above.

EXAMPLES

Example 1. Screening and Selection of Anti-DLL4 Antibodies

[00474] Antibodies to DLL4 {e.g., human DLL4) may be generated by any known method in the art including, for example, by hybridoma technology or by phage display. Methods for selecting antibodies may be designed as described herein.

[00475] In an exemplary method, DLL4 antibodies are generated using phage display or hybridoma technology. The generated antibodies are then examined for binding to DLL4. Next, antibodies that bind DLL4 are subject to a multi-tier selection to obtain DLL4 antibodies with desired characteristics. For example, antibodies may be first screened in a Notchl binding inhibition assay. Those antibodies that are able to inhibit DLL4 binding to Notchl may be selected for further screening. In a second screen, the antibodies obtained from the first screen may be examined in a Hes1 reporter assay. Those antibodies that are able to reduce the activation of Hes1 may be then selected for a third screen. In the third screen, the antibodies may be subject to a HUVEC proliferation assay. Antibodies that inhibit the proliferation of HUVEC cells may be selected for additional screening comprising one or more assays such as epitope binning, affinity determination, cross-reactivity determination, and tube formation. Antibodies may be selected from the additional screens that have one or more desired characteristics including, for example, bind to an epitope of DLL4 that is not bound by existing antibodies, bind to DLL4 with a higher affinity than existing DLL4 antibodies, do not exhibit cross- reactivity to other Notch 1 ligands and/or inhibit angiogenesis. Such assays mentioned above are described in the Examples that follow.

[00476] Hybridoma technology may be employed to generate antibodies to DLL4. In an exemplary method, 22 CD2F1 (Charles River, San Diego, CA) and 25 KM mice were immunized with his-DLL4 (R&D Systems, Minneapolis, MN) or 293F cells engineered to express DLL4. Mice were boosted 8 times (twice/week) in adjuvant Titer Max. Next, a lateral tarsal vein pre fusion boost (boost 9) was carried out without adjuvant -30 days following initial boost. Axillary, popliteal and inguinal nodes were then removed from the mice. Subsequently, lymphocytes were isolated and fused with myeloma P3U1 by BTX Electrofusion™. IgG producing hybridoma clones were picked by Clonepix™, split into single wells and then expanded in culture. Supernatants from the culture of expanded clones were then assayed for DLL4 binding in a FACS assay using 293F/DII4 cells as described below.

[00477] A clone, termed 1 C1 , was generated by hybridoma technology. The antibody expressed by 1 C1 comprised a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 53 and a light chain variable region sequence comprising the amino acid sequence of SEQ ID NO: 58. As determined by the method of Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991 ), these variable region sequences consisted of the following HCDRs and LCDRs: HCDR1 (GYSWH, SEQ ID NO: 1 ), HCDR2 (YIHYSGYTHYNPSLKS, SEQ ID NO: 4), HCDR3 (SDYSANEGAMAY, SEQ ID NO: 6), LCDR1 (RASESVDSFGNSFMH , SEQ ID NO: 8), LCDR2 (RTSNLQS, SEQ ID NO: 10) and LCDR3 (QQSNEDPWT, SEQ ID NO: 1 1 ). Alternatively, as determined by the method of Chothia (see, e.g., Chothia et al., J. Mol. Biol. 196: 901 -917 (1987) these variable region sequences comprised the following HCDRs and LCDRs: HCDR1 (DHSITSG, SEQ ID NO: 2), HCDR2 (YIHYSGYTHYNPSLKS, SEQ ID NO: 4) and HCDR3 (SDYSANEGAMAY, SEQ ID NO: 6); and/or wherein the light chain variable region comprises LCDR1 (RASESVDSFGNSFMH , SEQ ID NO: 8), LCDR2 (RTSNLQS, SEQ ID NO: 10) and LCDR3 (QQSNEDPWT, SEQ ID NO: 1 1 ). Alternatively, as determined by the ImMunoGeneTics (IMGT) methodology (see, e.g., Ruiz et al., "IMGT, the international ImMunoGeneTics database, "Nucleic Acids Res., 28:219-221 (2000)) these variable region sequences comprised the following HCDRs and LCDRs: HCDR1 (DHSITSGYS; SEQ ID NO: 3), HCDR2 (IHYSGY, SEQ ID NO: 5) and HCDR3 (AGSDYSANEGAMAY, SEQ ID NO: 7); and/or wherein the light chain variable region comprises LCDR1 (ESVDSFGNSF, SEQ ID NO: 9), LCDR2 (R) and LCDR3 (QQSNEDPWT, SEQ ID NO: 1 1 ).

[00478] Additionally or alternatively, phage display may be employed to obtain antibodies to DLL4. In an exemplary method, a phage display library was panned against biotinylated DLL4 or 293F cells expressing recombinant DII4 either separately or in combination. Both murine and human DLL4 were used. After several rounds of panning, individual scFv phage clones were selected. Subsequently, purified scFv preparations were obtained from the selected clones and characterized. Positive clones with selected attributes were reformatted into human lgG2 format, expressed as antibodies and purified prior to further characterization in functional assays and higher resolution kinetics of binding and epitope binning.

[00479] Additional clones identified by the above-described methods include 12.14, 4C9, 1A6, 12.55, 1A10, 1 E8, 5.1 D8, 5.6A9, 12.08, 12.02, 12.19, 12.16, 12.26, 12.24, 12.22, 12.50, 12.38, 12.40, 12.73, 1 C3, 1 E2, 1 D2, 2G10, 1 C10, 1 C8, 12.43, 3C4, 2E2, 1A4, 5B12, 2C1 1 , 2A2, 1 D10, 1 C2, 3A1 , and 1 D3.

[00480] For example, a clone, termed 12.73, was generated by phage display technology. The antibody expressed by 12.73 comprised a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 54 and a light chain variable region sequence comprising the amino acid sequence of SEQ ID NO: 59. As determined by the method of Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991 ), these variable region sequences consisted of the following HCDRs and LCDRs: HCDR1 (DEYWS, SEQ ID NO: 12), HCDR2 (EIHESGKTNYNPSLKP, SEQ ID NO: 15) and HCDR3 (LAYRDRWYGAFDV, SEQ ID NO: 17); and/or wherein the light chain variable region comprises LCDR1 (QASHDITNYIN, SEQ ID NO: 19), LCDR2 (HTSKLQT, SEQ ID NO: 21 ) and LCDR3 (QQFDNLLLT, SEQ ID NO: 22). Alternatively, as determined by the method of Chothia (see, e.g., Chothia et al., J. Mol. Biol. 196: 901-917 (1987) these variable region sequences comprised the following HCDRs and LCDRs: HCDR1 (GGSFND, SEQ ID NO: 13), HCDR2 (EIHESGKTNYNPSLKP, SEQ ID NO: 15) and HCDR3 (LAYRDRWYGAFDV, SEQ ID NO: 17); and/or wherein the light chain variable region comprises LCDR1 (QASHDITNYIN, SEQ ID NO: 19), LCDR2 (HTSKLQT, SEQ ID NO: 21 ) and LCDR3 (QQFDNLLLT, SEQ ID NO: 22). Alternatively, as determined by the ImMunoGeneTics (IMGT) methodology (see, e.g., Ruiz et al., "IMGT, the international ImMunoGeneTics database, "Nucleic Acids Res., 28:219-221 (2000)) these variable region sequences comprised the following HCDRs and LCDRs: HCDR1 (GGSFNDEY; SEQ ID NO: 14), HCDR2 (IHESGKT, SEQ ID NO: 16) and HCDR3 (ARLAYRDRWYGAFDV, SEQ ID NO: 18); and/or wherein the light chain variable region comprises LCDR1 (HDITNY, SEQ ID NO: 20), LCDR2 (H) and LCDR3 (QQFDNLLLT, SEQ ID NO: 22).

[00481] Additionally, for example, a clone, termed 12.08, was generated by phage display technology. The antibody expressed by 12.08 comprised a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 55 and a light chain variable region sequence comprising the amino acid sequence of SEQ ID NO: 60. As determined by the method of Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991 ), these variable region sequences consisted of the following HCDRs and LCDRs: HCDR1 (GYYMH, SEQ ID NO: 23), HCDR2 (WINPNSGGTNYAQ, SEQ ID NO: 26) and HCDR3 (GAGVAAYDY, SEQ ID NO: 28); and/or wherein the light chain variable region comprises LCDR1 (RASQSISSYLN, SEQ ID NO: 30), LCDR2 (AASSLQS, SEQ ID NO: 32) and LCDR3 (QQSYSTPVT, SEQ ID NO: 33). Alternatively, as determined by the method of Chothia (see, e.g., Chothia et al., J. Mol. Biol. 196: 901-917 (1987) these variable region sequences comprised the following HCDRs and LCDRs: HCDR1 (GYTFTG, SEQ ID NO: 24), HCDR2 (WINPNSGGTNYAQ, SEQ ID NO: 26) and HCDR3 (GAGVAAYDY, SEQ ID NO: 28); and/or wherein the light chain variable region comprises LCDR1 (RASQSISSYLN, SEQ ID NO: 30), LCDR2 (AASSLQS, SEQ ID NO: 32) and LCDR3 (QQSYSTPVT, SEQ ID NO: 33). Alternatively, as determined by the ImMunoGeneTics (IMGT) methodology (see, e.g., Ruiz et al., "IMGT, the international ImMunoGeneTics database, "Nucleic Acids Res., 28:219-221 (2000)) these variable region sequences comprised the following HCDRs and LCDRs: HCDR1 (GYTFTGYY; SEQ ID NO: 25), HCDR2 (INPNSGG, SEQ ID NO: 27) and HCDR3 (ARGAGVAAYDY, SEQ ID NO: 29); and/or wherein the light chain variable region comprises LCDR1 (QSISSY, SEQ ID NO: 31 ), LCDR2 (A) and LCDR3 (QQSYSTPVT, SEQ ID NO: 33).

[00482] Additionally, for example, a clone, termed 12.14, was generated by phage display technology. The antibody expressed by 12.14 comprised a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 56 and a light chain variable region sequence comprising the amino acid sequence of SEQ ID NO: 61 . As determined by the method of Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991 ), these variable region sequences consisted of the following HCDRs and LCDRs: HCDR1 (DYAMS, SEQ ID NO: 34), HCDR2 (TISGSGGDTFYADSVKG, SEQ ID NO: 37) and HCDR3 (DKNRGAYADAFD, SEQ ID NO: 39); and/or wherein the light chain variable region comprises LCDR1 (TLRSDINVGTYRIY, SEQ ID NO: 41 ), LCDR2 (YKSDTDK, SEQ ID NO: 43) and LCDR3 (MVWHKSAPI, SEQ ID NO: 45). Alternatively, as determined by the method of Chothia (see, e.g., Chothia et al., J. Mol. Biol. 196: 901 -917 (1987) these variable region sequences comprised the following HCDRs and LCDRs: HCDR1 (GFTFSD, SEQ ID NO: 35), HCDR2 (TISGSGGDTFYADSVKG, SEQ ID NO: 37) and HCDR3 (DKNRGAYADAFD, SEQ ID NO: 39); and/or wherein the light chain variable region comprises LCDR1 (TLRSDINVGTYRIY, SEQ ID NO: 41 ), LCDR2 (YKSDTDK, SEQ ID NO: 43) and LCDR3 (MVWHKSAPI, SEQ ID NO: 45). Alternatively, as determined by the ImMunoGeneTics (IMGT) methodology (see, e.g., Ruiz et al., "IMGT, the international ImMunoGeneTics database, "Nucleic Acids Res., 28:219-221 (2000)) these variable region sequences comprised the following HCDRs and LCDRs: HCDR1 (GFTFSDYA; SEQ ID NO: 36), HCDR2 (ISGSGGDT, SEQ ID NO: 38) and HCDR3 (AKDKNRGAYADAFD, SEQ ID NO: 40); and/or wherein the light chain variable region comprises LCDR1 (SDINVGTYR, SEQ ID NO: 42), LCDR2 (YKSDTD, SEQ ID NO: 44) and LCDR3 (MVWHKSAPI, SEQ ID NO: 45).

[00483] Additionally, for example, a clone, termed 12.55, was generated by phage display technology. The antibody expressed by 12.55 comprised a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 57 and a light chain variable region sequence comprising the amino acid sequence of SEQ ID NO: 62. As determined by the method of Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991 ), these variable region sequences consisted of the following HCDRs and LCDRs: HCDR1 (DYAMS, SEQ ID NO: 34), HCDR2 (TISGSGGDTFYADSVKG, SEQ ID NO: 37) and HCDR3 (DKNRGAYADAFDI, SEQ ID NO: 47); and/or wherein the light chain variable region comprises LCDR1 (TLRSDINVATYRIY, SEQ ID NO: 49), LCDR2 (YKSDSDK, SEQ ID NO: 51 ) and LCDR3 (MVWHKSAPI, SEQ ID NO: 45). Alternatively, as determined by the method of Chothia (see, e.g., Chothia et al., J. Mol. Biol. 196: 901 -917 (1987) these variable region sequences comprised the following HCDRs and LCDRs: HCDR1 (GFTFSD, SEQ ID NO: 35), HCDR2 (TISGSGGDTFYADSVKG, SEQ ID NO: 37) and HCDR3 (DKNRGAYADAFDI, SEQ ID NO: 47); and/or wherein the light chain variable region comprises LCDR1 (TLRSDINVATYRIY, SEQ ID NO: 49), LCDR2 (YKSDSDK, SEQ ID NO: 51 ) and LCDR3 (MVWHKSAPI, SEQ ID NO: 45). Alternatively, as determined by the ImMunoGeneTics (IMGT) methodology (see, e.g., Ruiz et al., "IMGT, the international ImMunoGeneTics database, "Nucleic Acids Res., 28:219-221 (2000)) these variable region sequences comprised the following HCDRs and LCDRs: HCDR1 (GFTFSDYA; SEQ ID NO: 36), HCDR2 (ISGSGGD, SEQ ID NO: 46) and HCDR3 (AKDKNRGAYADAFDI, SEQ ID NO: 48); and/or wherein the light chain variable region comprises LCDR1 (SDINVATYR, SEQ ID NO: 50), LCDR2 (YKSDSD, SEQ ID NO: 52) and LCDR3 (MVWHKSAPI, SEQ ID NO: 45).

Example 2. Expression ofAnti- DLL4 Antibodies

[00484] Expression plasmids may be constructed which contain one or more DLL4 antibody CDRs generated and obtained as described in Example 1 .

[00485] DLL4 antibody CDRs that may be inserted into expression plasmids include: HCDR1 (GYSWH, SEQ ID NO: 1 ; DHSITSG, SEQ ID NO: 2; or DHSITSGYS; SEQ ID NO: 3), HCDR2 (YIHYSGYTHYNPSLKS, SEQ ID NO: 4; or IHYSGY, SEQ ID NO: 5) and HCDR3 (SDYSANEGAMAY, SEQ ID NO: 6; or AGSDYSANEGAMAY, SEQ ID NO: 7); and/or wherein the light chain variable region comprises LCDR1 (RASESVDSFGNSFMH , SEQ ID NO: 8; or ESVDSFGNSF, SEQ ID NO: 9), LCDR2 (RTSNLQS, SEQ ID NO: 10; or R) and LCDR3 (QQSNEDPWT, SEQ ID NO: 1 1 ). Alternatively, expression plasmids may be constructed that comprise the heavy and/or light chain variable region of the DLL4 antibodies generated and obtained in Example 1 , including, for example, SEQ ID NO: 53 and SEQ ID NO: 58, respectively. An exemplary antibody comprising a heavy chain variable region that is SEQ ID NO: 53 and a light chain variable region that is SEQ ID NO: 58 is designated herein as 12.3 1 C1 or 3.1 C1 (see, e.g., Examples 3-1 1 ). Optionally, the antibody heavy and/or light chain variable regions may be configured for expression of an antibody including, for example, a full-length antibody.

[00486] Alternatively, DLL4 antibody CDRs that may be inserted into expression plasmids include: HCDR1 (DEYWS, SEQ ID NO: 12; GGSFND, SEQ ID NO: 13; or GGSFNDEY; SEQ ID NO: 14), HCDR2 (EIHESGKTNYNPSLKP, SEQ ID NO: 15; or IHESGKT, SEQ ID NO: 16) and HCDR3 (LAYRDRWYGAFDV, SEQ ID NO: 17; or ARLAYRDRWYGAFDV, SEQ ID NO: 18); and/or wherein the light chain variable region comprises LCDR1 (QASHDITNYIN, SEQ ID NO: 19; or HDITNY, SEQ ID NO: 20), LCDR2 (HHTSKLQT, SEQ ID NO: 21 ; or H) and LCDR3 (QQFDNLLLT, SEQ ID NO: 22). Such CDRs may alternatively include HCDR1 (GYYMH, SEQ ID NO: 23; GYTFTG, SEQ ID NO: 24; or GYTFTGYY; SEQ ID NO: 25), HCDR2 (WINPNSGGTNYAQ, SEQ ID NO: 26; or INPNSGG, SEQ ID NO: 27) and HCDR3 (GAGVAAYDY, SEQ ID NO: 28; or ARGAGVAAYDY, SEQ ID NO: 29); and/or wherein the light chain variable region comprises LCDR1 (RASQSISSYLN, SEQ ID NO: 30; or QSISSY, SEQ ID NO: 31 ), LCDR2 (AASSLQS, SEQ ID NO: 32; or A) and LCDR3 (QQSYSTPVT, SEQ ID NO: 33). Alternatively, expression plasmids may be constructed that comprise the heavy and/or light chain variable region of the DLL4 antibodies generated and obtained in Example 1 , including, for example, SEQ ID NO: 54 or SEQ ID NO: 55 and SEQ ID NO: 59 or SEQ ID NO: 60, respectively. An exemplary antibody comprising a heavy chain variable region that is SEQ ID NO: 54 and a light chain variable region that is SEQ ID NO: 59 is designated herein as 12.73. An additional exemplary antibody comprising a heavy chain variable region that is SEQ ID NO: 55 and a light chain variable region that is SEQ ID NO: 60 is designated herein as 12.08 (see, e.g., Examples 3-1 1 ). Optionally, the antibody heavy and/or light chain variable regions may be configured for expression of an antibody including, for example, a full-length antibody.

[00487] Alternatively, DLL4 antibody CDRs that may be inserted into expression plasmids include: HCDR1 (DYAMS, SEQ ID NO: 34; GFTFSD, SEQ ID NO: 35; or GFTFSDYA; SEQ ID NO: 36), HCDR2 (TISGSGGDTFYADSVKG, SEQ ID NO: 37; or ISGSGGDT, SEQ ID NO: 38) and HCDR3 (DKNRGAYADAFD, SEQ ID NO: 39; or AKDKNRGAYADAFD, SEQ ID NO: 40); and/or wherein the light chain variable region comprises LCDR1 (TLRSDINVGTYRIY, SEQ ID NO: 41 ; or SDINVGTYR, SEQ ID NO: 42), LCDR2 (YKSDTDK, SEQ ID NO: 43; or YKSDTD, SEQ ID NO: 44) and LCDR3 (MVWHKSAPI, SEQ ID NO: 45). Such CDRs may alternatively include HCDR1 (DYAMS, SEQ ID NO: 34; GFTFSD, SEQ ID NO: 35; or GFTFSDYA; SEQ ID NO: 36), HCDR2 (TISGSGGDTFYADSVKG, SEQ ID NO: 37; or ISGSGGD, SEQ ID NO: 46) and HCDR3 (DKNRGAYADAFDI, SEQ ID NO: 47; or AKD KN RGAYAD AFD I , SEQ ID NO: 48); and/or wherein the light chain variable region comprises LCDR1 (TLRSDINVATYRIY, SEQ ID NO: 49; or SDINVATYR, SEQ ID NO: 50), LCDR2 (YKSDSDK, SEQ ID NO: 51 ; or YKSDSD, SEQ ID NO: 52) and LCDR3 (MVWHKSAPI, SEQ ID NO: 45). Alternatively, expression plasmids may be constructed that comprise the heavy and/or light chain variable region of the DLL4 antibodies generated and obtained in Example 1 , including, for example, SEQ ID NO: 56 or SEQ ID NO: 57 and SEQ ID NO: 61 or SEQ ID NO: 62, respectively. An exemplary antibody comprising a heavy chain variable region that is SEQ ID NO: 56 and a light chain variable region that is SEQ ID NO: 61 is designated herein as 12.14. An additional exemplary antibody comprising a heavy chain variable region that is SEQ ID NO: 57 and a light chain variable region that is SEQ ID NO: 62 is designated herein as 12.55 (see, e.g., Examples 3-1 1 ). Optionally, the antibody heavy and/or light chain variable regions may be configured for expression of an antibody including, for example, a full- length antibody. [00488] Several reference DLL4 antibodies including, BA1 (alternatively designated herein as 012 BA1 ) and BA2 (alternatively designated herein as 012 BA2), were also expressed as described above. BA1 comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 65 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 64 (see, e.g., Figure 2A). The heavy and light chain variable region sequences of BA1 are described in WO 08/042236 and termed OPM-21 M18 H7 and OPM-21 M18 L2, respectively. BA2 comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 67 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 66 (see, e.g., Figure 2B). The BA2 antibody is described in WO 08/060705 and termed clone #26.82.

Example 3. Epitope Specificity of Anti-DLL4 Antibodies

[00489] DLL4 antibodies generated as described in Examples 1 and 2, such as antibody 12.3 1 C1 , 12.73, 12.08, 12.14, or 12.55, may be tested by any known method in the art including, for example, an epitope binning assay. Such as epitope binning assay may use a sandwich assay comparable to those used in ELISA studies. These assays may be used to determine if the antibodies bind to the same epitope or different epitope on DLL4 to which other known binding molecules bind DLL4 {e.g., a benchmark or reference antibody). Briefly, in an exemplary epitope binning assay, a benchmark DLL4 antibody is covalently immobilized to a solid surface. Next, human DLL4 is injected over the immobilized benchmark antibody and binds to the immobilized antibody. A test antibody is then injected over the DLL4-benchmark antibody complex. If the benchmark and test antibody do not share the same epitope {e.g., are in different epitope bins), then binding of the test antibody to the antigen can be observed. If both antibodies bind to the same region {e.g., are in the same epitope bin), then binding of the benchmark to antigen will preclude binding of the test antibody and no interaction will be observed.

[00490] In an exemplary epitope binning assay, using Biacore A100 instrumentation (Biacore Life Sciences, GE Healthcare, Piscataway, NJ), a benchmark antibody (012 BA1 ) was tested with 15 other antibodies including, 12.43, 12.2 3C4, 12.3 1 C1 , 12.3 1 E2, 12.3 2G10, 12.4 1A6, 012 BA2, 12.02, 12.08, 12.19, 12.22, 12.24, 12.26, 12.73 and 12.55. The antibodies were covalently immobilized on four of the five spots of the four flow cells of a CM5 sensor chip (Biacore). The fifth spot of each of flow cells was left unmodified and used as a reference spot. During each cycle, human DLL4 (100 nM) was injected over all of the immobilized antibodies. Comparable to a sandwich assay in an ELISA format, this was followed by injection of a single antibody (taken from the set of the 16 immobilized antibodies) at an antibody binding site concentration of 200 nM. Surfaces were regenerated using either phosphoric acid or glycine-HCI pH 2.5. This cycle was then repeated substituting different antibodies or buffer injection for each of the different cycles. Sensorgrams of injected antibody were then processed using standard single and double- referencing techniques and scored for either the absence or presence of an interaction with antigen bound to immobilized antibody. From these assays, the 16 anti-DLL4 antibodies initially fell into one of seven bins (see, Figure 3). Further, of the possible 256 interactions that were observed in these assays {e.g., the interaction of 16 injected antibodies with 16 different antibody-DLL4 surfaces), the interaction of 12.3 1A1 with 012 BA1 - and 12.3 1 C1 - DLL4 surfaces is shown in a graphic display in Figure 4. Figure 4 shows that injected 12.3 1A1 does not interact with 012 BA1 -DLL4 surfaces (top panel) but does with 12.3 1 C1 - DLL4 surfaces (bottom panel).

[00491] The epitope binning assay shows that 012 BA1-DLL4 surfaces preclude the binding of 12.3 1 C1 and vice versa, that surfaces of 12.3.1 C1- DLL4 preclude the binding of 012 BA1 , suggesting similarity in the epitope binding region of these two antibodies are the same (see, e.g., Figure 3). Further epitope binning assays were performed with 012 BA1 , 012 BA2, 12.3 1A1 , 12.3 1 C1 , 12.3 1 C8, 12.3 1 C10, 12.3 1 D2, 12.5 1 D8, 12.5 1 G9, 12.5 6A9, 12.5 10C1 , PD-12.17, PD-12.18, PD-12.23, PD-12.33 and PD-12.7 to further investigate the epitope specificity of 012 BA1 and 12.3 1 C1. Such assays indicated that 12.3 1A1 can interact with DLL4 bound to 12.3 1 C1 but not to DLL4 bound to 012 BA1 . Further, the absence or presence of an interaction with injected 12.3 1A1 on 012 BA1 and 12.3 1 C1 -DLL4 surfaces shows that the epitope binding regions of 012 BA1 and 12.3 1 C1 are not identical (see, Figure 5). Also shown in Figure 5 are controls showing lack of response for 012 BA1 binding to 012 BA1-DLL4 surfaces and of 12.3 1 C1 binding to 12.3 1 C1 -DLL4 surfaces. Also shown in Figure 5 are controls showing the interaction of 12.5 6A9 with these two surfaces demonstrating that the 012 BA1 -DLL4 surface is active. As such, the epitope binding regions of 012 BA1 and 12.3 1 C1 are not identical and were grouped into separate bins (see, Figure 5). Thus, the epitope specificity of 12.3 1 C1 was unexpected, including in view of the epitope specificity of several benchmark antibodies.

[00492] Additionally, the epitope binning assay shows that 012 BA1-DLL4 and 012

BA2-DLL4 surfaces do not preclude the binding of 12.73 or 12.08 and vice versa, that surfaces of 12.73-DLL4 or 12.08-DLL4 do not preclude the binding of 012 BA1 or 012 BA2, suggesting that the epitope binding regions of these antibodies are not the same (see, e.g., Figure 3). As such, the epitope specificity of 12.73 and 12.08 were unexpected, including in view of the epitope specificity of several benchmark antibodies. [00493] Additionally, the epitope binning assay shows that 012 BA1-DLL4 and 012 BA2-DLL4 surfaces do not preclude the binding of 12.14 or 12.55 and vice versa, that surfaces of 12.55-DLL4 do not preclude the binding of 012 BA1 or 012 BA2, suggesting that the epitope binding regions of these antibodies are not the same (see, e.g., Figure 3). As such, the epitope specificity of 12.14 and 12.55 were unexpected, including in view of the epitope specificity of several benchmark antibodies.

Example 4. Binding of DLL4 Antibodies to DLL4

[00494] DLL4 antibodies generated as described in Examples 1 and 2 may be tested for their binding to DLL4 by a variety of methods including, for example, by fluorescence activated cell sorting (FACS) or by enzyme-linked immunosorbent assays (ELISA).

[00495] In an exemplary ELISA binding assay, DLL4 was immobilized to the wells of a 96 well plate by adding 50 μΙ of 1 μg/ml DLL4 protein (R&D Systems, Minneapolis, MN) per well of a 96 well plate and incubated at 4°C overnight. Next, plates were washed 4 times with phosphate buffered saline containing 0.05% Tween (PBS-T), were blocked with blocker (2% BSA-PBS), followed by the addition of 50 μΙ of goat anti-human Fcg fragment specific Ab-HRP (JIR) in 1 :10,000 dilution in 0.2% BSA-PBS. The plates were then incubated for 1.5 hours at room temperature after which they were washed six times with PBS-T. Subsequently, 50 μΙ of TMB Peroxidase EIA Substrate™ (Bio-Rad) was added to the wells and the plates were then incubated for 5 minutes at room temperature. Next, 50 μΙ of 1 M H2S04 was added to the wells to stop the reaction. Absorbance at 450 nm was then measured for each well by using a microplate assay reader SpectraMax (Molecular Devices, Sunnyvale, CA). Binding (EC50 (nM)) of several anti-DLL4 antibodies generated as described in Example 1 to DLL4 as determined by ELISA is shown in Table 1.

Table 1: Binding of DLL4 Antibodies to DLL4 (ELISA detection)

Figure imgf000136_0001
[00496] The binding of additional DLL4 antibodies to DLL4 was also determined by ELISA and their EC50 values determined {e.g., 4C9 (137.5 nM), 12.16 (18.0 nM), 12.26 (18.3 nM), 12.24 (23.2 nM), 12.22 (43.4 nM), 12.50 (40.0 nM), 12.38 (15.1 nM), 12.40 (42.8 nM), 1 C3 (17.9 nM), 1 D2 (48.7 nM), 2G10 (18.5 nM), 1 C10 (50.6 nM), 1 C8 (62.2 nM), 3C4 (81.4 nM), 2E2 (7144 nM), 1A4 (159.7 nM), 5B12 (310.6 nM), and 2C1 1 (333.3 nM)).

[00497] In an exemplary FACS binding assay, 293F cells that had been engineered to express DLL4 (DLL4/293F) were employed. Briefly, 1.0 x 105 DII4/293F cells were added to 96 well V bottom plates. After removing supernatant, 50 μΙ of DLL4 antibody in PBS containing 1 % FBS (FCM buffer) was added to the wells and the plates were then incubated for 30 minutes on ice. Next, cells were washed 2 times with FCM buffer. Subsequently, 50 μΙ of 2 μg/ml of Alexa Fluor 488™ goat anti-human IgG antibody (Invitrogen, Carlsbad, CA) in FCM buffer was added to the plates. After a 30 minute incubation on ice, the cells were washed 2 times with FCM buffer. Fluorescence was then measured by FACS CANT II™ (BD Biosciences, San Jose, CA). Binding (EC50 (nM)) of several anti-DLL4 antibodies generated as described in Example 1 to DLL4 as determined by FACS is shown in Table 2.

Table 2: Binding of DLL4 Antibodies to DLL4 (FACS detection)

Figure imgf000137_0001

[00498] The binding of additional DLL4 antibodies to DLL4 was also determined by FACS and their EC50 values determined {e.g., 4C9 (weak), 12.16 (2.0 nM), 12.26 (1 .5 nM), 12.24 (1.6 nM), 12.22 (1.9 nM), 12.50 (3.8 nM), 12.38 (1.0 nM), 12.40 (4.0 nM), 1 C3 (1 .5 nM), 1 D2 (4.0 nM), 2G10 (2.0 nM), 1 C10 (3.7 nM), 1 C8 (3.4 nM), 3C4 (3.8 nM), 2E2 (weak), 1A4 (8.7 nM), 5B12 (20.7 nM), and 2C1 1 (38.5 nM)).

Example 5. Binding Kinetics and Affinity of DLL4 Antibodies

[00499] DLL4 antibodies generated as described in Examples 1 and 2 may be tested for their binding kinetics and affinity to DLL4 by any method known in the art including, for example, Biacore (e.g., a Biacore A100 instrument), and the determined binding kinetics and affinity may be compared to other known DLL4 antibodies.

[00500] In an exemplary method employing Biacore, goat anti-human antibody surfaces or rabbit anti-mouse surface were constructed on flow cells on a CM5 chip using standard amine coupling chemistry. Carboxymethyldextran surfaces were first activated with NHS/EDC for 10 minutes prior to a 10 minute injection of either goat anti-human antibody (Caltag, Code#: H 10500, Invitrogen Carlsbad, CA) diluted to 20 g/mL using 10 mM NaOAc pH 5.0, or rabbit anti-mouse antibody (GE Healthcare, Piscataway, NJ, Cat. # BR-1008-38) also diluted to 20 μg/mL. Subsequently, excess activated carboxyls were blocked with 1 M ethanolamine, pH 8.5 with spot 3 on each of the four flow cells left unmodified. Immobilizations were then performed at 22 °C in HBS-N buffer (10 mM HEPES pH 7.4, 150 mM NaCI). Approximately 7000-7700 RU of goat anti-human antibody or 3000-4000 RU of rabbit anti-mouse was immobilized on a given spot on a flow cell. Experiments were initially performed to test anti-DLL4 antibody concentrations that would lead to optimal capture levels. The final experiment included 30 second captures during each cycle of anti-DLL4 antibody. Captures were then followed by a 3 minute stabilization period before injection of DLL4 for 5 minutes followed by 45 minutes of dissociation. Next, DLL4 was prepared at a concentration of 286 nM and 1 1 two-fold serial dilutions thereof and then injected randomly in triplicate. Buffer blanks were injected every fifth injection for double referencing purposes. At the end of each cycle, goat anti-human surfaces were regenerated with two 20 second pulses of 146 mM H3P04 (1/100 dilution of concentrated phosphoric acid) and RAM surfaces with two 20 second pulses of 10 mM glycine, pH 1 .7 from GE Healthcare (Cat #: BR-1008-38). Data was then processed and globally fit to a 1 :1 model with a trial version of Scrubber software designed for A100 data. Binding affinity (Kd (nM)) for several anti-DLL4 antibodies generated as described in Example 1 are shown in Table 3.

Table 3: Binding affinity of DLL4 Antibodies

Figure imgf000138_0001
Example 6. In Vitro Studies of DLL4 Antibodies

[00501] DLL4 antibodies including, for example, anti-DLL4 antibodies as described in Examples 1 and 2, may be tested for their ability to bind and affect DLL4 mediated processes in vitro. For example, the anti-DLL4 antibodies described herein may be tested for their ability to affect Notchl signaling, affect HES1 gene activation, affect cell proliferation and affect the induction of angiogenesis. Exemplary assays are described in Examples 7-10. Example 7. Inhibition of Notch and DLL4 Interaction with DLL4 Antibodies

[00502] DLL4 antibodies generated as described in Examples 1 and 2, such as antibody 3.1 C1 , 12.73, 12.08, 12.14, or 12.55, may be tested for their ability to affect interaction of DLL4 with Notch {e.g., Notch 1 and/or Notch 2) by any method known in the art including, for example, a Notch inhibition assay.

[00503] In an exemplary Notch inhibition assay, Notch binding to DLL4 was determined by quantifying the amount of Notch 1-Fc Alexa 647 bound to 293/DLL4 cells (see, e.g., Example 4) in the presence of varying concentrations of anti-DLL4 monoclonal antibodies. Briefly, 1 .0 x 105 DII4/293F cells were added to 96 well V bottom plates. After removing supernatant, 50 μΙ of anti-DLL4 antibody in PBS containing 1 % FBS (FCM buffer) was added and the plates were then incubated for 30 minutes on ice. Subsequently, 50 μΙ of Notchl -Fc-647 in 1 :800 dilution in FCM buffer was added to the plates, followed by an incubation for 30 minutes. Next, the cells were washed 2 times with FCM buffer and fluorescence was then measured by FACS CANT II™ (BD Biosciences; see also, Example 4). Notch 1 inhibition (IC50 (nM)) for several anti-DLL4 antibodies generated as described in Example 1 are shown in Table 4.

Table 4: Notch 1 inhibition with DLL4 Antibodies

Figure imgf000139_0001

[00504] The capacity of several additional DLL4 antibodies to inhibit interaction of DLL4 with Notch was also assayed and their IC50 values determined {e.g., 12.16 (14.1 nM), 12.26 (3.3 nM), 12.24 (8.9 nM), 12.22 (6.3 nM), 12.50 (16.9 nM), 12.38 (9.4 nM), 12.40 (22.2 nM), 1 C3 (4.3 nM), 1 D2 (16.6 nM), 2G10 (4.8 nM), 1 C10 (18.1 nM), 1 C8 (16.2 nM), 3C4 (15.4 nM), 2E2 (6.6 nM), 1A4 (28.0 nM), 5B12 (83.8 nM), and 2C1 1 (754.3 nM)). Example 8. Inhibition of Gene Activation with DLL4 Antibodies

[00505] DLL4 antibodies generated as described in Examples 1 and 2, such as antibody 3.1 C1 , 12.73, 12.08, 12.14, or 12.55, may be tested for their ability to affect DLL4 from activating its downstream effector targets such as HES1.

[00506] In an exemplary assay, HELA cells (ATCC) were seeded into a well of a six well plate at 2.5 x 105 cells per well and, the next day, were transiently transfected with 1 μg HES1 Luciferase reporter construct (Switchgear Genomics, Menlo Park, CA). The following day, cells were harvested and seeded into 96 well plates that had previously been coated with 50 μΙ of 1 μg/ml of DLL4 overnight at 4°C. Anti-DLL4 antibodies were then added to the wells and the plates were incubated for an additional 24 hours. After incubation, luciferase expression was determined by incubation of the wells with SteadyGlo™ (Promega, Madison, Wl). The capacity of several anti-DLL4 antibodies generated as described in Example 1 for affecting HES1 expression is shown in Table 5.

Table 5: HES1 Expression in the Presence of DLL4 Antibodies*

Figure imgf000140_0001

* Indicated EC50 values are the average of those values obtained from three independent experiments

[00507] The capacity of several additional DLL4 antibodies to influence HES1 expression was also assayed and their EC50 values determined {e.g., 4C3 (0.67 nM), 1A6 (10.4 nM), 12.55 (21.85 nM), 1A10 (24.4 nM), 1 E8 (68 nM), 12.16 (1 .95 nM), 12.26 (3.95 nM), 12.24 (1 1 .55 nM), 12.22 (16.95 nM), 12.50 (23.8 nM), 12.38 (43.7 nM), 12.40 (1 19.5 nM), 1 C3 (0.15 nM), 1 D2 (0.98 nM), 2G10 (0.22 nM), 1 C10 (1.65 nM), 1 C8 (2.15 nM), 3C4 (1 .21 nM), 2E2 (0.8 nM), 1A4 (2.38 nM), 5B12 (0.54 nM), 2C1 1 (29.6 nM), 2A2 (1.8 nM), 1 D10 (13.67 nM), 1 C2 (1 .8 nM), 3A1 (5.6 nM), 1 D3 (1.97 nM). Example 9. Inhibition of Cell Proliferation with DLL4 Antibodies

[00508] DLL4 antibodies generated as described in Examples 1 and 2, such as antibody 3.1 C1 , 12.73, 12.08, 12.14, or 12.55, may be tested for their ability to inhibit DLL4 mediated cell proliferation by any method in the art including, for example, a HUVEC proliferation assay.

[00509] In an exemplary HUVEC cell proliferation assay, DLL4 proteins (1 g/ml) were immobilized onto 96 wells plates. Next, HUVEC cells were obtained that had been plated and cultured in HUVEC medium (Invitrogen, Carlsbad CA) in the presence of 12.5 ng/ml VEGF. The HUVEC cells 3.1 C1 were then added to the 96 well plates with DLL4 antibody and cell proliferation was measured on day 3 using CellTiter Glo™ (Promega, Madison, Wl). Responses were then normalized to the presence or absence of DLL4. The 3.1 C1 antibody significantly inhibited HUVEC proliferation (EC50 (nM) of 3.9) as compared to two benchmark anti-DLL4 antibodies BA1 and BA2 (EC50 (nM) of 60 and 45). The proliferation of HUVEC cells in the presence of anti-DLL4 antibodies generated, as described in Example 1 is shown in Table 6.

Table 6: Proliferation of HUVEC Cells in the Presence of DLL4 Antibodies*

Figure imgf000141_0001

* Indicated EC50 values are the average of those values obtained from three independent experiments

[00510] The capacity of several additional DLL4 antibodies to influence HUVEC proliferation was also assayed and their EC50 values determined {e.g., 4C3 (369.49 nM), 1A6 (78.06 nM), 1 C3 (4.31 nM), 1 E2 (5.55 nM), 1 D2 (21 .37 nM), 2G10 (41 .1 nM), 1 C10 (70.07 nM), 1 C8 (52.87 nM), 3C4 (19.83 nM), 2E2 (3.35 nM), 1A4 (72.6 nM), 5B12 (108.97 nM), 2C1 1 (42 nM), 2A2 (92.97 nM), 1 C2 (192.6 nM), and 3A1 (154 nM).

Example 10. Inhibition of Anqioqenesis with DLL4 Antibodies

[0051 1] DLL4 antibodies generated as described in Examples 1 and 2, such as antibody 3.1 C1 , 12.73, 12.08, 12.14, or 12.55, may be tested in a HUVEC tube formation assay to study the capacity of the DLL4 antibody to inhibit the induction of angiogenesis. The tube formation assay is based on the ability of endothelial cells to form three- dimensional capillary-like tubular structures when cultured on a gel of basement membrane extract (BME).

[00512] In an exemplary HUVEC tube formation assay, Cytodex-3™ beads coated with HUVEC cells were embedded in a fibrin gel and cultured overnight. Next, Detroit 551 fibroblasts and DLL4 antibodies were added the next day and cultured for another 10 days with media changes every 2 days. On day 10, cultures were stained with anti-CD31 FITC. Images were acquired on ImageXpress Micro™ at 4x magnification with 72 images/well. Vessel features were extracted and tube length and branch points quantified using the ImageXpress software. The capacity of several anti-DLL4 antibodies generated as described in Example 1 to inhibit angiogenesis is shown in Table 7. The 3.1 C1 antibody was able to reduce angiogenesis branching (IC50 (nM) of 0.37) as compared to two benchmark anti-DLL antibodies BA1 and BA2 (IC50 (nM) of 2.37 and 0.1 1 ).

Table 7: Inhibition of Angiogenesis with DLL4 Antibodies

Figure imgf000142_0001
[00513] The capacity of several additional DLL4 antibodies to influence angiogenesis was also assayed. The following DLL4 antibodies were able to inhibit angiogenesis: 1A1 , 12.26, 1 C3, 1 D2, 2G10, 1 C10, 1 C8, 3C4, and 1A4.

Example 11. Cross Reactivity of DLL4 Antibodies with DLL4

[00514] DLL4 antibodies generated as described in Examples 1 and 2, such as antibody 3.1 C1 , 12.73, 12.08, 12.14, or 12.55, may be tested for their ability to cross-react with DLL4 obtained from various species {e.g., murine DLL4) and/or other ligands for Notch receptors such as DLL1 , Jaggedl , and Jagged2 by any method known in the art.

[00515] In an exemplary method employing Biacore as described in Example 5, cross-reactivity of the DLL4 antibodies with murine DLL4 may be determined. Briefly, goat anti-human antibody surfaces were constructed on flow cells 1 and 2 and rabbit anti-mouse surfaces on flow cells 3 and 4 on a CM5 chip using standard amine coupling chemistry. Carboxymethyldextran surfaces were first activated with NHS/EDC for 10 minutes prior to a 10 minute injection of either goat anti-human antibody (Caltag, Code#: H 10500) or rabbit anti-mouse antibody (GE Healthcare, BR-1008-38) diluted to 10 g/mL in 10 mM NaOAc, pH 5.0. Next, excess activated carboxyls were blocked with 1 M ethanolamine pH 8.5 with spot 3 on each of the four flow cells left unmodified. Subsequently, immobilizations were performed at 22 °C in HBS-N buffer (10 mM HEPES pH 7.4, 150 mM NaCI). Between 3250-3800 RU of goat anti-human antibody was immobilized on flow cells 1 and 2 and between 1870 and 2600 RU on flow cells 3 and 4. For each cycle separate anti-DLL4 antibody was captured on Spots 1 and 5 of each flow cell using Spots 2 and 4 as reference surfaces. Thus for each cycle, a total of 8 anti-DLL4 antibodies were captured. Anti-DLL4 antibody at concentrations of ~ 10 μg/mL (diluted in running buffer from mAb stocks) were captured for 90 seconds at a flow rate of 10 μΙ_/Γηίη. Next, binding of DLL-4 from various species was studied by injecting over captured anti-DLL4 antibodies either 100 nM murine DLL-4 (R&D Systems, 1389-D4/CF), cyno DLL4 (R&D Systems), or human DLL4 (R&D Systems, 1506-D4/CF) and characterized for binding activity based on KinExa (Sapidyne, Boise, ID) studies. DLL-4 was injected for a contact time of 120 seconds and dissociation was followed for 600 seconds at a flow rate of 30 μί/ηηίη. Goat anti-human surfaces were then regenerated with two 20 second pulses of 146 mM phosphoric acid and rabbit anti- mouse surfaces with two 20 second pulses of 10 mM glycine, pH 1 .7 (GE Healthcare, Cat # BR-1008-38). For each anti-DLL4 antibody, buffer was injected after anti-DLL4 antibody capture and used for double-referencing purposes. Data was collected at 22 °C using HBS-P (10 mM HEPES pH 7.4, 150 mM NaCI, 0.005% P-20) supplemented with BSA (Fraction V, Fisher Scientific, Cat #BP1605-100) to a final concentration of 100 μg/mL. Cross-reactivity of DLL4 antibodies with murine DLL4 was scored as a yes (Y) or a no (N) binding (see, e.g., Table 8).

[00516] In an exemplary method, cross-reactivity of the DLL4 antibodies with Jagged 1 , Jagged 3 and DLL1 may be determined. Briefly, high and low density surfaces of human DLL-1 , human Jagged-1 Fc Chimera and human Jagged-2 Fc Chimera were constructed on flow cells 1-3 of a CM5 chip using standard amine coupling chemistry. All reagents were obtained from R&D Systems, (Minneapolis, MN); DLL-1 (Cat #: 1818-D) and Jagged-2 (Cat#: 1726-JG) were reconstituted to 500 μg/mL in 20 mM KH2P04, pH 6.5; Human Jagged-1 (Cat#: 1277-JG) was reconstituted to 500 μg/mL in PBS. Next, high density surfaces were constructed on spots 1 and 2, and low density surfaces on spots 4 and 5 of flow cells 1 -3. Surfaces were first activated with NHS/EDC for 10 minutes prior to a 10 minute injection of mAbs diluted using 10 mM NaOAc pH 5.0 to final concentrations of 20 and 5 μg/mL for high and low density surfaces of human DLL-1 and Jagged-2 and 20 and 10 μg/mL for high and low density surfaces of human Jagged-1. Excess activated carboxyls were then blocked with 1 M ethanolamine pH 8.5. Spot 3 on each of the four flow cells was activated and deactivated but otherwise left unmodified. Subsequently, immobilizations were performed at 22 °C in HBS-N buffer (10 mM HEPES pH 7.4, 150 mM NaCI). For each cycle, mAb was injected at a molecular concentration of 100 nM for association and dissociation times of 30 and 600 seconds using flow rates of 30 μΙ_/Γηίη. Human DLL-1 surfaces were regenerated with a 20 second pulse of 10 mM NaOH, and human Jagged-1 and Jagged-2 surfaces with two 20 second pulses of 10 mM glycine-HCI 2.0 (GE Healthcare Cat: BR-1003-55) and 10 mM glycine 1.7 (GE Healthcare BR-1008- 38), respectively. Each injection of mAb was followed with an injection of buffer for double referencing purposes. The positive controls {e.g., anti-human DLL-1 (Cat#: MAB1818) reconstituted to 1 mg/mL in PBS, anti-human Jagged-1 , a biotinylated antibody used in previous human Jagged-1 cross reactivity experiments (Cat#: BAF 1277) reconstituted to 50 μg/mL in Tris-buffered saline, and anti-human Jagged-2 (Cat#: AF1726) reconstituted to 1 mg/mL in PBS) were also injected at a molecular concentration of 100 nM (and therefore an antibody binding site concentration of 200 nM). Data was collected at 22 °C using HBS- P (10 mM HEPES pH 7.4, 150 mM NaCI, 0.005% P-20) supplemented with BSA (Fraction V, Fisher Scientific, Cat #: BP1605-100) to a final concentration of 100 g/mL. The resulting sensorgrams were processed using the "Kinetics and Affinity" module in the Biacore A100 Evaluation software package. Cross-reactivity of DLL4 antibodies with Jagged 1 , DLL1 and Jagged 2 was scored as a yes (Y) or a no (N) binding (see, e.g., Table 8).

Table 8: Cross-Reactivity of DLL4 Antibodies

Figure imgf000144_0001

Example 12. Isolation of Immune Cells

[00517] Immune cells (e.g., human T cells and/or dendritic cells) for use in the following Examples may be obtained and isolated from any source including, for example, from the Buffy coat existing after density gradient centrifugation of a human blood sample. [00518] In an exemplary method, Buffy coats were obtained from the blood of healthy adult volunteer donors participating in the Stanford Medical School Blood Center blood donation program. Next, CD45RA+ CD4 T cells were isolated from Buffy coats by depletion using CD4 RosetteSep™ (Stem Cell Technologies, Vancouver, Canada) followed by depletion using AutoMACs of CD45RO+ T cells using anti-CD45RO MACS™ beads (Miltenyi Biotec, Auburn, CA). Likewise, dendritic cells were isolated using the Blood Dendritic Cell Isolation Kit™ (Miltenyi Biotec) or by using the MDC1 Isolation Kit™ (Miltenyi Biotec). Example 13. Cytokine Expression in DLL4 Activated T Cells

[00519] The expression of cytokines such as IL-17, may be determined in DLL4 activated T cells by any method known in the art including, for example, using a Cytometric Bead Array™ or by intracellular cytokine staining.

[00520] In an exemplary method, recombinant DLL4 at 2.5 μg/ml in PBS was immobilized overnight at 4°C onto flat bottom 96 well plates. The following day, the plates were washed twice and naive CD45RA+ CD4+ T cells obtained in Example 1 were cultured at 1 x 105 cells per well. Next, naive CD45RA+ CD4+ T cells obtained in Example 1 were activated with T activator beads (0.5 x 106 beads per well, Invitrogen, Carlsbad, CA) coated with anti-CD3/anti-CD28 in the presence of immobilized DLL4 or PBS vehicle control. Cell populations were subject to different Th cell polarization conditions such as: IL-12 (10 ng/ml); pro-alFN-γ (5 g/ml) + alL-4 (5 g/ml); and TGF-β (1 ng/ml) + pro-alFN-γ (5 g/ml) + alL-4 (5 g/ml). Five days after activation of the T cells, IL-17A concentration in the conditioned media was measured by Cytometric Bead Array™ (BD Biosciences). Activation of T cells in the presence of the vehicle control and exogenous pro (IL-6, I L- 1 β , and IL-23) induced little IL-17A expression (see, e.g., Figure 6A). The expression of IL- 17A was enhanced in the T cells activated in the presence of the vehicle control by the addition of TGF-β. T cells activated in the presence of immobilized DLL4 exhibited substantially enhanced (approximately 2-fold increase) in IL-17A production (see, e.g., Figure 6A). Given that IL-17A expression was below the level of detection in either media or IL-12 treated samples, IL-17A expression was also assessed by intracellular cytokine staining. Briefly, cells were restimulated with 50 ng/ml PMA (Sigma), 1 μg/ml ionomycin (Sigma), and GolgiStop™ (Pharmingen) for 5 hours. Cells were then fixed with Cytofix/Cytoperm™ (Pharmingen) and stained with anti-CD4 and an antibody to IL-17A (eBiosciences). Intracellular staining detected by FACS showed that IL-17A expression increased in those T cells activated in the presence of DLL4 and IL-12 (approximately 4- fold) as compared to T cells activated in the presence of DLL4 and the vehicle control (see, e.g., Figure 6B).

Example 14. Cell Proliferation of DLL4 Activated T Cells

[00521] The proliferation of activated T cells may be determined by any method known in the art including, for example, a carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution assay employing flow cytometry.

[00522] In an exemplary method, naive CD4+ T cells were labeled with CFSE and activated under ThO or Th17 conditions with or without immobilized DLL4 as described in Example 13. Five days after T cell activation, CFSE dilution profiles were assessed by flow cytometry. No substantial differences in cell proliferation were observed in cells activated in media alone or cells activated in the presence of TGF-β, pro, alFN-γ and al L-4 suggesting that DLL4 enhances Th17 differentiation through a proliferation-independent mechanism (see, e.g., Figure 6C).

Example 15. Impact of DLL4 on Th17 Differentiation

[00523] The capacity of DLL4 to impact Th17 differentiation in T cells such as naive or activated T cells may be determined by any method known in the art including, for example, assays employing the use of a Cytometric Bead Array™ to evaluate IL-17A expression in the T cells.

[00524] In an exemplary method, naive (CD45RA+) and memory/activated (CD45RO+) CD4 T cells were obtained from eight human donors and were activated as described in Example 1. After 5 days, IL-17A expression in the conditioned media was measured as described in Example 13. In the tested donors, DLL4 enhanced IL-17A production by naive CD4 T cells activated with polarizing conditions (e.g., pro + alFN-γ + al L-4; and TGF-β + pro + alFN-γ + al L-4) by an average of -80-100 fold (see, e.g., Figure 6D). Fold change was measured relative to PBS condition. For naive (CD45RA+) and memory/activated (CD45RO+) CD4 T cells the fold increase in IL-17A expression with media alone or IL-12 was below the level of detection. Further, DLL4 also enhanced IL-17 production from CD45RO+ memory/activated CD4 T cells with polarizing conditions (e.g., pro + alFN-γ + alL-4; and TGF-β + pro + alFN-γ + al L-4) and non-polarizing conditions (media alone and IL-12), although the average fold induction was 2-3 fold lower. Example 16. Th17 Cell Activation and/or Differentiation Upon Substitution of a Pro- Inflammatory Cytokine with DLL4

[00525] The capacity for DLL4 to substitute for any of the cytokines necessary for Th17 differentiation (e.g., pro-inflammatory cytokines such as IL-6, IL-Ι β, or IL-23) may be tested by any method known in the art including, for example, assays employing the use of a Cytometric Bead Array™.

[00526] In an exemplary method, naive human CD4 T cells were activated with anti-CD3/anti-CD28 beads as described above in Example 13 except that the cells were grown in a chemically-defined serum-free media (X-Vivo 20) and in the presence of IL-6; IL-1 β; IL-23; TGF-β; IL-6 + TGF-β; IL-1 β + TGF-β; IL-23 + TGF-β; pro; TGF-β + pro or anti- INF-γ + anti-IL-4. Next, IL-17A expression in conditioned media was measured on day 5 using a Cytometric Bead Array™. DLL4 in combination with neutralizing anti-IFN-γ and anti-IL-4 did not induce IL-17 production (see, e.g., Figure 7). In conditions where TGF-β was not added, IL-6, I L- 1 β , IL-23, either alone or in combination, did not induce IL-17A production in the presence of DLL4, indicating that DLL4 could not substitute for TGF- β signaling. DLL4 was also not sufficient to induce IL-17 production in the presence of TGF- β alone, suggesting DLL4 could not substitute for the pro-inflammatory cytokines. DLL4 did enhance IL-17 production when TGF-β was present in combination with either IL-6, IL- 1 β, IL-23, or all three.

Example 17. Effect of DLL4 on Cytokine Expression

[00527] The effect of DLL4 on the expression of cytokines such as IL-22, IFN-γ, and IL-4 may be assessed by any method known in the art including, for example, assays that employ the use of a Cytometric Bead Array™ or ELISA.

[00528] In an exemplary method, naive human CD4 T cells were activated with anti-CD3/anti-CD28 beads in wells previously coated with DLL4 or PBS without any polarizing cytokines (see, e.g., Figure 8A). IL-22, IFN-γ, and IL-4 expression were then examined in conditioned media after day 5 of activation. IL-22 expression in the conditioned media was measured by ELISA using IL-22 Duoset (R&D Systems) while IFN- Y and IL-4 expression were measured with a Cytometric Bead Array™. DLL4 induced expression of IFN-γ and IL-22 but not IL-4. Additionally, naive and memory/Ag experienced human CD4 T cells from 8 individual donors were activated in wells coated with either DLL4 or PBS along with anti-CD3/anti-CD28 beads in the presence of media alone; IL-12; pro, anti-INF-γ + anti-IL-4; or TGF-β + pro + anti-INF-γ + anti-IL-4. Again cytokine expression in conditioned media was measured on day 5 as described above. A consistent increase in IL-22 expression (6-7 fold) was observed when CD45RA+ cells were activated in media with IL-12 (Th1 ), or pro, alFN-γ and alL-4, or TGF-β, pro, alFN-γ and alL-4 (Th17) polarizing conditions (see, e.g., Figure 8B). This effect on IL-22 was more pronounced for naive cells as compared to memory/antigen experienced cells which exhibited less than a 1.5 fold change under the tested polarizing conditions, suggesting a more critical role for DLL4 during initial Th22 differentiation. In comparison, the effects of DLL4 signaling on IFN-γ in the absence of polarizing cytokines were less than on DLL4, resulting in a modest increase (2.5 -3 fold) for both naive and memory/activated cells activated without any polarizing cytokines. This suggests that this effect is not specific for the initial differentiation of Th1 cells. No changes in IFN-γ production were observed in the presence of polarizing cytokines. In addition to increasing IL-22 and IFN-γ, DLL4 modestly inhibited TNF-a production (2-3 fold) across all conditions for naive cells.

[00529] Based on the above assays, additional assays were conducted to examine the effect of an anti-Notch 1 (e.g., at 10 g/ml) on DLL4 induced cytokine expression in T cells from two donors (e.g., Donor 1 and Donor 2) that were activated under ThO, Th1 , and Th17 conditions. DLL4 increased IL-17A expression in both Donor 1 and Donor 2 under Th17 conditions. DLL4 also increased IFN-γ expression under Th1 conditions. DLL4 inhibited TNF-a and IL-6 production under Th1 and Th17 conditions. Thus, addition of a Notchl antibody inhibited DLL4 mediated changes in IL-17A, IFN-γ, TNF-a, IL-10, and IL-6, resulting in cytokine production similar to T cells cultured alone in the absence of DLL4, suggesting that DLL4 signals through at least Notch 1 receptor.

Example 18. Regulation of IL-10 Expression by DLL4

[00530] DLL4 may be tested for its effects on IL-10 expression by any method known in the art including, for example, assays employing the use of a Cytometric Bead Array™ to evaluate IL-10 expression in the T cells.

[00531] In an exemplary method, naive and memory/activated human CD4 T cells from 8 individual donors were activated with anti-CD3/anti-CD28 beads in the presence of the indicated cytokines and antibodies in wells coated with either DLL4 or PBS (see, e.g., Figure 9A). IL-10 expression in conditioned media was measured on day 5. IL-10 production by naive T cells was enhanced by DLL4 (8-13 fold), with the highest production from IL-12 activated T cells. DLL4 also increased IL-10 production from memory/activated cells but to a lower extent. The induction of IL-10 by DLL4 under almost all conditions tested suggested that this IL-10 may not be selective to Th1 or Th17 cells. To examine if IL-10 is co-produced by either a IFN-γ or IL-17A producing population, intracellular cytokine staining under Th1 and Th17 polarizing conditions with and without DLL4 was performed (see, e.g., Figure 9B). Whereas IL-10 is co-secreted with IFN-γ in Th1 cells, co-expression of IL-10 with IL-17A was not observed.

Example 19. Induction of Cytokine Expression in Activated T Cells with Notch Liqands

[00532] Notch ligands such as DLL4, DLL1 , Jaggedl , and Jagged2, may be tested by any method known in the art for their ability to regulate cytokine expression including, for example, a HES1 luciferase reporter assay.

[00533] In an exemplary assay, 50 μΙ of 1 μg/ml recombinant DLL4, DLL1 , Jaggedl , and Jagged2 were immobilized onto wells of a 96 well/plate overnight at 4° C. Next, HELA cells (ATCC) were transiently transfected with 1 μg HES1 Luciferase reporter construct (Switchgear Genomics, Menlo Park, CA) and Fugene6 (Roche Diagnostics). The following day, the HELA cells were harvested and seeded onto the 96 well plates coated with PBS, DLL4, DLL1 , Jaggedl , or Jagged2. After a 24 hour incubation, luciferase expression was determined by incubation with SteadyGlo (Promega, Madison, Wl) following the manufacturer's directions. DLL4, DLL1 , Jaggedl , and Jagged2 each induced HES1 gene transcription from HELA cells, indicating that these proteins are all active (see, e.g., Figure 10A). Next, naive human CD4 T cells were activated with anti-CD3/anti-CD28 beads in the presence of TGF-β, IL-1 β , IL-6, and IL-23 in wells previously coated with DLL4, DLL1 , Jaggedl , or Jagged2. Cytokine expression was then measured in conditioned media on day 5. Under these conditions, DLL4 was the only Notch ligand that enhanced expression of IL-17A, IL-22 and IL-10 while reducing expression of TNF-a (see, e.g., Figure 10B).

Example 20. Expression of Notch Receptors on Activated T cells

[00534] The expression of Notch receptors on naive CD4 T cells may be determined and compared to the expression of Notch receptors on activated T cells by any method known in the art including, for example, an assay employing monoclonal antibodies specific for Notchl , 2, 3, or 4.

[00535] In an exemplary method, the expression of Notchl , Notch2, Notch3, and Notch4 was determined on naive CD4 T cells and on day 5 CD4 T cells activated with anti- CD3/anti-CD28 beads. Briefly, Notchl Fc and Notch2 Fc were immobilized onto plates. Next, soluble DLL4-biotin was added to each well at the indicated concentration and binding of DLL4 was determined using a monoclonal antibody to each of Notchl , Notch2, Notch3, and Notch4. None of the assayed Notch receptors were found on resting naive CD4 T cells (see, e.g., Figure 1 1 A). However, activation of T cells with anti-CD3/CD28 induced Notch 1 , Notch2, and Notch4 expression whereas no Notch3 was detected (see, e.g., Figure 1 1A).

Example 21. Interaction of DLL4 with Notch2

[00536] Interactions between DLL4 and any of the Notch receptors including, for example, Notch2, may be determined by any method known in the art such as a Notch Fc- DLL4 binding assay.

[00537] In an exemplary method to test for any interaction between Notch2 and DLL4, 1 μg/ml of Notchl and Notch2 Fc were immobilized onto half well 96 well plates overnight. After washing with PBS-T and blocking with BSA, biotinylated DLL4 was added at the indicated concentrations and incubated for 2 hours at room temperature. Bound DLL4 was detected using Streptavidin HRP (Thermo, Worcester, MA) and TMB substrate (Biorad, Hercules, CA). To test the specificity of this interaction, 200 ng/ml of biotinylated DLL4 was incubated with anti-DLL4 or mlgG1 at the indicated concentrations before adding to Notchl Fc or Notch2 Fc immobilized plates. After washing the plates, DLL4 bound to Notchl or Notch2 was detected by streptavidin HRP. DLL4 bound strongly with Notchl and weakly bound Notch2 (see, e.g., Figure 1 1 B). In an effort to determine if the interactions between Notchl and Notch2 with DLL4 are specific, a neutralizing antibody to DLL4 generated in Example 1 was employed. This antibody was identified through binding and cell based functional assays, including inhibiting DLL4 mediated HES1 activation in HELA cells (see, e.g., Figure 1 1 C) and inhibited DLL4 mediated IL-17 production from Th17 cells (see, e.g., Figure 1 1 D). This antibody did not cross react with other paralogs, including DLL1 , Jaggedl , and Jagged 2. When this antibody was tested in the Notch Fc- DLL4 binding assay, both Notchl and Notch2 binding to DLL4 was completely inhibited. These data suggest a specific interaction between Notchl and Notch2 and DLL4 and demonstrate a novel finding that DLL4 is also ligand for the Notch 2 receptor.

Example 22. Expression of DLL4 on Human PBMCs

[00538] The expression of DLL4 on human PBMCs may be determined by any method known in the art including, for example, assays employing the use of FACS analysis.

[00539] In an exemplary method, PBMCs were activated with 100 ng/ml LPS for 2 days and then stained for DLL4 and CD14 expression with antibodies to both DLL4 and CD14, respectively. Treatment of total PBMCs with LPS led to an increase in DLL4 expression on a small percentage of CD14" cells (see, e.g., Figure 12A). Similarly, expression of DLL4 was also observed on CD83+ cells (see, e.g., Figure 12B). Next, pDCs and mDCs were purified from whole blood and cultured with 100 ng/ml LPS for 2 days. Activation with LPS induced DLL4 expression on CD1 c+ mDCs but not on BDCA-2+ pDCs, confirming expression of DLL4 on a subset of activated dendritic cells (see, e.g., Figure 12C).

Example 23. In Vivo Alteration of Cytokine Production with Anti-DLL4 Antibodies

[00540] The ability of an anti-DLL4 antibody to neutralize and affect cytokine expression in vivo may be determined by any method known in the art including, for example, an assay employing the use of a mixed lymphocyte reaction.

[00541] In an exemplary assay to determine if neutralizing endogenous DLL4 in a mixed lymphocyte reaction with human LPS activated mDC1 s and naive CD4 T cells results in altered cytokine secretion, CD1 c+ mDC1 cells were purified and activated with LPS for 2 days. Next, naive CD4 T cells were co-cultured with CD1 c+ mDC1 cells for 5 days with polarizing cytokines and antibodies such as media alone; IL-12; pro, anti-INF-γ + anti-IL-4; or TGF-β + pro + anti-INF-γ + anti-IL-4. Anti-DLL4 or relevant isotype control was used at 10 μg/ml. After 7 days, cytokine production was measured as described above. IL- 17A, DLL4, and IL-10 expression substantially decreased in the presence of neutralizing anti-DLL4 antibody (see, e.g., Figure 12D). Anti-DLL4 treatment also led to a decrease in IL-17A and IL-22 expression as detected by intracellular cytokine staining of T cells activated with Th17 polarizing conditions (see, e.g., Figure 12E). In contrast, no effects on IFN-γ production were observed with anti-DLL4, even in the absence of IL-12. TNF-β expression was consistently increased in the presence of anti-DLL4, suggesting that DLL4 plays a role in regulating TNF-β production by T cells. Whether the enhanced TNF-β production observed with neutralizing anti-DLL4 antibodies was indirectly due to the associated decrease in IL-10 was also evaluated. Briefly, a neutralizing anti-IL-10 antibody in MLR cultures was used to inhibit IL-10 signaling. Neutralization of IL-10 did not have any direct impact on TNF-β, IL-17A, or DLL4 production in these MLR cultures, suggesting that the increased TNF-β production observed here is independent of the immunosuppressive effects of IL-10

[00542] For the disclosure herein, the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[00543] The terms "a," "an," "the" and similar referents used in the context of describing the exemplary embodiments (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language {e.g., "such as") provided herein is intended merely to better illuminate the exemplary embodiments and does not pose a limitation on the scope of the exemplary embodiments otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the exemplary embodiments.

[00544] Groupings of alternative elements or embodiments disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[00545] Certain embodiments are described herein, including the best mode known to the inventors for carrying out the exemplary embodiments. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. Skilled artisans are expected to employ such variations as appropriate, and the embodiments are intended to be practiced otherwise than specifically described herein. Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the disclosure unless otherwise indicated herein or otherwise clearly contradicted by context.

[00546] Furthermore, numerous references have been made to patents and printed publications. Each of the above-cited references is individually incorporated herein by reference in their entirety. [00547] Specific embodiments disclosed herein can be further limited in the claims using consisting of or and consisting essentially of language. When used in the claims, whether as filed or added per amendment, the transition term "consisting of excludes any element, step, or ingredient not specified in the claims. The transition term "consisting essentially of" limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s). Exemplary embodiments so claimed are inherently or expressly described and enabled herein.

[00548] In closing, it is to be understood that the exemplary embodiments disclosed herein are illustrative of the principles of the present disclosure. Other modifications that can be employed are within the scope of the disclosure. Thus, by way of example, but not of limitation, alternative configurations of the present exemplary embodiments can be utilized in accordance with the teachings herein. Accordingly, the present exemplary embodiments are not limited to that precisely as shown and described.

Claims

Claims:
1. An isolated delta-like ligand 4 (DLL4) antibody comprising a heavy chain variable region and a light chain variable region, wherein:
i. ) the heavy chain variable region comprises HCDR1 (GYSWH, SEQ ID NO: 1 ; DHSITSG, SEQ ID NO: 2; or DHSITSGYS; SEQ ID NO: 3), HCDR2
(YIHYSGYTHYNPSLKS, SEQ ID NO: 4; or IHYSGY, SEQ ID NO: 5) and HCDR3 (SDYSANEGAMAY, SEQ ID NO: 6; or AGS DYS AN E GAM AY, SEQ ID NO: 7); and/or the light chain variable region comprises LCDR1 (RASESVDSFGNSFMH , SEQ ID NO: 8; or ESVDSFGNSF, SEQ ID NO: 9), LCDR2 (RTSNLQS, SEQ ID NO: 10; or R) and LCDR3 (QQSNEDPWT, SEQ ID NO: 1 1 );
ii. ) the heavy chain variable region comprises HCDR1 (DEYWS, SEQ ID NO: 12; GGSFND, SEQ ID NO: 13; or GGSFNDEY; SEQ ID NO: 14), HCDR2 (EIHESGKTNYNPSLKP, SEQ ID NO: 15; or IHESGKT, SEQ ID NO: 16) and HCDR3 (LAYRDRWYGAFDV, SEQ ID NO: 17; or ARLAYRDRWYGAFDV, SEQ ID NO: 18); and/or the light chain variable region comprises LCDR1 (QASHDITNYIN, SEQ ID NO: 19; or HDITNY, SEQ ID NO: 20), LCDR2 (HHTSKLQT, SEQ ID NO: 21 ; or H) and LCDR3 (QQFDNLLLT, SEQ ID NO: 22);
iii. ) the heavy chain variable region comprises HCDR1 (GYYMH, SEQ ID NO: 23; GYTFTG, SEQ ID NO: 24; or GYTFTGYY; SEQ ID NO: 25), HCDR2 (WINPNSGGTNYAQ, SEQ ID NO: 26; or INPNSGG, SEQ ID NO: 27) and HCDR3 (GAGVAAYDY, SEQ ID NO: 28; or ARGAGVAAYDY, SEQ ID NO: 29); and/or the light chain variable region comprises LCDR1 (RASQSISSYLN, SEQ ID NO: 30; or QSISSY, SEQ ID NO: 31 ), LCDR2 (AASSLQS, SEQ ID NO: 32; or A) and LCDR3 (QQSYSTPVT, SEQ ID NO: 33);
iv. ) the heavy chain variable region comprises HCDR1 (DYAMS, SEQ ID NO: 34; GFTFSD, SEQ ID NO: 35; or GFTFSDYA; SEQ ID NO: 36), HCDR2
(TISGSGGDTFYADSVKG, SEQ ID NO: 37; or ISGSGGDT, SEQ ID NO: 38) and HCDR3 (DKNRGAYADAFD, SEQ ID NO: 39; or AKDKNRGAYADAFD, SEQ ID NO: 40); and/or the light chain variable region comprises LCDR1 (TLRSDINVGTYRIY, SEQ ID NO: 41 ; or SDINVGTYR, SEQ ID NO: 42), LCDR2 (YKSDTDK, SEQ ID NO: 43; or YKSDTD, SEQ ID NO: 44) and LCDR3 (MVWHKSAPI, SEQ ID NO: 45); or
v. ) the heavy chain variable region comprises HCDR1 (DYAMS, SEQ ID NO: 34; GFTFSD, SEQ ID NO: 35; or GFTFSDYA; SEQ ID NO: 36), HCDR2 (TISGSGGDTFYADSVKG, SEQ ID NO: 37; or ISGSGGD, SEQ ID NO: 46) and HCDR3 (DKNRGAYADAFDI, SEQ ID NO: 47; or AKDKNRGAYADAFDI, SEQ ID NO: 48); and/or the light chain variable region comprises LCDR1 (TLRSDINVATYRIY, SEQ ID NO: 49; or SDINVATYR, SEQ ID NO: 50), LCDR2 (YKSDSDK, SEQ ID NO: 51 ; or YKSDSD, SEQ ID NO: 52) and LCDR3 (MVWHKSAPI, SEQ ID NO: 45).
2. The isolated DLL4 antibody of claim 1 , wherein:
the antibody in part i.) comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 53;
the antibody in part ii.) comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 54;
the antibody in part iii.) comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 55;
the antibody in part iv.) comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 56; or
the antibody in part v.) comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 57.
3. The isolated DLL4 antibody of claim 1 , wherein:
the antibody in part i.) comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58;
the antibody in part ii.) comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 59;
the antibody in part iii.) comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 60;
the antibody in part iv.) comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 61 ; or
the antibody in part v.) comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 62.
4. The isolated DLL4 antibody of claim 1 , wherein:
the antibody in part i.) comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 53 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58;
the antibody in part ii.) comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 54 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 59; the antibody in part iii.) comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 55 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 60;
the antibody in part iv.) comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 56 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 61 ; or
the antibody in part v.) comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 57 and a light chain variable region comprising the amino acid sequence SEQ ID NO: 62.
5. An isolated delta-like ligand 4 (DLL4) antibody that binds to the same epitope that an antibody comprising a heavy chain variable region and a light chain variable region binds to, wherein:
i. ) the heavy chain variable region comprises HCDR1 (GYSWH, SEQ ID NO: 1 ; DHSITSG, SEQ ID NO: 2; or DHSITSGYS; SEQ ID NO: 3), HCDR2
(YIHYSGYTHYNPSLKS, SEQ ID NO: 4; or IHYSGY, SEQ ID NO: 5) and HCDR3 (SDYSANEGAMAY, SEQ ID NO: 6; or AGS DYS AN E GAM AY, SEQ ID NO: 7); and/or the light chain variable region comprises LCDR1 (RASESVDSFGNSFMH , SEQ ID NO: 8; or ESVDSFGNSF, SEQ ID NO: 9), LCDR2 (RTSNLQS, SEQ ID NO: 10; or R) and LCDR3 (QQSNEDPWT, SEQ ID NO: 1 1 );
ii. ) the heavy chain variable region comprises HCDR1 (DEYWS, SEQ ID NO: 12; GGSFND, SEQ ID NO: 13; or GGSFNDEY; SEQ ID NO: 14), HCDR2 (EIHESGKTNYNPSLKP, SEQ ID NO: 15; or IHESGKT, SEQ ID NO: 16) and HCDR3 (LAYRDRWYGAFDV, SEQ ID NO: 17; or ARLAYRDRWYGAFDV, SEQ ID NO: 18); and/or the light chain variable region comprises LCDR1 (QASHDITNYIN, SEQ ID NO: 19; or HDITNY, SEQ ID NO: 20), LCDR2 (HHTSKLQT, SEQ ID NO: 21 ; or H) and LCDR3 (QQFDNLLLT, SEQ ID NO: 22);
iii. ) the heavy chain variable region comprises HCDR1 (GYYMH, SEQ ID NO: 23; GYTFTG, SEQ ID NO: 24; or GYTFTGYY; SEQ ID NO: 25), HCDR2 (WINPNSGGTNYAQ, SEQ ID NO: 26; or INPNSGG, SEQ ID NO: 27) and HCDR3 (GAGVAAYDY, SEQ ID NO: 28; or ARGAGVAAYDY, SEQ ID NO: 29); and/or the light chain variable region comprises LCDR1 (RASQSISSYLN, SEQ ID NO: 30; or QSISSY, SEQ ID NO: 31 ), LCDR2 (AASSLQS, SEQ ID NO: 32; or A) and LCDR3 (QQSYSTPVT, SEQ ID NO: 33);
iv. ) the heavy chain variable region comprises HCDR1 (DYAMS, SEQ ID NO: 34; GFTFSD, SEQ ID NO: 35; or GFTFSDYA; SEQ ID NO: 36), HCDR2
(TISGSGGDTFYADSVKG, SEQ ID NO: 37; or ISGSGGDT, SEQ ID NO: 38) and HCDR3 (DKNRGAYADAFD, SEQ ID NO: 39; or AKDKNRGAYADAFD, SEQ ID NO: 40); and/or the light chain variable region comprises LCDR1 (TLRSDINVGTYRIY, SEQ ID NO: 41 ; or SDINVGTYR, SEQ ID NO: 42), LCDR2 (YKSDTDK, SEQ ID NO: 43; or YKSDTD, SEQ ID NO: 44) and LCDR3 (MVWHKSAPI, SEQ ID NO: 45); or
v.) the heavy chain variable region comprises HCDR1 (DYAMS, SEQ ID NO: 34;
GFTFSD, SEQ ID NO: 35; or GFTFSDYA; SEQ ID NO: 36), HCDR2 (TISGSGGDTFYADSVKG, SEQ ID NO: 37; or ISGSGGD, SEQ ID NO: 46) and HCDR3 (DKNRGAYADAFDI, SEQ ID NO: 47; or AKDKNRGAYADAFDI, SEQ ID NO: 48); and/or the light chain variable region comprises LCDR1 (TLRSDINVATYRIY, SEQ ID NO: 49; or SDINVATYR, SEQ ID NO: 50), LCDR2 (YKSDSDK, SEQ ID NO: 51 ; or YKSDSD, SEQ ID NO: 52) and LCDR3 (MVWHKSAPI, SEQ ID NO: 45).
6. An isolated delta-like ligand 4 (DLL4) antibody that binds to the same epitope that an antibody comprising:
i.) a heavy chain variable region comprising the amino acid sequence of SEQ ID
NO: 53 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58 binds to;
ii. ) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 54 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 59 binds to;
iii. ) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 55 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 60 binds to;
iv. ) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 56 and a light chain variable region comprising the amino acid sequence of
SEQ ID NO: 61 binds to; or
v. ) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 57 and a light chain variable region comprising the amino acid sequence SEQ ID NO: 62 binds to.
7. An isolated delta-like ligand 4 (DLL4) antibody that binds to human DLL4 (SEQ ID NO: 63) and competes with the binding of an antibody comprising a heavy chain variable region and a light chain variable region, wherein:
i.) the heavy chain variable region comprises HCDR1 (GYSWH, SEQ ID NO: 1 ; DHSITSG, SEQ ID NO: 2; or DHSITSGYS; SEQ ID NO: 3), HCDR2 (YIHYSGYTHYNPSLKS, SEQ ID NO: 4; or IHYSGY, SEQ ID NO: 5) and HCDR3 (SDYSANEGAMAY, SEQ ID NO: 6; or AGS DYS AN E GAM AY, SEQ ID NO: 7); and/or the light chain variable region comprises LCDR1 (RASESVDSFGNSFMH , SEQ ID NO: 8; or ESVDSFGNSF, SEQ ID NO: 9), LCDR2 (RTSNLQS, SEQ ID NO: 10; or R) and LCDR3 (QQSNEDPWT, SEQ ID NO: 1 1 );
ii.) the heavy chain variable region comprises HCDR1 (DEYWS, SEQ ID NO: 12;
GGSFND, SEQ ID NO: 13; or GGSFNDEY; SEQ ID NO: 14), HCDR2 (EIHESGKTNYNPSLKP, SEQ ID NO: 15; or IHESGKT, SEQ ID NO: 16) and HCDR3 (LAYRDRWYGAFDV, SEQ ID NO: 17; or ARLAYRDRWYGAFDV, SEQ ID NO: 18); and/or the light chain variable region comprises LCDR1 (QASHDITNYIN, SEQ ID NO: 19; or HDITNY, SEQ ID NO: 20), LCDR2 (HHTSKLQT, SEQ ID NO: 21 ; or H) and LCDR3 (QQFDNLLLT, SEQ ID NO: 22);
iii. ) the heavy chain variable region comprises HCDR1 (GYYMH, SEQ ID NO: 23; GYTFTG, SEQ ID NO: 24; or GYTFTGYY; SEQ ID NO: 25), HCDR2 (WINPNSGGTNYAQ, SEQ ID NO: 26; or INPNSGG, SEQ ID NO: 27) and HCDR3 (GAGVAAYDY, SEQ ID NO: 28; or ARGAGVAAYDY, SEQ ID NO: 29); and/or the light chain variable region comprises LCDR1 (RASQSISSYLN, SEQ ID NO: 30; or QSISSY, SEQ ID NO: 31 ), LCDR2 (AASSLQS, SEQ ID NO: 32; or A) and LCDR3 (QQSYSTPVT, SEQ ID NO: 33);
iv. ) the heavy chain variable region comprises HCDR1 (DYAMS, SEQ ID NO: 34; GFTFSD, SEQ ID NO: 35; or GFTFSDYA; SEQ ID NO: 36), HCDR2 (TISGSGGDTFYADSVKG, SEQ ID NO: 37; or ISGSGGDT, SEQ ID NO: 38) and HCDR3 (DKNRGAYADAFD, SEQ ID NO: 39; or AKDKNRGAYADAFD, SEQ ID NO: 40); and/or the light chain variable region comprises LCDR1 (TLRSDINVGTYRIY, SEQ ID NO: 41 ; or SDINVGTYR, SEQ ID NO: 42), LCDR2 (YKSDTDK, SEQ ID NO: 43; or YKSDTD, SEQ ID NO: 44) and LCDR3 (MVWHKSAPI, SEQ ID NO: 45); or
v.) the heavy chain variable region comprises HCDR1 (DYAMS, SEQ ID NO: 34;
GFTFSD, SEQ ID NO: 35; or GFTFSDYA; SEQ ID NO: 36), HCDR2 (TISGSGGDTFYADSVKG, SEQ ID NO: 37; or ISGSGGD, SEQ ID NO: 46) and HCDR3 (DKNRGAYADAFDI, SEQ ID NO: 47; or AKDKNRGAYADAFDI, SEQ ID NO: 48); and/or the light chain variable region comprises LCDR1 (TLRSDINVATYRIY, SEQ ID NO: 49; or SDINVATYR, SEQ ID NO: 50), LCDR2 (YKSDSDK, SEQ ID NO: 51 ; or YKSDSD, SEQ ID NO: 52) and LCDR3 (MVWHKSAPI, SEQ ID NO: 45).
8. An isolated delta-like ligand 4 (DLL4) antibody that binds to human DLL4 (SEQ ID NO: 63) and competes with the binding of an antibody comprising: i. ) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 53 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58;
ii. ) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 54 and a light chain variable region comprising the amino acid sequence of SEQ ID
NO: 59;
iii. ) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 55 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 60;
iv.) a heavy chain variable region comprising the amino acid sequence of
SEQ ID NO: 56 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 61 ; or
v.) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 57 and a light chain variable region comprising the amino acid sequence SEQ ID NO: 62.
9. The isolated DLL4 antibody of any one of claims 1-8, wherein the heavy chain variable region comprises HCDR1 (GYSWH, SEQ ID NO: 1 ), HCDR2 (YIHYSGYTHYNPSLKS, SEQ ID NO: 4) and HCDR3 (SDYSANEGAMAY, SEQ ID NO: 6); and/or wherein the light chain variable region comprises LCDR1 (RASESVDSFGNSFMH, SEQ ID NO: 8), LCDR2 (RTSNLQS, SEQ ID NO: 10) and LCDR3 (QQSNEDPWT, SEQ ID NO: 1 1 ).
10. The isolated DLL4 antibody of any one of claims 1-8, wherein the heavy chain variable region comprises HCDR1 (DHSITSG, SEQ ID NO: 2), HCDR2
(YIHYSGYTHYNPSLKS, SEQ ID NO: 4) and HCDR3 (SDYSANEGAMAY, SEQ ID NO: 6); and/or wherein the light chain variable region comprises LCDR1 (RASESVDSFGNSFMH, SEQ ID NO: 8), LCDR2 (RTSNLQS, SEQ ID NO: 10) and LCDR3 (QQSNEDPWT, SEQ ID NO: 1 1 ).
1 1 . The isolated DLL4 antibody of any one of claims 1-8, wherein the heavy chain variable region comprises HCDR1 (DHSITSGYS; SEQ ID NO: 3), HCDR2 (IHYSGY, SEQ ID NO: 5) and HCDR3 (AGSDYSANEGAMAY, SEQ ID NO: 7); and/or wherein the light chain variable region comprises LCDR1 (ESVDSFGNSF, SEQ ID NO: 9), LCDR2 (R) and LCDR3 (QQSNEDPWT, SEQ ID NO: 1 1 ).
12. The isolated DLL4 antibody of any one of claims 1-8, wherein the heavy chain variable region comprises HCDR1 (DEYWS, SEQ ID NO: 12), HCDR2 (EIHESGKTNYNPSLKP, SEQ ID NO: 15) and HCDR3 (LAYRDRWYGAFDV, SEQ ID NO: 17); and/or wherein the light chain variable region comprises LCDR1 (QASHDITNYIN, SEQ ID NO: 19), LCDR2 (HTSKLQT, SEQ ID NO: 21 ) and LCDR3 (QQFDNLLLT, SEQ ID NO: 22).
13. The isolated DLL4 antibody of any one of claims 1-8, wherein the heavy chain variable region comprises HCDR1 (GGSFND, SEQ ID NO: 13), HCDR2 (EIHESGKTNYNPSLKP, SEQ ID NO: 15) and HCDR3 (LAYRDRWYGAFDV, SEQ ID NO: 17); and/or wherein the light chain variable region comprises LCDR1 (QASHDITNYIN, SEQ ID NO: 19), LCDR2 (HTSKLQT, SEQ ID NO: 21 ) and LCDR3 (QQFDNLLLT, SEQ ID NO: 22).
14. The isolated DLL4 antibody of any one of claims 1-8, wherein the heavy chain variable region comprises HCDR1 (GGSFNDEY; SEQ ID NO: 14), HCDR2 (IHESGKT, SEQ ID NO: 16) and HCDR3 (ARLAYRDRWYGAFDV, SEQ ID NO: 18); and/or wherein the light chain variable region comprises LCDR1 (HDITNY, SEQ ID NO: 20), LCDR2 (H) and LCDR3 (QQFDNLLLT, SEQ ID NO: 22).
15. The isolated DLL4 antibody of any one of claims 1-8, wherein the heavy chain variable region comprises HCDR1 (GYYMH, SEQ ID NO: 23), HCDR2 (WINPNSGGTNYAQ, SEQ ID NO: 26) and HCDR3 (GAGVAAYDY, SEQ ID NO: 28); and/or wherein the light chain variable region comprises LCDR1 (RASQSISSYLN, SEQ ID NO: 30), LCDR2 (AASSLQS, SEQ ID NO: 32) and LCDR3 (QQSYSTPVT, SEQ ID NO: 33).
16. The isolated DLL4 antibody of any one of claims 1-8, wherein the heavy chain variable region comprises HCDR1 (GYTFTG, SEQ ID NO: 24), HCDR2 (WINPNSGGTNYAQ, SEQ ID NO: 26) and HCDR3 (GAGVAAYDY, SEQ ID NO: 28); and/or wherein the light chain variable region comprises LCDR1 (RASQSISSYLN, SEQ ID NO: 30), LCDR2 (AASSLQS, SEQ ID NO: 32) and LCDR3 (QQSYSTPVT, SEQ ID NO: 33).
17. The isolated DLL4 antibody of any one of claims 1-8, wherein the heavy chain variable region comprises HCDR1 (GYTFTGYY; SEQ ID NO: 25), HCDR2 (INPNSGG, SEQ ID NO: 27) and HCDR3 (ARGAGVAAYDY, SEQ ID NO: 29); and/or wherein the light chain variable region comprises LCDR1 (QSISSY, SEQ ID NO: 31 ), LCDR2 (A) and LCDR3 (QQSYSTPVT, SEQ ID NO: 33).
18. The isolated DLL4 antibody of any one of claims 1-8, wherein the heavy chain variable region comprises HCDR1 (DYAMS, SEQ ID NO: 34), HCDR2 (TISGSGGDTFYADSVKG, SEQ ID NO: 37) and HCDR3 (DKNRGAYADAFD, SEQ ID NO: 39); and/or wherein the light chain variable region comprises LCDR1 (TLRSDINVGTYRIY, SEQ ID NO: 41 ), LCDR2 (YKSDTDK, SEQ ID NO: 43) and LCDR3 (MVWHKSAPI, SEQ ID NO: 45).
19. The isolated DLL4 antibody of any one of claims 1-8, wherein the heavy chain variable region comprises HCDR1 (GFTFSD, SEQ ID NO: 35), HCDR2 (TISGSGGDTFYADSVKG, SEQ ID NO: 37) and HCDR3 (DKNRGAYADAFD, SEQ ID NO: 39); and/or wherein the light chain variable region comprises LCDR1 (TLRSDINVGTYRIY, SEQ ID NO: 41 ), LCDR2 (YKSDTDK, SEQ ID NO: 43) and LCDR3 (MVWHKSAPI, SEQ ID NO: 45).
20. The isolated DLL4 antibody of any one of claims 1-8, wherein the heavy chain variable region comprises HCDR1 (GFTFSDYA; SEQ ID NO: 36), HCDR2
(ISGSGGDT, SEQ ID NO: 38) and HCDR3 (AKDKNRGAYADAFD, SEQ ID NO: 40); and/or wherein the light chain variable region comprises LCDR1 (SDINVGTYR, SEQ ID NO: 42), LCDR2 (YKSDTD, SEQ ID NO: 44) and LCDR3 (MVWHKSAPI, SEQ ID NO: 45).
21 . The isolated DLL4 antibody of any one of claims 1-8, wherein the heavy chain variable region comprises HCDR1 (DYAMS, SEQ ID NO: 34), HCDR2 (TISGSGGDTFYADSVKG, SEQ ID NO: 37) and HCDR3 (DKNRGAYADAFDI, SEQ ID NO: 47); and/or wherein the light chain variable region comprises LCDR1 (TLRSDINVATYRIY, SEQ ID NO: 49), LCDR2 (YKSDSDK, SEQ ID NO: 51 ) and LCDR3 (MVWHKSAPI, SEQ ID NO: 45).
22. The isolated DLL4 antibody of any one of claims 1-8, wherein the heavy chain variable region comprises HCDR1 (GFTFSD, SEQ ID NO: 35), HCDR2 (TISGSGGDTFYADSVKG, SEQ ID NO: 37) and HCDR3 (DKNRGAYADAFDI, SEQ ID NO: 47); and/or wherein the light chain variable region comprises LCDR1 (TLRSDINVATYRIY, SEQ ID NO: 49), LCDR2 (YKSDSDK, SEQ ID NO: 51 ) and LCDR3 (MVWHKSAPI, SEQ ID NO: 45).
23. The isolated DLL4 antibody of any one of claims 1-8, wherein the heavy chain variable region comprises HCDR1 (GFTFSDYA; SEQ ID NO: 36), HCDR2 (ISGSGGD, SEQ ID NO: 46) and HCDR3 (AKDKNRGAYADAFDI, SEQ ID NO: 48); and/or wherein the light chain variable region comprises LCDR1 (SDINVATYR, SEQ ID NO: 50), LCDR2 (YKSDSD, SEQ ID NO: 52) and LCDR3 (MVWHKSAPI, SEQ ID NO: 45).
24. The isolated DLL4 antibody of any one of claims 1 -23, wherein the antibody is humanized.
25. The isolated DLL4 antibody of any one of claims 1 -23, wherein the antibody is a full length antibody.
26. The isolated DLL4 antibody of any one of claims 1 -23, wherein the antibody is a human IgG.
27. The isolated DLL4 antibody of any one of claims 1 -23, wherein the antibody is an antibody fragment.
28. The isolated DLL4 antibody of claim 27, wherein the antibody fragment is a Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2 or SCA (single chain antibody).
29. The isolated DLL4 antibody of any one of claims 1 -23, wherein the antibody is bound to a detectable label.
30. An immobilized antibody comprising an isolated DLL4 antibody of any one of claims 1-23 bound to a solid phase.
31 . A conjugate comprising an isolated DLL4 antibody of any one of claims 1 -23 bound to a cytotoxic or non-cytotoxic agent.
32. A method for determining the presence of DLL4 protein, the method comprising: exposing a sample suspected of containing the DLL4 protein to an isolated DLL4 antibody of any one of claims 1 -23 and determining binding of the antibody to the sample.
33. A kit comprising an isolated DLL4 antibody of any one of claims 1 -23 and instructions for using the DLL4 antibody to detect the DLL4 protein.
34. An isolated nucleic acid sequence encoding an isolated DLL4 antibody of any one of claims 1 -23.
35. A vector comprising the nucleic acid sequence of claim 34.
36. A host cell comprising the nucleic acid sequence of claim 34 or the vector of claim 35.
37. A process of producing a DLL4 antibody comprising culturing a host cell comprising the nucleic acid sequence of claim 34 so that the nucleic acid sequence is expressed.
38. The process of claim 37 further comprising recovering the DLL4 antibody from the host cell culture.
39. A composition comprising an isolated DLL4 antibody of any one of claims 1- 23 and one or more pharmaceutically acceptable carriers or diluents.
40. A method for treating a DLL4-associated disease or disorder in a subject, the method comprising administering to the subject a therapeutically effective amount of an isolated DLL4 antibody of any one of claims 1 -23.
41 . The method of claim 40, wherein the disease or disorder is a tumor, a cancer, a cell proliferative disorder, a pathological condition associated with angiogenesis or a non-neoplastic disorder.
42. The method of claim 40 further comprising administering another agent or another therapy before, after or simultaneously with the isolated DLL4 antibody.
43. The method of claim 42, wherein the other agent is a chemotherapeutic agent, a cytotoxic agent, a growth inhibitory agent, doxorubicin, an anti-angiogenesis agent, or combinations thereof.
44. The method of claim 42, wherein the other therapy is a cancer therapy selected from the group of surgery, radiological treatment, chemotherapy, treatment with anti-cancer agents and combinations thereof.
45. A method for detection of DLL4 comprising assaying a sample with an amount of an isolated DLL4 antibody of any one of claims 1-23 to detect DLL4 present in the sample.
46. The method of claim 45, wherein the sample is assayed by (a) obtaining the sample from a desired source; (b) admixing the sample with the isolated DLL4 antibody to allow the antibody to form antibody/DLL4 complex with DLL4 present in the mixture; and (c) detecting antibody/DLL4 complex present in the mixture.
47. The method of claim 45, wherein the sample is a biological sample.
48. A method for diagnosing a disorder associated with DLL4 expression and/or activity comprising, detecting DLL4, DLL4 antibody or DLL4/DLL4 antibody complex in a sample from a subject having or suspected of having the disorder.
49. A method of treating a Th17 cell-mediated disease or disorder in a human, the method comprising: administering a therapeutically effective amount of a DLL4 antibody or fragment thereof to the human.
50. A method of treating an autoimmune disease or disorder in a human, the method comprising: administering a therapeutically effective amount of a DLL4 antibody or fragment thereof to the human.
51 . A method of treating lupus in a human, the method comprising: administering a therapeutically effective amount of a DLL4 antibody or fragment thereof to the human.
52. A method for reducing activation and/or differentiation of human Th17 cells, the method comprising: contacting human T cells with a DLL4 antibody or fragment thereof.
53. A method for reducing cytokine production by Th17 cells, the method comprising: contacting the Th17 cells with a DLL4 antibody or fragment thereof.
54. A method for modulating cytokine production by human Th17 cells, the method comprising: activating the Th17 cells in the presence of a DLL4 antibody or fragment thereof.
55. The method of any one of claims 49-54, wherein additionally or alternatively a DLL4 antagonist is used instead of the DLL4 antibody or fragment thereof.
56. The method of any one of claims 49-54, wherein the antibody fragment is a
Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2 or SCA (single chain antibody).
57. The method of any one of claims 49-54, wherein the antibody is humanized or a human antibody.
58. The method of any one of claims 49-54, wherein the DLL4 antibody is the isolated DLL4 antibody of any one of claims 1 -23.
59. The method of claim 54, wherein production of the cytokine is reduced.
60. The method of claim 54, wherein production of the cytokine is increased.
61 . The method of claim 59, wherein the cytokine is IL-22, IL-17 or IFN-γ.
62. The method of claim 60, wherein the cytokine is TNF-a.
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