CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. provisional patent application serial numbers 61/361,312, filed July 2, 2010, and 61/497,289, filed June 15, 2011; the entire contents of each of which are incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The field of the invention is molecular biology, immunology and oncology. More particularly, the field is antibodies that bind human Notch 1.

BACKGROUND

[0003] Notch pathway signaling is involved in numerous cellular processes, including cell fate determination, differentiation, proliferation, apoptosis, migration and angiogenesis. In mammals, there are four Notch proteins (sometimes called "Notch receptors"), designated Notch 1 -No tch4. All four Notch proteins have a similar domain structure, which includes an extracellular domain, a negative regulatory (NRR) domain, a single-pass transmembrane domain, and an intracellular domain. The extracellular domain contains a series of EGF-like repeats that are involved in ligand binding. During maturation, the Notch polypeptide is cleaved by a furin-like protease. This cleavage divides the Notch protein into two subunits that are held together by the NRR. In the absence of ligand binding, the NRR domain functions to keep the Notch protein in a protease-resistant conformation. The intracellular domain is a transcription factor called Notch intracellular domain (NICD), which is released upon proteolytic cleavage by gamma secretase, in response to binding of the Notch protein by a ligand. In mammals, the Notch ligands are Delta-like and Jagged. When the NICD is released, it travels to the nucleus, where it activates transcription of the Notch-responsive genes, HES1, HES5, NRARP, Deltexl and c-MYC. For reviews of Notch-related biology, see, e.g., Bray, 2006, NATURE REVIEWS 7:678-689; Kopan et al., 2009, CELL 137:216-233.

[0004] While Notch proteins play crucial roles in normal development, dysregulation of the Notch proteins is associated with various types of cancer, including T-cell acute lymphatic leukemia/lymphoma (T-All), breast cancer, colon cancer, ovarian cancer and lung cancer. See, e.g., Miele et al, 2006, CURRENT CANCER DRUG TARGETS 6:313-323. Accordingly, one therapeutic approach for the treatment of cancer is inhibition of Notch pathway signaling. Inhibition of Notch pathway signaling has been achieved using monoclonal antibodies (Wu et al, 2010, NATURE 464: 1052-1057; Aste-Amezaga et al, 2010, PLOS ONE 5: 1-13 e9094).

[0005] Naturally-occurring antibodies are multimeric proteins that contain four polypeptide chains (FIG. 1). Two of the polypeptide chains are called immunoglobulin heavy chains (H chains), and two of the polypeptide chains are called immunoglobulin light chains (L chains). The immunoglobulin heavy and light chains are connected by an interchain disulfide bond. The immunoglobulin heavy chains are connected by interchain disulfide bonds. A light chain consists of one variable region (V _L in FIG. 1) and one constant region (C _L in FIG. 1). The heavy chain consists of one variable region (V _H in FIG. 1) and at least three constant regions (CHi, CH ₂ and CH ₃ in FIG. 1). The variable regions determine the specificity of the antibody.

[0006] Each variable region contains three hypervariable regions known as

complementarity determining regions (CDRs) flanked by four relatively conserved regions known as framework regions (FRs). The three CDRs, referred to as CDR _l5 CDR ₂ , and CDR ₃ , contribute to the antibody binding specificity. Naturally occurring antibodies have been used as starting material for engineered antibodies, such as chimeric antibodies and humanized antibodies.

[0007] There is a need for improved antibodies that neutralize the biological activity of human Notch 1 and that can be used as therapeutic agents to treat human patients.

SUMMARY OF THE INVENTION

[0008] The invention is based on the discovery of a family of antibodies that specifically bind human Notch 1. Antibodies disclosed herein contain Notch 1 binding sites based on the CDRs of the anti-Notch 1 antibodies described herein. The disclosed antibodies prevent or inhibit activation of human Notch 1. They do so by inhibiting Notch 1 from binding to Notch ligands, i.e., Jagl, Jag2, DLLl, and DLL4. The disclosed antibodies can be used to inhibit the proliferation of tumor cells in vitro and/or in vivo. When administered to a human cancer patient, the antibodies inhibit or reduce tumor growth in the human patient. [0009] These and other aspects and advantages of the invention are illustrated by the following figures, detailed description and claims. As used herein, "including" means without limitation, and examples cited are non-limiting.

DESCRIPTION OF THE DRAWINGS

[0010] The invention can be more completely understood with reference to the following drawings.

[0011] FIG. 1 (prior art) is a schematic representation of a typical naturally-occurring antibody.

[0012] FIG. 2 is a sequence alignment showing the amino acid sequence of the complete immunoglobulin heavy chain variable region of the antibodies designated 2G10, 2E6, 2A11 and 2D11. The amino acid sequences for each antibody are aligned against one another, and Complementary Determining Sequences (CDR) (Kabat definition), CDR _l5 CDR ₂ , and CDR ₃ are identified in boxes. The unboxed sequences represent framework (FR) sequences.

[0013] FIG. 3 is a sequence alignment showing the CDR _l5 CDR ₂ , and CDR ₃ sequences (Kabat definition) for each of the variable region sequences shown in FIG. 2.

[0014] FIG. 4 is a sequence alignment showing the amino acid sequence of the complete immunoglobulin light chain variable region of antibodies 2G10, 2E6, 2A11 and 2D11. The amino acid sequences for each antibody are aligned against one another, and CDR _l5 CDR ₂ , and CDR ₃ sequences (Kabat definition) are identified in boxes. The unboxed sequences represent framework (FR) sequences.

[0015] FIG. 5 is a sequence alignment showing the CDR _l5 CDR ₂ , and CDR ₃ sequences (Kabat definition) for each of the variable region sequences shown in FIG. 4.

[0016] FIG. 6 is a histogram summarizing results of an experiment to determine specificity of antibody binding to human Notch 1 on the surface of CHO-Flpln-Notchl cells. Antibodies 2G10, 2E6 (also referred to herein as antibody 2E06), 2A11, and 2D11 are shown from left to right.

[0017] FIG. 7A is a histogram summarizing results from a Notch 1- specific reporter assay showing that antibody 2E6 (also referred to herein as antibody 2E06) inhibits Notch 1- dependent reporter gene expression in the presence of Jagl. The Notch 1- specific (Nl-specific) control inhibitor is an anti-Notchl polyclonal antibody (AF1057, R&D Systems). The Notch2- specific (N2-specific) control inhibitor is an anti-Notch2 polyclonal antibody (AF1190, R&D Systems). The Notch3- specific (N3-specific) control inhibitor is an anti-Notch3 polyclonal antibody (AF1559, R&D Systems).

[0018] FIG. 7B is a histogram summarizing results from a No tch2- specific reporter assay showing that antibody 2E6 does not inhibit Notch2-dependent reporter gene expression in the presence of Jagl. The Notch 1 -specific (Nl -specific) control inhibitor is an anti-Notch 1 polyclonal antibody (AF1057, R&D Systems). The No tch2- specific (N2-specific) control inhibitor is an anti-Notch2 polyclonal antibody (AF1190, R&D Systems). The Notch3- specific (N3-specific) control inhibitor is an anti-Notch3 polyclonal antibody (AF1559, R&D Systems).

[0019] FIG. 7C is a histogram summarizing results from a Notch3- specific reporter assay showing that antibody 2E6 does not inhibit No tch3 -dependent reporter gene expression in the presence of Jagl. The Notch 1 -specific (Nl -specific) control inhibitor is an anti-Notch 1 polyclonal antibody (AF1057, R&D Systems). The No tch2- specific (N2-specific) control inhibitor is an anti-Notch2 polyclonal antibody (AF1190, R&D Systems). The Notch3- specific (N3-specific) control inhibitor is an anti-Notch3 polyclonal antibody (AF1559, R&D Systems).

[0020] FIG. 8A is a histogram summarizing results of Notch 1- specific reporter assays showing that antibody 2E6 inhibits Notch 1 -dependent reporter gene expression induced by the ligands Jagl, Jag2, DLL1 and DLL4. Reporter activity in the absence of any activating ligand (Fc + mlgG) was defined as 100% inhibition, and activity in the presence of ligand and treated with mouse IgG (Ligand alone + mlgG) was defined as 0% inhibition.

[0021] FIG. 8B is a histogram summarizing results of Notch 1 -specific reporter assays showing that antibody 2A11 inhibits Notch 1 -dependent reporter gene expression induced by the ligands Jagl, Jag2, DLL1 and DLL4. Reporter activity in the absence of any activating ligand (Fc + mlgG) was defined as 100% inhibition, and activity in the presence of ligand and treated with mouse IgG (Ligand alone + mlgG) was defined as 0% inhibition.

[0022] FIG. 8C is a histogram summarizing results of Notch 1- specific reporter assays showing that antibody 2D11 inhibits Notch 1 -dependent reporter gene expression induced by the ligands Jagl, Jag2, DLL1 and DLL4. Reporter activity in the absence of any activating ligand (Fc + mlgG) was defined as 100% inhibition, and activity in the presence of ligand and treated with mouse IgG (Ligand alone + mlgG) was defined as 0% inhibition. [0023] FIG. 9A is a histogram showing the effect of DBZ (dibenzazipine; a gamma secretase inhibitor dosed at 10 μΜοΙ/kg once daily) and antibody 2E6 (dosed at 40, 100, or 150 mg/kg (abbreviated "mpk") three times per week) on thymocyte population in mice.

[0024] FIG. 9B is a graph showing mouse body weight over time, for mice treated with DBZ or antibody 2E6. This shows that DBZ at 30 μπιοΐ/kg (A ) or 10 μπιοΐ/kg (■) induces weight loss in mice in 4 days and 17 days, respectively. By contrast, 40 mg/kg (abbreviated as mpk) (*), 100 mg/kg (·), or 150 mg/kg (+) of antibody 2E6 does not induce weight loss in mice (vehicle, (♦) and murine IgG (x)).

[0025] FIG. 10 is a histogram summarizing data from an experiment to assess the effects of antibody 2E6 on functional angiogenesis in vivo induced by bFGF, with hemoglobin content serving as a surrogate indicator of functional angiogenesis.

[0026] FIG. 11 is a histogram summarizing data from an experiment to assess the effects of antibody 2E6 on functional angiogenesis in vivo induced by human cancer cell lines (pancreatic cancer (SW1990) cells, breast cancer (MDA-MB-231 cells), and human lung cancer (Calu-6) cells), with hemoglobin content serving as a surrogate indicator of functional angiogenesis.

[0027] FIG. 12 is a schematic diagram showing the amino acid sequences of the complete immunoglobulin heavy chain variable region of 2E6 (SEQ ID NO: 12) and the complete humanized heavy chain variable regions denoted as Hu2E6_Hvl (SEQ ID NO: 103),

Hu2E6_Hvl T57A (SEQ ID NO: 105), Hu2E6_Hv2 (SEQ ID NO: 107), and Hu2E6_Hv2 T57A (SEQ ID NO: 109). The amino acid sequences for each heavy chain variable region are aligned against one another, and Complementary Determining Sequences (CDR) (Kabat definition), CDR _l5 CDR ₂ , and CDR _3> are identified in boxes. The unboxed sequences represent framework (FR) sequences.

[0028] FIG. 13 is a schematic diagram showing the CDR _l5 CDR ₂ , and CDR ₃ sequences (Kabat definition) for each of the variable region sequences shown in FIG. 12.

[0029] FIG. 14 is a schematic diagram showing the amino acid sequences of the complete light chain variable region of 2E6 (SEQ ID NO: 14) and the complete humanized light chain variable regions denoted as Hu2E6_Kvl (SEQ ID NO: 111) and Hu2E6_Kv2 (SEQ ID NO: 113). The amino acid sequences for each light chain variable region are aligned against one another, and CDR _{l 5} CDR ₂ , and CDR ₃ sequences (Kabat definition) are identified in boxes. The unboxed sequences represent framework (FR) sequences.

[0030] FIG. 15 is a sequence alignment showing the CDR _l5 CDR ₂ , and CDR ₃ sequences (Kabat definition) for each of the variable region sequences shown in FIG. 14. [0031] Fig. 16 is a graph summarizing results from a Notch- 1 specific reporter assay showing inhibition of DLL4-induced Notch- 1 dependent reporter gene expression by antibodies mu2E6 (■), Hu2E6-62 ( A ), A2-NRR1 ( T ), and a murine IgG control (·). Reporter activity in the absence of any activating ligand (Fc + mlgG) was defined as 100% inhibition, and activity in the presence of ligand and treated with mouse IgG (Ligand alone + mlgG) was defined as 0% inhibition.

[0032] Fig. 17 A is a histogram summarizing results from a Notch- 1 specific reporter assay showing that antibody Hu2E6-62 inhibits Notch- 1 dependent reporter gene expression in the presence of DLL4. Fig. 17B are histograms demonstrating inhibition of endogenous Notch 1- target genes by the Hu2E6-62 antibody. [0033] Fig. 18 is a histogram showing the effect of the Hu2E6-62 antibody dosed at 20 mg/kg (abbreviated "mpk") three times per week on thymocyte population in mice.

[0034] Fig. 19A is a graph showing mouse body weight over time, for mice treated with the antibodies Hu2E6-62 (■), A2-NRR1 ( A ) and a human IgG ( )control. Fig. 19B are photographs showing alcian blue staining of small intestine sections after treatment with antibodies Hu2E6-62, A2-NRR1 and a human IgG control.

[0035] Fig. 20 is a histogram summarizing data from an experiment to assess the effects of antibody Hu2E6-62 on functional angiogenesis in vivo induced by bFGF, with hemoglobin content serving as a surrogate indicator for functional angiogenesis.

[0036] Fig. 21 is a histogram summarizing data from an experiment to assess the effects of antibody Hu2E6-62 on functional angiogenesis in vivo induced by the human lung cancer cell line Calu-6, with hemoglobin content serving as a surrogate indicator of functional

angiogenesis.

DETAILED DESCRIPTION

[0037] The antibodies of the invention are based on the antigen binding sites of certain monoclonal antibodies that have been selected on the basis of binding and neutralizing the activity of human Notch 1. The antibodies contain immunoglobulin variable region CDR sequences that define a binding site for human Notch 1.

[0038] Because of the neutralizing activity of these antibodies, they are useful for inhibiting the growth and/or proliferation of certain cancer cells and tumors. The antibodies can be engineered to minimize or eliminate an immune response when administered to a human patient. Various features and aspects of the invention are discussed in more detail below.

[0039] As used herein, unless otherwise indicated, the term "antibody" means an intact antibody (e.g., an intact monoclonal antibody) or antigen-binding fragment of an antibody (e.g., an antigen-binding fragment of a monoclonal antibody), including an intact antibody or antigen-binding fragment that has been modified, engineered or chemically conjugated.

Examples of antibodies that have been modified or engineered are chimeric antibodies, humanized antibodies, and multispecific antibodies (e.g., bispecific antibodies). Examples of antigen-binding fragments include Fab, Fab', F(ab') ₂ , Fv, single chain antibodies (e.g., scFv), minibodies, and diabodies. An antibody conjugated to a toxin moiety is an example of a chemically conjugated antibody.

I. Antibodies that Bind Human Notchl

[0040] As disclosed herein, the antibodies may comprise: (a) an immunoglobulin heavy chain variable region comprising the structure CDRHI-CDRH ₂ -CDRH3 and (b) an

immunoglobulin light chain variable region comprising the structure CDR _L1 -CDR _L2 -CDR _L3 , wherein the heavy chain variable region and the light chain variable region together define a single binding site for binding human Notchl .

[0041] In some embodiments, the antibody comprises: (a) an immunoglobulin heavy chain variable region comprising the structure CDR _H i-CDR _H2 -CDR _H3 and (b) an immunoglobulin light chain variable region, wherein the heavy chain variable region and the light chain variable region together define a single binding site for binding human Notchl . A CDRHI comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5 (2G10), SEQ ID NO: 38 (2G10), SEQ ID NO: 15 (2E6), SEQ ID NO: 40 (2E6), SEQ ID NO: 25 (2A11), SEQ ID NO: 42 (2A11), SEQ ID NO: 32 (2D11), and SEQ ID NO: 44 (2D11); a CDR _H2 comprises an amino acid sequence selected from the group consisting SEQ ID NO: 6 (2G10), SEQ ID NO: 16 (2E6), SEQ ID NO: 26 (2A11), SEQ ID NO: 33 (2D11), SEQ ID NO: 94

(Hu2E6_Hvl T57A), SEQ ID NO: 95 (Hu2E6_Hv2), and SEQ ID NO: 96 (Hu2E6_Hv2 T57A); and a CDRH ₃ comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 7 (2G10), SEQ ID NO: 17 (2E6), SEQ ID NO: 27 (2A11), and SEQ ID NO: 34 (2D11). Throughout the specification a particular SEQ ID NO. is followed in parentheses by the antibody that was the origin of that sequence. For example, "SEQ ID NO: 5 (2G10)" means that SEQ ID NO: 5 comes from antibody 2G10.

[0042] In some embodiments, the antibody comprises an immunoglobulin heavy chain variable region comprising a CDR _H i comprising the amino acid sequence of SEQ ID NO: 5 (2G10) or SEQ ID NO: 38 (2G10), a CDR _H2 comprising the amino acid sequence of SEQ ID NO: 6 (2G10), and a CDR _H3 comprising the amino acid sequence of SEQ ID NO: 7 (2G10).

[0043] In some embodiments, the antibody comprises an immunoglobulin heavy chain variable region comprising a CDR _H i comprising the amino acid sequence of SEQ ID NO: 15 (2E6) or SEQ ID NO: 40 (2E6), a CDR _H2 comprising the amino acid sequence of SEQ ID NO: 16 (2E6), and a CDRH ₃ comprising the amino acid sequence of SEQ ID NO: 17 (2E6).

[0044] In some embodiments, the antibody comprises an immunoglobulin heavy chain variable region comprising a CDR _H i comprising the amino acid sequence of SEQ ID NO: 25 (2A11) or SEQ ID NO: 42 (2A11), a CDR _H2 comprising the amino acid sequence of SEQ ID NO: 26 (2A11), and a CDR _H3 comprising the amino acid sequence of SEQ ID NO: 27 (2A11).

[0045] In some embodiments, the antibody comprises an immunoglobulin heavy chain variable region comprising a CDR _H i comprising the amino acid sequence of SEQ ID NO: 32 (2D11) or SEQ ID NO: 44 (2D11), a CDR _H2 comprising the amino acid sequence of SEQ ID NO: 33 (2D11), and a CDR _H3 comprising the amino acid sequence of SEQ ID NO: 34 (2D11).

[0046] In some embodiments, the antibody comprises an immunoglobulin heavy chain variable region comprising a CDR _H i comprising the amino acid sequence of SEQ ID NO: 15 (2E6), a CDR _H2 comprising the amino acid sequence of SEQ ID NO: 94 (Hu2E6_Hvl T57A), SEQ ID NO: 95 (Hu2E6_Hv2), or SEQ ID NO: 96 (Hu2E6_Hv2 T57A), and a CDR _H3 comprising the amino acid sequence of SEQ ID NO: 17 (2E6).

[0047] In some embodiments, the antibody comprises an immunoglobulin heavy chain variable region comprising a CDR _H i comprising the amino acid sequence of SEQ ID NO: 15 (2E6), a CDR _H2 comprising the amino acid sequence of SEQ ID NO: 94 (Hu2E6_Hvl T57A), and a CDRH ₃ comprising the amino acid sequence of SEQ ID NO: 17 (2E6). [0048] Preferably, the CDR _H I, CDR _H2 , and CDR _H3 sequences are interposed between human or humanized immunoglobulin FRs. The antibody can be an intact antibody or an antigen-binding antibody fragment.

[0049] In other embodiments, the antibody comprises (a) an immunoglobulin light chain variable region comprising the structure CDR _L I-CDR _L2 -CDR _L3 , and (b) an immunoglobulin heavy chain variable region, wherein the IgG light chain variable region and the IgG heavy chain variable region together define a single binding site for binding human Notch 1. A CDR _L1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8 (2G10), SEQ ID NO: 18 (2E6, 2A11), SEQ ID NO: 35 (2D11), and SEQ ID NO: 99

(Hu2E6_Kvl, Hu2E6_Kv2); a CDR _L2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 9 (2G10), SEQ ID NO: 19 (2E6, 2A11), SEQ ID NO: 36 (2D11), SEQ ID NO: 100 (Hu2E6_Kvl), and SEQ ID NO: 101 (Hu2E6_Kv2); and a CDR _L3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 10

(2G10), SEQ ID NO: 20 (2E6, 2A11), and SEQ ID NO: 37 (2D11).

[0050] In one embodiment, the antibody comprises an immunoglobulin light chain variable region comprising a CDR _L1 comprising the amino acid sequence of SEQ ID NO: 8 (2G10); a CDR _L2 comprising the amino acid sequence of SEQ ID NO: 9 (2G10); and a CDR _L3 comprising the amino acid sequence of SEQ ID NO: 10 (2G10).

[0051] In one embodiment, the antibody comprises an immunoglobulin light chain variable region comprising a CDR _L1 comprising the amino acid sequence of SEQ ID NO: 18 (2E6, 2A11); a CDR _L2 comprising the amino acid sequence of SEQ ID NO: 19 (2E6, 2A11); and a CDR _L3 comprising the amino acid sequence of SEQ ID NO: 20 (2E6, 2A11).

[0052] In one embodiment, the antibody comprises an immunoglobulin light chain variable region comprising a CDR _L1 comprising the amino acid sequence of SEQ ID NO: 35 (2D11); a CDR _L2 comprising the amino acid sequence of SEQ ID NO: 36 (2D11); and a CDR _L3 comprising the amino acid sequence of SEQ ID NO: 37 (2D11).

[0053] In one embodiment, the antibody comprises an immunoglobulin light chain variable region comprising a CDR _L1 comprising the amino acid sequence of SEQ ID NO: 99

(Hu2E6_Kvl, Hu2E6_Kv2), a CDR _L2 comprising the amino acid sequence of SEQ ID NO: 100 (Hu2E6_Kvl) or SEQ ID NO: 101 (Hu2E6_Kv2), and a CDR _L3 comprising the amino acid sequence of SEQ ID NO: 20 (2E6). [0054] In one embodiment, the antibody comprises an immunoglobulin light chain variable region comprising a CDR _L1 comprising the amino acid sequence of SEQ ID NO: 99

(Hu2E6_Kvl, Hu2E6_Kv2), a CDR _L2 comprising the amino acid sequence of SEQ ID NO:

100 (Hu2E6_Kvl), and a CDR _L3 comprising the amino acid sequence of SEQ ID NO: 20

(2E6).

[0055] In one embodiment, the antibody comprises an immunoglobulin light chain variable region comprising a CDR _L1 comprising the amino acid sequence of SEQ ID NO: 99

(Hu2E6_Kvl, Hu2E6_Kv2), a CDR _L2 comprising the amino acid sequence of SEQ ID NO:

101 (Hu2E6_Kv2), and a CDR _L3 comprising the amino acid sequence of SEQ ID NO: 20

(2E6).

[0056] Preferably, the CDR _L1 , CDR _L2 , and CDR _L3 sequences are interposed between human or humanized immunoglobulin FRs. The antibody can be an intact antibody or an antigen-binding antibody fragment.

[0057] In some embodiments, the antibody comprises: (a) an immunoglobulin heavy chain variable region comprising the structure CDR _H i-CDR _H2 -CDR _H3 and (b) an immunoglobulin light chain variable region comprising the structure CDR _L1 -CDR _L2 -CDR _L3 , wherein the heavy chain variable region and the light chain variable region together define a single binding site for binding human Notch 1. The CDR _H I is an amino acid sequence selected from the group consisting of SEQ ID NO: 5 (2G10), SEQ ID NO: 38 (2G10), SEQ ID NO: 15 (2E6), SEQ ID NO: 40 (2E6), SEQ ID NO: 25 (2A11), SEQ ID NO: 42 (2A11), SEQ ID NO: 32 (2D11), and SEQ ID NO: 44 (2D11); the CDR _H2 is an amino acid sequence selected from the group consisting SEQ ID NO: 6 (2G10), SEQ ID NO: 16 (2E6), SEQ ID NO: 26 (2A11), SEQ ID NO: 33 (2D11), SEQ ID NO: 94 (Hu2E6_Hvl T57A), SEQ ID NO: 95 (Hu2E6_Hv2), and SEQ ID NO: 96 (Hu2E6_Hv2 T57A); and the CDR _H3 is an amino acid sequence selected from the group consisting of SEQ ID NO: 7 (2G10), SEQ ID NO: 17 (2E6), SEQ ID NO: 27 (2A11), and SEQ ID NO: 34 (2D11). The CDR _L I is an amino acid sequence selected from the group consisting of SEQ ID NO: 8 (2G10), SEQ ID NO: 18 (2E6, 2A11), SEQ ID NO: 35 (2D11), and SEQ ID NO: 99 (Hu2E6_Kvl, Hu2E6_Kv2); the CDR _L2 is an amino acid sequence selected from the group consisting of SEQ ID NO:9 (2G10), SEQ ID NO: 19 (2E6, 2A11), SEQ ID NO: 36 (2D11), SEQ ID NO: 100 (Hu2E6_Kvl), and SEQ ID NO: 101 (Hu2E6_Kv2); and the CDR _L3 is an amino acid sequence selected from the group consisting of SEQ ID NO: 10 (2G10), SEQ ID NO: 20 (2E6, 2A11), and SEQ ID NO: 37 (2D11).

[0058] In some embodiments, the antibody comprises an immunoglobulin heavy chain variable region selected from the group consisting of SEQ ID NO: 2 (2G10), SEQ ID NO: 12 (2E6), SEQ ID NO: 22 (2A11), SEQ ID NO: 29 (2D11), SEQ ID NO: 103 (Hu2E6_Hvl), SEQ ID NO: 105 (Hu2E6_Hvl T57A), SEQ ID NO: 107 (Hu2E6_Hv2), and SEQ ID NO: 109 (Hu2E6_Hv2 T57A), and an immunoglobulin light chain variable region selected from the group consisting of SEQ ID NO: 4 (2G10), SEQ ID NO: 14 (2E6), SEQ ID NO: 24 (2A11), SEQ ID NO: 31 (2D11), SEQ ID NO: 111 (Hu2E6_Kvl), and SEQ ID NO: 113

(Hu2E6_Kv2).

[0059] In another embodiment, the antibody comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 (2G10), and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 4 (2G10).

[0060] In another embodiment, the antibody comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 12 (2E6), and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 14 (2E6).

[0061] In another embodiment, the antibody comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 22 (2A11), and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 24 (2A11).

[0062] In another embodiment, the antibody comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 29 (2D11), and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 31 (2D11).

[0063] In another embodiment, the antibody comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 105 (Hu2E6_Hvl T57A), and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 111 (Hu2E6_Kvl). [0064] In another embodiment, the antibody comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 105 (Hu2E6_Hvl T57A), and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 113 (Hu2E6_Kv2).

[0065] In other embodiments, the antibody comprises (i) an immunoglobulin heavy chain selected from the group consisting of SEQ ID NO: 69 (2G10), SEQ ID NO: 73 (2E6), SEQ ID NO: 77 (2A11), SEQ ID NO: 81 (2D11), SEQ ID NO: 120 (2E6 Chimeric Heavy IgGl), SEQ ID NO: 124 (Hu2E6_Hvl IgGl), SEQ ID NO: 126 (Hu2E6_Hvl T57A IgGl), SEQ ID NO: 128 (Hu2E6_Hv2 IgGl), and SEQ ID NO: 130 (Hu2E6_Hv2 T57A IgGl), and (ii) an immunoglobulin light chain selected from the group consisting of SEQ ID NO: 71 (2G10), SEQ ID NO: 75 (2E6), SEQ ID NO: 79 (2A11), SEQ ID NO: 83 (2D11), SEQ ID NO: 122 (2E6 Chimeric Kappa), SEQ ID NO: 132 (Hu2E6_Kvl Kappa), and SEQ ID NO: 134

(Hu2E6_Kv2 Kappa).

[0066] In another embodiment, the antibody comprises an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 69 (2G10), and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 71 (2G10).

[0067] In some embodiments, the antibody comprises an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 73 (2E6), and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 75 (2E6).

[0068] In some embodiments, the antibody comprises an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 77 (2A11), and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 79 (2A11).

[0069] In some embodiments, the antibody comprises an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 81 (2D11), and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 83 (2D11).

[0070] In some embodiments, the antibody comprises an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 126 (Hu2E6_Hvl T57A IgGl), and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 132

(Hu2E6_Kvl Kappa). [0071] In some embodiments, the antibody comprises an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 126 (Hu2E6_Hvl T57A IgGl), and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 134

(Hu2E6_Kv2 Kappa).

[0072] In certain embodiments, an isolated antibody comprises an immunoglobulin heavy chain variable region comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the entire variable region or the framework region sequence of SEQ ID NO: 2 (2G10), SEQ ID NO: 12 (2E6), SEQ ID NO: 22 (2A11), SEQ ID NO: 29 (2D11), SEQ ID NO: 103 (Hu2E6_Hvl), SEQ ID NO: 105 (Hu2E6_Hvl T57A), SEQ ID NO: 107 (Hu2E6_Hv2), or SEQ ID NO: 109 (Hu2E6_Hv2 T57A).

[0073] In certain embodiments, an isolated antibody comprises an immunoglobulin light chain variable region comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the entire variable region or the framework region sequence of SEQ ID NO: 4 (2G10), SEQ ID NO: 14 (2E6), SEQ ID NO: 24 (2A11), SEQ ID NO: 31 (2D11), SEQ ID NO: 1 1 1 (Hu2E6_Kvl), or SEQ ID NO: 1 13 (Hu2E6_Kv2).

[0074] Homology or identity may be determined 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. BLAST (Basic Local Alignment Search Tool) analysis using the algorithm employed by the programs blastp, blastn, blastx, tblastn and tblastx (Karlin et al, ( 1990) PROC. NATL. ACAD. SCI. USA 87, 2264-2268; Altschul, (1993) J. MOL.

EVOL. 36, 290-300; Altschul et al, (1997) NUCLEIC ACIDS RES. 25, 3389-3402, incorporated by reference) are tailored for sequence similarity searching. The approach used by the BLAST program is to first consider similar segments between a query sequence and a database sequence, then to evaluate the statistical significance of all matches that are identified and finally to summarize only those matches which satisfy a preselected threshold of significance. For a discussion of basic issues in similarity searching of sequence databases see Altschul et al, ( 1994) NATURE GENETICS 6, 1 19- 129 which is fully incorporated by reference. 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. The search parameters for histogram, descriptions, alignments, expect (i.e., the statistical significance threshold for reporting matches against database sequences), cutoff, matrix and filter are at the default settings. The default scoring matrix used by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff et al, (1992) PROC. NATL. ACAD. SCI. USA 89, 10915-10919, fully incorporated by reference). Four blastn parameters may be adjusted as follows: Q=10 (gap creation penalty); R=10 (gap extension penalty); wink=l (generates word hits at every wink.sup.th position along the query); and gapw=16 (sets the window width within which gapped alignments are generated). The equivalent Blastp parameter settings may be Q=9; R=2; wink=l; and gapw=32. Searches may also be conducted using the NCBI (National Center for Biotechnology Information) BLAST Advanced Option parameter (e.g.: -G, Cost to open gap [Integer]: default = 5 for nucleotides/ 11 for proteins; -E, Cost to extend gap [Integer]: default = 2 for nucleotides/ 1 for proteins; -q, Penalty for nucleotide mismatch [Integer]: default = -3; -r, reward for nucleotide match [Integer]: default = 1; -e, expect value [Real]: default = 10; -W, wordsize [Integer]: default = 11 for nucleotides/ 28 for megablast/ 3 for proteins; -y, Dropoff (X) for blast extensions in bits: default = 20 for blastn/ 7 for others; -X, X dropoff value for gapped alignment (in bits): default = 15 for all programs, not applicable to blastn; and -Z, final X dropoff value for gapped alignment (in bits): 50 for blastn, 25 for others). ClustalW for pairwise protein alignments may also be used (default parameters may include, e.g., Blosum62 matrix and Gap Opening Penalty = 10 and Gap Extension Penalty = 0.1). A Bestfit comparison between sequences, available in the GCG package version 10.0, uses DNA parameters GAP=50 (gap creation penalty) and LEN=3 (gap extension penalty) and the equivalent settings in protein comparisons are GAP=8 and LEN=2.

[0075] In each of the foregoing embodiments, it is contemplated herein that

immunoglobulin heavy chain variable region sequences and/or light chain variable region sequences that together bind human Notchl may contain amino acid alterations (e.g., at least 1, 2, 3, 4, 5, or 10 amino acid substitutions, deletions, or additions) in the framework regions of the heavy and/or light chain variable regions.

[0076] In certain embodiments, an isolated antibody binds human Notchl with a K _D of 100 nM, 50 nM, 20 nM, 15 nM, 12 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM or lower. Unless otherwise specified, K _D values are determined by surface plasmon resonance methods under the conditions described in Examples 3 and 14.

[0077] Antibody Hu2E6-62 binds human Notchl with a K _D of 10 nM, 9 nM, 8 nM, 7 nM, 5 nM, 4 nM, 2 nM, 1 nM or lower as measured by surface plasmon resonance methods under the conditions described in Examples 3 and 14. In an exemplary embodiment, antibody Hu2E6-62 binds human Notch 1 with a K _D of 8 nM or lower as measured by surface plasmon resonance methods at 37°C under the conditions described in Examples 3 and 14.

[0078] Antibody Hu2E6-74 binds human Notchl with a K _D of 10 nM, 9 nM, 8 nM, 7 nM, 5 nM, 4 nM, 2 nM, 1 nM or lower as measured by surface plasmon resonance methods under the conditions described in Examples 3 and 14. In an exemplary embodiment, antibody Hu2E6-74 binds human Notchl with a K _D of 8 nM or lower as measured by surface plasmon resonance methods at 37°C under the conditions described in Examples 3 and 14.

II. Antibody Production

[0079] Methods for producing antibodies of the invention are known in the art. For example, DNA molecules encoding light chain variable regions and/or heavy chain variable regions can be chemically synthesized using the sequence information provided herein.

Synthetic DNA molecules can be ligated to other appropriate nucleotide sequences, including, e.g., constant region coding sequences, and expression control sequences, to produce conventional gene expression constructs encoding the desired antibody. Production of defined gene constructs is within routine skill in the art. Alternatively, the sequences provided herein can be cloned out of hybridomas by conventional hybridization techniques or polymerase chain reaction (PCR) techniques, using synthetic nucleic acid probes whose sequences are based on sequence information provided herein, or prior art sequence information regarding genes encoding the heavy and light chains of murine antibodies in hybridoma cells.

[0080] Nucleic acids encoding desired antibodies can be incorporated (ligated) into expression vectors, which can be introduced into host cells through conventional transfection or transformation techniques. Exemplary host cells are E. coli cells, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and myeloma cells that do not otherwise produce IgG protein. Transformed host cells can be grown under conditions that permit the host cells to express the genes that encode the immunoglobulin light and/or heavy chain variable regions.

[0081] Specific expression and purification conditions will vary depending upon the expression system employed. For example, if a gene is to be expressed in E. coli, it is first cloned into an expression vector by positioning the engineered gene downstream from a suitable bacterial promoter, e.g. , Trp or Tac, and a prokaryotic signal sequence. The expressed secreted protein accumulates in refractile or inclusion bodies, and can be harvested after disruption of the cells by French press or sonication. The refractile bodies then are solubilized, and the proteins refolded and cleaved by methods known in the art. [0082] If the engineered gene is to be expressed in eukayotic host cells, e.g., CHO cells, it is first inserted into an expression vector containing a suitable eukaryotic promoter, a secretion signal, IgG enhancers, and various introns. This expression vector optionally contains sequences encoding all or part of a constant region, enabling an entire, or a part of, a heavy or light chain to be expressed. The gene construct can be introduced into eukaryotic host cells using conventional techniques. The host cells express VL or VH fragments, VL-VH

heterodimers, VH-VL or VL-VH single chain polypeptides, complete heavy or light

immunoglobulin chains, or portions thereof, each of which may be attached to a moiety having another function (e.g. , cytotoxicity). In some embodiments, a host cell is transfected with a single vector expressing a polypeptide expressing an entire, or part of, a heavy chain (e.g., a heavy chain variable region) or a light chain (e.g., a light chain variable region). In other embodiments, a host cell is transfected with a single vector encoding (a) a polypeptide comprising a heavy chain variable region and a polypeptide comprising a light chain variable region, or (b) an entire immunoglobulin heavy chain and an entire immunoglobulin light chain. In still other embodiments, a host cell is co-transfected with more than one expression vector (e.g., one expression vector encoding a polypeptide comprising an entire, or part of, a heavy chain or heavy chain variable region, and another expression vector encoding a polypeptide comprising an entire, or part of, a light chain or light chain variable region).

[0083] A polypeptide comprising an immunoglobulin heavy chain variable region or light chain variable region can be produced by growing a host cell transfected with an expression vector encoding such variable region, under conditions that permit expression of the polypeptide. Following expression, the polypeptide can be harvested and purified using techniques well known in the art, e.g., affinity tags such as glutathione-S-transferase (GST) and histidine tags.

[0084] A monoclonal antibody that binds human Notch 1, or an antigen-binding fragment of the antibody, can be produced by growing a host cell transfected with: (a) an expression vector that encodes a complete or partial immunoglobulin heavy chain, and a separate expression vector that encodes a complete or partial immunoglobulin light chain; or (b) a single expression vector that encodes both chains (e.g., complete or partial heavy and light chains), under conditions that permit expression of both chains. The intact antibody (or antigen-binding fragment of the antibody) can be harvested and purified using techniques well known in the art, e.g., Protein A, Protein G, affinity tags such as glutathione-S-transferase (GST) and histidine tags. It is within ordinary skill in the art to express the heavy chain and the light chain from a single expression vector or from two separate expression vectors.

III. Antibody Modifications

[0085] Methods for reducing or eliminating the antigenicity of antibodies and antibody fragments are known in the art. When the antibodies are to be administered to a human, the antibodies preferably are "humanized" to reduce or eliminate antigenicity in humans.

Preferably, the humanized antibodies have the same, or substantially the same, affinity for the antigen as the non-humanized mouse antibody from which it was derived.

[0086] In one humanization approach, chimeric proteins are created in which mouse immunoglobulin constant regions are replaced with human immunoglobulin constant regions. See, e.g., Morrison et al, 1984, PROC. NAT. ACAD. SCI. 81 :6851-6855, Neuberger et al, 1984, NATURE 312:604-608; U.S. Patent Nos. 6,893,625 (Robinson); 5,500,362 (Robinson); and 4,816,567 (Cabilly).

[0087] In an approach known as CDR grafting, the CDRs of the light and heavy chain variable regions are grafted into frameworks from another species. For example, murine CDRs can be grafted into human FRs. In some embodiments of the invention, the CDRs of the light and heavy chain variable regions of an anti-Notch 1 antibody are grafted into human FRs or consensus human FRs. To create consensus human FRs, FRs from several human heavy chain or light chain amino acid sequences are aligned to identify a consensus amino acid sequence. CDR grafting is described in U.S. Patent Nos. 7,022,500 (Queen); 6,982,321 (Winter);

6, 180,370 (Queen); 6,054,297 (Carter); 5,693,762 (Queen); 5,859,205 (Adair); 5,693,761 (Queen); 5,565,332 (Hoogenboom); 5,585,089 (Queen); 5,530, 101 (Queen); Jones et al. ( 1986) NATURE 321 : 522-525; Riechmann et al. ( 1988) NATURE 332: 323-327; Verhoeyen et al.

(1988) SCIENCE 239: 1534- 1536; and Winter ( 1998) FEBS LETT 430: 92-94.

[0088] In an approach called "SUPERHUMANIZATION™," human CDR sequences are chosen from human germline genes, based on the structural similarity of the human CDRs to those of the mouse antibody to be humanized. See, e.g., U.S. Patent No. 6,881,557 (Foote); and Tan et al., 2002, J. IMMUNOL 169: 1119-1125.

[0089] Other methods to reduce immunogenicity include "reshaping,"

"hyperchimerization," and "veneering/resurfacing." See, e.g., Vaswami et ah, 1998, ANNALS OF ALLERGY, ASTHMA, & IMMUNOL. 81 : 105; Roguska et al. , 1996, PROT. ENGINEER 9:895-904; and U.S. Patent No. 6,072,035 (Hardman). In the veneering/resurfacing approach, the surface accessible amino acid residues in the murine antibody are replaced by amino acid residues more frequently found at the same positions in a human antibody. This type of antibody resurfacing is described, e.g., in U.S. Patent No. 5,639,641 (Pedersen).

[0090] Another approach for converting a mouse antibody into a form suitable for medical use in humans is known as ACTIVMAB™ technology (Vaccinex, Inc., Rochester, NY), which involves a vaccinia virus-based vector to express antibodies in mammalian cells. High levels of combinatorial diversity of IgG heavy and light chains are said to be produced. See, e.g., U.S. Patent Nos. 6,706,477 (Zauderer); 6,800,442 (Zauderer); and 6,872,518 (Zauderer).

[0091] Another approach for converting a mouse antibody into a form suitable for use in humans is technology practiced commercially by KaloBios Pharmaceuticals, Inc. (Palo Alto, CA). This technology involves the use of a proprietary human "acceptor" library to produce an "epitope focused" library for antibody selection.

[0092] Another approach for modifying a mouse antibody into a form suitable for medical use in humans is HUMAN ENGINEERING™ technology, which is practiced commercially by XOMA (US) LLC. See, e.g., PCT Publication No. WO 93/11794 and U.S. Patent Nos.

5,766,886 (Studnicka); 5,770,196 (Studnicka); 5,821,123 (Studnicka); and 5,869,619

(Studnicka).

[0093] Any suitable approach, including any of the above approaches, can be used to reduce or eliminate human immunogenicity of an antibody of the invention.

[0094] The antibody can be conjugated to an effector moiety such as a small molecule toxin or a radionuclide using standard in vitro conjugation chemistries. If the effector moiety is a polypeptide, the antibody can be chemically conjugated to the effector or joined to the effector as a fusion protein. Construction of fusion proteins is within ordinary skill in the art. IV. Use of Antibodies

[0095] Antibodies disclosed herein can be used to treat various forms of cancer, e.g., breast, ovarian, prostate, cervical, colorectal, lung, pancreatic, gastric, and head and neck cancers. The cancer cells are exposed to a therapeutically effective amount of the antibody so as to inhibit or reduce proliferation of the cancer cells. In some embodiments, the antibodies inhibit cancer cell proliferation by at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100%.

[0096] In some embodiments, the disclosed antibodies (e.g., 2E6, 2G10, 2A11, 2D11, Hu2E6) may inhibit or reduce proliferation of a tumor cell by inhibiting binding of human Notchl to a ligand, e.g., Jagl, Jag2, DLL1, and DLL4. The antibodies (e.g., 2E6, 2G10, 2A11, 2D11, Hu2E6) can also be used in therapy. The disclosed antibodies (e.g., 2E6, 2G10, 2A11, 2D11, Hu2E6) can be used in a method to inhibit tumor growth in a mammal (e.g., a human patient). The method comprises administering to the mammal a therapeutically effective amount of the antibody.

[0097] In certain embodiments, antibody Hu2E6-62 is used in therapy. For example, antibody Hu2E6-62 can be used for inhibiting or reducing proliferation of a tumor cell.

Antibody Hu2E6-62 can also be used for inhibiting or reducing tumor growth in a mammal.

[0098] In other embodiments, antibody Hu2E6-74 is used in therapy. For example, antibody Hu2E6-74 can be used for inhibiting or reducing proliferation of a tumor cell.

Antibody Hu2E6-74 can also be used for inhibiting or reducing tumor growth in a mammal.

[0099] Cancers associated with Notchl overexpression and/or activation include breast cancer, ovarian cancer, prostate cancer, cervical cancer, lung cancer, brain cancers (e.g., glioblastoma, astrocytoma, neuroblastoma), melanomas, gastrointestinal cancers (e.g., colorectal, pancreatic, and gastric), head and neck cancer, and hematopoietic cell cancers, (e.g., multiple myeloma, leukemia, e.g., precursor T acute lymphoblastic leukemia (T-ALL), precursor B acute lymphoblastic leukemia (B-ALL) and B-cell chronic lymphoblastic leukemia (B-CLL)).

[00100] As used herein, "treat", "treating" and "treatment" mean the treatment of a disease in a mammal, e.g., in a human. This includes: (a) inhibiting the disease, i.e., arresting its development; and (b) relieving the disease, i.e., causing regression of the disease state; and (c) curing the disease. [00101] Generally, a therapeutically effective amount of active component is in the range of 0.1 mg/kg to 100 mg/kg, e.g., 1 mg/kg to 100 mg/kg, 1 mg/kg to 10 mg/kg. The amount administered will depend on variables such as the type and extent of disease or indication to be treated, the overall health of the patient, the in vivo potency of the antibody, the pharmaceutical formulation, and the route of administration. The initial dosage can be increased beyond the upper level in order to rapidly achieve the desired blood-level or tissue-level. Alternatively, the initial dosage can be smaller than the optimum, and the daily dosage may be progressively increased during the course of treatment. Human dosage can be optimized, e.g., in a conventional Phase I dose escalation study designed to run from 0.5 mg/kg to 20 mg/kg.

Dosing frequency can vary, depending on factors such as route of administration, dosage amount and the disease being treated. Exemplary dosing frequencies are once per day, once per week and once every two weeks. A preferred route of administration is parenteral, e.g., intravenous infusion. Formulation of monoclonal antibody-based drugs is within ordinary skill in the art. In some embodiments, a monoclonal antibody is lyophilized and reconstituted in buffered saline at the time of administration.

[00102] For therapeutic use, an antibody preferably is combined with a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" means buffers, carriers, and excipients suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. The carrier(s) should be "acceptable" in the sense of being compatible with the other ingredients of the formulations and not deleterious to the recipient. Pharmaceutically acceptable carriers include buffers, solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art.

[00103] Pharmaceutical compositions containing antibodies disclosed herein can be presented in a dosage unit form and can be prepared by any suitable method. A pharmaceutical composition should be formulated to be compatible with its intended route of administration. Examples of routes of administration are intravenous (IV), intradermal, inhalation, transdermal, topical, transmucosal, and rectal administration. A preferred route of administration for monoclonal antibodies is IV infusion. Useful formulations can be prepared by methods well known in the pharmaceutical art. For example, see Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990). Formulation components suitable for parenteral administration include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose.

[0100] For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). The carrier should be stable under the conditions of manufacture and storage, and should be preserved against microorganisms. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol), and suitable mixtures thereof.

[0101] Pharmaceutical formulations preferably are sterile. Sterilization can be

accomplished by any suitable method, e.g., filtration through sterile filtration membranes. Where the composition is lyophilized, filter sterilization can be conducted prior to or following lyophilization and reconstitution.

EXAMPLES

[0102] The following Examples are merely illustrative and are not intended to limit the scope or content of the invention in any way.

Example 1: Production of Anti-hNotchl Monoclonal Antibodies

[0103] Immunizations, fusions, and primary screens were conducted at Maine

Biotechnology Services Inc. following the Repetitive Immunization Multiple Sites (RIMMS) protocol. Five A J mice and five Balb/c mice were immunized with a concatemeric protein containing 4 repeats of amino acids 413-488 of human Notchl linked by two non-Notchl amino acids, i.e., alanine and glycine (AG linker) or glutamine and phenylalanine (QF linker). Subsequent boosts alternated between concatemers containing the AG linker and the QF linker. Two AJ mice and 2 Balb/c mice having sera displaying high binding to immunogen by Enzyme Linked Immunosorbent Assay (ELISA) were chosen for subsequent fusion. Spleens and lymph nodes from the selected mice were harvested. B cells were harvested and fused with a myeloma line. Fusion products from AJ mice and Balb/c mice were serially diluted in forty 96- well plates to near clonality. A total of 10,560 supernatants from the cell fusions were screened for binding to human Notchl on the surface of CHO cells, using a Mesoscale

electrochemiluminescence assay (MSD). Three hundred supernatants that bound human Notchl in this assay were identified from each of the AJ and Balb/c fusions. These 600 fusion products were further characterized by in vitro biochemical and cell-based assays, as discussed below. A panel of hybridomas was selected, the hybridomas were subcloned, and monoclonal hybridomas were expanded. Hybridoma cell lines were transferred to BioXCell (West Lebanon, NH) for antibody expression and purification by affinity chromatography on Protein G resin under standard conditions. Example 2: Antibody Sequence Analysis

[0104] The light chain isotype and heavy chain isotype of each monoclonal antibody in Example 1 was determined using the IsoStrip™ Mouse Monoclonal Antibody Isotyping Kit according to the kit vendor's instructions (Roche Applied Science, Indianapolis, IN). All antibodies were found to be kappa light chain and IgGl or IgG2b heavy chain.

[0105] The heavy and light chain variable regions of the mouse monoclonal antibodies were sequenced using 5' RACE (Rapid Amplification of cDNA Ends). Total RNA was extracted from each monoclonal hybridoma cell line using the RNeasy ^® Miniprep kit according to the vendor's instructions (Qiagen, Valencia, CA). Full-length first strand cDNA containing 5' ends was generated using either the GeneRacer™ Kit (Invitrogen, Carlsbad, California) or SMARTer™ RACE cDNA Amplification Kit (Clontech, Mountain View, CA) according to the kit vendor's instructions, using random primers for 5' RACE.

[0106] The variable regions of the kappa and heavy (IgGlor IgG2b) chains were amplified by PCR, using KOD Hot Start Polymerase (EMD Chemicals, Gibbstown, NJ), Expand High Fidelity PCR System (Roche Applied Science), or Advantage 2 Polymerase Mix (Clontech, Mountain View, CA) according to the vendor's instructions. For amplification of 5' cDNA ends in conjunction with the GeneRacer Kit, the GeneRacer 5' Primer, 5'

cgactggagcacgaggacactga 3' (SEQ ID NO: 84) (Invitrogen) was used as a 5' primer. For amplification of 5' cDNA ends in conjunction with the SMARTer™ RACE cDNA

Amplification Kit, the Universal Primer Mix A primer (Clontech), a mix of

5 ' CTAATACGACTC ACTATAGGGCAAGC AGTGGTATCAACGCAGAGT 3' (SEQ ID NO: 85) and 5' CTAATACGACTCACTATAGGGC 3' (SEQ ID NO: 86), was used as a 5' primer. Heavy chain variable regions were amplified using the above 5' primers and a 3' IgGl constant region specific primer, either 5' TATGCAAGGCTTACAACCACA 3' (SEQ ID NO: 87) or 5' GCCAGTGGATAGACAGATGGGGGTGTCG 3' (SEQ ID NO: 88). IgG2b sequences were amplified with 5' GGCCAGTGGATAGACTGATGGGGGTGTTGT 3' (SEQ ID NO: 89). Kappa chain variable regions were amplified with the above 5' primers and a 3' kappa constant region specific primer, either 5' CTCATTCCTGTTGAAGCTCTTGACAAT 3' (SEQ ID NO: 90) or 5' CGACTGAGGCACCTCCAGATGTT 3' (SEQ ID NO: 91).

[0107] Individual PCR products were isolated by agarose gel electrophoresis and purified using the Qiaquick ^® Gel Purification kit, according to the kit vendor's instructions (Qiagen). The PCR products were subsequently cloned into the pCR ^® 4Blunt plasmid or pCR2. l ^® TOPO plasmid using the Zero Blunt ^® TOPO ^® PCR Cloning Kit or the TOPO ^® TA Cloning Kit, respectively, according to the kit vendor's instructions (Invitrogen) and transformed into DH5- α bacteria (Invitrogen), using standard molecular biology techniques. Plasmid DNA isolated from transformed bacterial clones was sequenced using Ml 3 Forward

(5 ' GTAA AACGACGGCCAGT 3') (SEQ ID NO: 92) and M13 Reverse primers (5'

CAGGAAACAGCTATGACC 3') (SEQ ID NO: 93) by Beckman Genomics (Danvers, MA), using standard dideoxy DNA sequencing methods to identify the sequence of the variable region sequences. The sequences were analyzed using Vector NTI software (Invitrogen) and the IMGT/V-Quest web server (imgt.cines.fr) to identify and confirm variable region sequences.

[0108] The nucleic acid sequences encoding and the protein sequences defining variable regions of the murine monoclonal antibodies are summarized below (amino terminal signal peptide sequences are not shown). CDR sequences (Kabat definition) are shown in

bold/underlined in the amino acid sequences.

[0109] Nucleic Acid Sequence of Heavy Chain Variable Region of Antibody 2G10 (SEQ

ID NO: 1)

1 gatgtgcagc tggtggagtc tgggggagtc ttagtgcagc ctggagggtc ccggaaactc

61 tcctgtactg cctctggatt cactttcagt agctttggaa tgcactgggt tcgtcaggct 121 ccagagaagg ggctggagtg ggtcgcatac attagtagtg gcagtaaaac catctactat 181 gcagacacaa tgaagggccg attcaccatc tccagagaca atcccaagaa caccctgttc 241 ctgcaaatga cgagtctaag gtctgaggac acggccatat attactgtgc aagatcctac 301 gggtacttcg atgtctgggg cgcagggacc acggtcaccg tctcctca [0110] Protein Sequence of Heavy Chain Variable Region of Antibody 2G10 (SEQ ID NO: 2)

1 dvqlvesggv lvqpggsrkl sctasgftfs sfgmhwvrqa pekglewvay_ issgsktiyy 61 adtmkgrfti srdnpkntlf lqmtslrsed taiyycarsy_ gyfdvwgagt tvtvss

[0111] Nucleic Acid Sequence Encoding Kappa Chain Variable Region of Antibody 2G10 (SEQ ID NO: 3)

1 gacattgtga tgacccagtc tcaaaaattc atgtccacat cagtaggaga cagggtcagc

61 gtcacctgca aggccagtca gaatgtgggt actaatgtgg cctggtatca acagaaacca

121 ggacaatctc ctaaagtgct gatttactcg gcatcctacc ggtacagtgg agtccctgat

181 cgcttcacag gcagtggatc tgggacagat ttcactctca ccatcgccaa tgtgcagtct

241 gaagacttgg cagagtattt ctgtcagcaa tacgacagct atcctcggac gttcggtgga 301 gtcaccaagc tggaaatcaa a

[0112] Protein Sequence of Kappa Chain Variable Region of Antibody 2G10 (SEQ ID NO:

4)

1 divmtqsqkf mstsvgdrvs vtckasqnvg tnvawyqqkp gqspkvliys asyrysgvpd 61 rftgsgsgtd ftltianvqs edlaeyfcqcj ydsyprt fgg vtkleik

[0113] Nucleic Acid Sequence Encoding Heavy Chain Variable Region of Antibody 2E6 (SEQ ID NO: 11)

1 gaggttcagc tccagcagtc tggggctgag ctggcaagac ctggggcttc agtgaagatg

61 tcctgcaagg cttctggcta cacctttacc agctactgga tgcactgggt aaaacagagg

121 cctggacagg gtctggaatg gattggcgct gtttatccta gaaacaatga tactacttac

181 aatcagaagt tcaagggcaa ggccaagctg actgctgtca catccgccag cactgcctac

241 atggcactca gcagcctaac aaatgaggac tctgcggtct attactgtct ttattttaac

301 tacaactttg actactgggg ccaaggcacc actctcacag tctcctca

[0114] Protein Sequence of Heavy Chain Variable Region of Antibody 2E6 (SEQ ID NO: 12)

1 evqlqqsgae larpgasvkm sckasgytft sywmhwvkqr pgqglewiga vyprnndtty 61 nqkfkgkakl tavtsastay malssltned savyyclyf n ynfdywgqgt tltvss

[0115] Nucleic Acid Sequence Encoding Kappa Chain Variable Region of Antibody 2E6 (SEQ ID NO: 13)

1 caaattgttc tcacccagtc tccagcaatc atgtctgctt ctccagggga gaaggtcacc

61 atgacctgca gtgccagctc aagtgtaagt tacatgcact ggtaccagca gaagccagga

121 tcctccccca gactcctgat ttatgacaca tccaacctgg cttctggagt ccctgtgcac

181 ttcagtggca gtgggtctgg gacctcttac tctctcacaa tcatccgaat ggaggctgaa

241 gatgctgcca cttattactg ccagcagtgg agtagttacc cgtacacgtt cggagggggg 301 accaagctgg aaataaaa [0116] Protein Sequence of Kappa Chain Variable Region of Antibody 2E6 (SEQ ID NO: 14)

1 qivltqspai msaspgekvt mtcsasssvs ymhwyqqkpq ssprlliydt snlasgvpvh 61 fsgsgsgtsy sltiirmeae daatyycqqw ssypytfqqq tkleik

[0117] Nucleic Acid Sequence Encoding Heavy Chain Variable Region of Antibody 2A11 (SEQ ID NO: 21)

1 caggttcagc tgcagcagtc tggacctgag ctggtgaagc ctggggcctc agtgaagatt

61 tcctgcaagg cttctggcta tgcattcagt agctcctgga tgaactgggt gaagcagagg

121 cctggaaagg gtcttgagtg gattggacgg atttatcctg gagatggaga tactaactac

181 aatgggaaat tcaagggcaa ggccacactg actgcagaca aatcctccag cacagcctac

241 atgcaactca gcagcctgac atctgaggac tctgcggtct acttctgtgc aagatcgggc

301 tccatctact atggtaacca cggggactac tttgactact ggggccaagg caccactctc 361 acagtctcct ca

[0118] Protein Sequence Defining Heavy Chain Variable Region of Antibody 2A1 1 (SEQ ID NO: 22)

1 qvqlqqsgpe lvkpgasvki sckasgyafs sswmnwvkqr pgkglewigr iypgdgdtny

61 ngkfkgkatl tadkssstay mqlssltsed savyfcarsg_ siyygnhgdy fdywqqqttl

121 tvss

[0119] Nucleic Acid Sequence Encoding Kappa Chain Variable Region of Antibody 2A11 (SEQ ID NO: 23)

1 caaattgttc tcacccagtc tccagcaatc atgtctgctt ctccagggga gaaggtcacc

61 atgacctgca gtgccagctc aagtgtaagt tacatgcact ggtaccagca gaagccagga

121 tcctccccca gactcctgat ttatgacaca tccaacctgg cttctggagt ccctgtgcac

181 ttcagtggca gtgggtctgg gacctcttac tctctcacaa tcatccgaat ggaggctgaa

241 gatgctgcca cttattactg ccagcagtgg agtagttacc cgtacacgtt cggagggggg 301 accaagctgg aaataaaa

[0120] Protein Sequence of Kappa Chain Variable Region of Antibody 2A11 (SEQ ID NO: 24)

1 qivltqspai msaspgekvt mtcsasssvs ymhwyqqkpq ssprlliydt snlasgvpvh 61 fsgsgsgtsy sltiirmeae daatyycqqw ssypytfggg tkleik

[0121] Nucleic Acid Sequence Encoding Heavy Chain Variable Region of Antibody 2D11 (SEQ ID NO: 28)

1 gaggttcagc tccagcagtc tggggctgag ctggcaagac ctggggcttc agtgaagatg

61 tcctgcaagg cttctggcta cacctttacc aggtactgga tgcactgggt aaaacagagg

121 cctggacagg gtctggaatg gattggcgct atttatcctg gaaatagtga tactacctac

181 aatcagaagt tcaagggcaa ggccaaactg actgcagtca catccgccag cactgcctac

241 atggagctca gcagcctaac aaatgaggac tctgcggtct attactgtat atacccctat

301 gattaccttg actactgggg ccaaggcacc actctcacag tctcctca [0122] Protein Sequence Defining Heavy Chain Variable Region of Antibody 2D11 (SEQ ID NO: 29)

1 evqlqqsgae larpgasvkm sckasgytft rywmhwvkqr pgqglewiga iypgnsdtty 61 nqkfkgkakl tavtsastay melssltned savyyciypy dyldywgqgt tltvss

[0123] Nucleic Acid Sequence Encoding Kappa Chain Variable Region of Antibody 2D11 (SEQ ID NO: 30)

1 caaattgttc tcacccagtc tccagcaatc atgtctgcat ctccagggga gaaggtcacc 61 atgacctgca gtgccagctc aagtttaagt tacatgcact ggtaccagca gaagccaggc 121 acctccccca aaagatgggt ttatgacaca tccaaactgg cttctggagt ccctgctcgc

181 ttcagtggca gtgggtctgg gacctcttat tctctcacaa tcagcagcat ggaggctgaa 241 gatgctgcca cttattactg ccatcagcgg agtagttacc cgtacacgtt cggagggggg 301 accaagctgg aaataaaa

[0124] Protein Sequence Defining Kappa Chain Variable Region of Antibody 2D11 (SEQ ID NO: 31)

1 qivltqspai msaspgekvt mtcsasssls ymhwyqqkpg tspkrwvydt sklasgvpar 61 fsgsgsgtsy sltissmeae daatyychqr ssypyt fggg tkleik [0125] The amino acid sequences defining the immunoglobulin heavy chain variable regions for the antibodies in Example 1 are aligned in FIG. 2. Amino terminal signal peptide sequences (for proper expression/secretion) are not shown. CDR _l5 CDR ₂ , and CDR ₃ (Kabat definition) are identified by boxes. FIG. 3 shows an alignment of the separate CDR _l5 CDR ₂ , and CDR ₃ sequences for each antibody.

[0126] The amino acid sequences defining the immunoglobulin light chain variable regions for the antibodies in Example 1 are aligned in FIG. 4. Amino terminal signal peptide sequences (for proper expression/secretion) are not shown. CDR _l5 CDR ₂ and CDR ₃ are identified by boxes. FIG. 5 shows an alignment of the separate CDR _l5 CDR ₂ , and CDR ₃ sequences for each antibody.

[0127] Table 1 shows the SEQ ID NO. of each sequence discussed in this Example. Table 1

[0128] Mouse monoclonal antibody heavy chain CDR sequences (Kabat, Chothia, and IMGT definitions) are shown in Table 2.

Table 2

[0129] Mouse monoclonal antibody Kappa light chain CDR sequences (Kabat, Chothia, and IMGT definitions) are shown in Table 3. Table 3

[0130] To create the complete heavy or kappa chain antibody sequences, each variable sequence above is combined with its respective constant region. For example, a complete heavy chain comprises a heavy variable sequence followed by the murine IgGl or IgG2b heavy chain constant sequence, and a complete kappa chain comprises a kappa variable sequence followed by the murine kappa light chain constant sequence.

[0131] Nucleic Acid Sequence Encoding Murine IgGl Heavy Chain Constant Region (SEQ ID NO: 62)

1 gccaaaacga cacccccatc tgtctatcca ctggcccctg gatctgctgc ccaaactaac

61 tccatggtga ccctgggatg cctggtcaag ggctatttcc ctgagccagt gacagtgacc 121 tggaactctg gatccctgtc cagcggtgtg cacaccttcc cagctgtcct gcagtctgac 181 ctctacactc tgagcagctc agtgactgtc ccctccagca cctggcccag cgagaccgtc 241 acctgcaacg ttgcccaccc ggccagcagc accaaggtgg acaagaaaat tgtgcccagg 301 gattgtggtt gtaagccttg catatgtaca gtcccagaag tatcatctgt cttcatcttc 361 cccccaaagc ccaaggatgt gctcaccatt actctgactc ctaaggtcac gtgtgttgtg 421 gtagacatca gcaaggatga tcccgaggtc cagttcagct ggtttgtaga tgatgtggag 481 gtgcacacag ctcagacgca accccgggag gagcagttca acagcacttt ccgctcagtc 541 agtgaacttc ccatcatgca ccaggactgg ctcaatggca aggagttcaa atgcagggtc 601 aacagtgcag ctttccctgc ccccatcgag aaaaccatct ccaaaaccaa aggcagaccg 661 aaggctccac aggtgtacac cattccacct cccaaggagc agatggccaa ggataaagtc 721 agtctgacct gcatgataac agacttcttc cctgaagaca ttactgtgga gtggcagtgg 781 aatgggcagc cagcggagaa ctacaagaac actcagccca tcatggacac agatggctct 841 tacttcgtct acagcaagct caatgtgcag aagagcaact gggaggcagg aaatactttc 901 acctgctctg tgttacatga gggcctgcac aaccaccata ctgagaagag cctctcccac 961 tctcctggta aa

[0132] Protein Sequence of Murine IgGl Heavy Chain Constant Region (SEQ ID NO: 63)

1 akttppsvyp lapgsaaqtn smvtlgclvk gyfpepvtvt wnsgslssgv htfpavlqsd

61 lytlsssvtv psstwpsetv tcnvahpass tkvdkkivpr dcgckpcict vpevssvfif

121 ppkpkdvlti tltpkvtcvv vdiskddpev qfswfvddve vhtaqtqpre eqfnstfrsv

181 selpimhqdw lngkefkcrv nsaafpapie ktisktkgrp kapqvytipp pkeqmakdkv

241 sltcmitdff peditvewqw ngqpaenykn tqpimdtdgs yfvysklnvq ksnweagntf

301 tcsvlheglh nhhtekslsh spgk

[0133] Nucleic Acid Sequence Encoding Murine IgG2b Heavy Chain Constant Region (SEQ ID NO: 64)

1 gccaaaacaa cacccccatc agtctatcca ctggcccctg ggtgtggaga tacaactggt 61 tcctccgtga cctctgggtg cctggtcaag gggtacttcc ctgagccagt gactgtgact 121 tggaactctg gatccctgtc cagcagtgtg cacaccttcc cagctctcct gcagtctgga 181 ctctacacta tgagcagctc agtgactgtc ccctccagca cctggccaag tcagaccgtc 241 acctgcagcg ttgctcaccc agccagcagc accacggtgg acaaaaaact tgagcccagc 301 gggcccattt caacaatcaa cccctgtcct ccatgcaagg agtgtcacaa atgcccagct 361 cctaacctcg agggtggacc atccgtcttc atcttccctc caaatatcaa ggatgtactc 421 atgatctccc tgacacccaa ggtcacgtgt gtggtggtgg atgtgagcga ggatgaccca 481 gacgtccaga tcagctggtt tgtgaacaac gtggaagtac acacagctca gacacaaacc 541 catagagagg attacaacag tactatccgg gtggtcagca ccctccccat ccagcaccag 601 gactggatga gtggcaagga gttcaaatgc aaggtgaaca acaaagacct cccatcaccc 661 atcgagagaa ccatctcaaa aattaaaggg ctagtcagag ctccacaagt atacactttg 721 ccgccaccag cagagcagtt gtccaggaaa gatgtcagtc tcacttgcct ggtcgtgggc 781 ttcaaccctg gagacatcag tgtggagtgg accagcaatg ggcatacaga ggagaactac 841 aaggacaccg caccagttct tgactctgac ggttcttact tcatatatag caagctcaat 901 atgaaaacaa gcaagtggga gaaaacagat tccttctcat gcaacgtgag acacgagggt 961 ctgaaaaatt actacctgaa gaagaccatc tcccggtctc cgggtaaa

[0134] Protein Sequence of Murine IgG2b Heavy Chain Constant Region (SEQ ID NO: 65)

1 akttppsvyp lapgcgdttg ssvtsgclvk gyfpepvtvt wnsgslsssv htfpallqsg

61 lytmsssvtv psstwpsqtv tcsvahpass ttvdkkleps gpistinpcp pckechkcpa

121 pnleggpsvf ifppnikdvl misltpkvtc vvvdvseddp dvqiswfvnn vevhtaqtqt

181 hredynstir vvstlpiqhq dwmsgkefkc kvnnkdlpsp iertiskikg lvrapqvytl

241 pppaeqlsrk dvsltclvvg fnpgdisvew tsnghteeny kdtapvldsd gsyfiyskln

301 mktskwektd sfscnvrheg lknyylkkti srspgk

[0135] Nucleic Acid Sequence Encoding Murine Kappa Light Chain Constant Region

(SEQ ID NO: 66)

1 cgggctgatg ctgcaccaac tgtatccatc ttcccaccat ccagtgagca gttaacatct

61 ggaggtgcct cagtcgtgtg cttcttgaac aacttctacc ccaaagacat caatgtcaag

121 tggaagattg atggcagtga acgacaaaat ggcgtcctga acagttggac tgatcaggac

181 agcaaagaca gcacctacag catgagcagc accctcacgt tgaccaagga cgagtatgaa

241 cgacataaca gctatacctg tgaggccact cacaagacat caacttcacc cattgtcaag

301 agcttcaaca ggaatgagtg t [0136] Protein Sequence of Murine Kappa Light Chain Constant Region (SEQ ID NO: 67)

1 radaaptvsi fppsseqlts ggasvvcfln nfypkdinvk wkidgserqn gvlnswtdqd 61 skdstysmss tltltkdeye rhnsytceat hktstspivk sfnrnec [0137] The following sequences represent the actual or contemplated full length heavy and light chain sequence (i.e., containing both the variable and constant regions sequences) for each antibody described in this Example. Signal sequences for proper secretion of the antibodies (e.g., signal sequences at the 5' end of the DNA sequences or the amino terminal end of the protein sequences) are not shown in the full length heavy and light chain sequences disclosed herein and are not included in the final secreted protein. Also not shown are stop codons for termination of translation required at the 3' end of the DNA sequences. It is within ordinary skill in the art to select a signal sequence and/or a stop codon for expression of the disclosed full length IgG heavy chain and light chain sequences. It is also contemplated that the variable region sequences can be ligated to other constant region sequences to produce active full length IgG heavy and light chains.

[0138] Nucleic Acid Sequence Encoding the Full Length Heavy Chain Sequence (Heavy Chain Variable Region and IgGl Constant Region) of 2G10 (SEQ ID NO: 68)

1 gatgtgcagc tggtggagtc tgggggagtc ttagtgcagc ctggagggtc ccggaaactc 61 tcctgtactg cctctggatt cactttcagt agctttggaa tgcactgggt tcgtcaggct 121 ccagagaagg ggctggagtg ggtcgcatac attagtagtg gcagtaaaac catctactat

181 gcagacacaa tgaagggccg attcaccatc tccagagaca atcccaagaa caccctgttc 241 ctgcaaatga cgagtctaag gtctgaggac acggccatat attactgtgc aagatcctac 301 gggtacttcg atgtctgggg cgcagggacc acggtcaccg tctcctcagc caaaacgaca 361 cccccatctg tctatccact ggcccctgga tctgctgccc aaactaactc catggtgacc 421 ctgggatgcc tggtcaaggg ctatttccct gagccagtga cagtgacctg gaactctgga

481 tccctgtcca gcggtgtgca caccttccca gctgtcctgc agtctgacct ctacactctg 541 agcagctcag tgactgtccc ctccagcacc tggcccagcg agaccgtcac ctgcaacgtt 601 gcccacccgg ccagcagcac caaggtggac aagaaaattg tgcccaggga ttgtggttgt 661 aagccttgca tatgtacagt cccagaagta tcatctgtct tcatcttccc cccaaagccc 721 aaggatgtgc tcaccattac tctgactcct aaggtcacgt gtgttgtggt agacatcagc

781 aaggatgatc ccgaggtcca gttcagctgg tttgtagatg atgtggaggt gcacacagct 841 cagacgcaac cccgggagga gcagttcaac agcactttcc gctcagtcag tgaacttccc 901 atcatgcacc aggactggct caatggcaag gagttcaaat gcagggtcaa cagtgcagct 961 ttccctgccc ccatcgagaa aaccatctcc aaaaccaaag gcagaccgaa ggctccacag 1021 gtgtacacca ttccacctcc caaggagcag atggccaagg ataaagtcag tctgacctgc

1081 atgataacag acttcttccc tgaagacatt actgtggagt ggcagtggaa tgggcagcca 1141 gcggagaact acaagaacac tcagcccatc atggacacag atggctctta cttcgtctac 1201 agcaagctca atgtgcagaa gagcaactgg gaggcaggaa atactttcac ctgctctgtg 1261 ttacatgagg gcctgcacaa ccaccatact gagaagagcc tctcccactc tcctggtaaa [0139] Protein Sequence Defining the Full Length Heavy Chain Sequence (Heavy Chain Variable Region and IgGl Constant Region) of 2G10 (SEQ ID NO: 69)

1 dvqlvesggv lvqpggsrkl sctasgftfs sfgmhwvrqa pekglewvay issgsktiyy 61 adtmkgrfti srdnpkntlf lqmtslrsed taiyycarsy gyfdvwgagt tvtvssaktt 121 ppsvyplapg saaqtnsmvt Igclvkgyfp epvtvtwnsg slssgvhtfp avlqsdlytl 181 sssvtvpsst wpsetvtcnv ahpasstkvd kkivprdcgc kpcictvpev ssvfifppkp 241 kdvltitltp kvtcvvvdis kddpevqfsw fvddvevhta qtqpreeqfn stfrsvselp 301 imhqdwlngk efkcrvnsaa fpapiektis ktkgrpkapq vytipppkeq makdkvsltc 361 mitdffpedi tvewqwngqp aenykntqpi mdtdgsyfvy sklnvqksnw eagntftcsv 421 lheglhnhht ekslshspgk

[0140] Nucleic Acid Sequence Encoding the Full Length Light Chain Sequence (Kappa

Chain Variable Region and Constant Region) of 2G10 (SEQ ID NO: 70)

1 gacattgtga tgacccagtc tcaaaaattc atgtccacat cagtaggaga cagggtcagc

61 gtcacctgca aggccagtca gaatgtgggt actaatgtgg cctggtatca acagaaacca

121 ggacaatctc ctaaagtgct gatttactcg gcatcctacc ggtacagtgg agtccctgat

181 cgcttcacag gcagtggatc tgggacagat ttcactctca ccatcgccaa tgtgcagtct

241 gaagacttgg cagagtattt ctgtcagcaa tacgacagct atcctcggac gttcggtgga

301 gtcaccaagc tggaaatcaa acgggctgat gctgcaccaa ctgtatccat cttcccacca

361 tccagtgagc agttaacatc tggaggtgcc tcagtcgtgt gcttcttgaa caacttctac

421 cccaaagaca tcaatgtcaa gtggaagatt gatggcagtg aacgacaaaa tggcgtcctg

481 aacagttgga ctgatcagga cagcaaagac agcacctaca gcatgagcag caccctcacg

541 ttgaccaagg acgagtatga acgacataac agctatacct gtgaggccac tcacaagaca

601 tcaacttcac ccattgtcaa gagcttcaac aggaatgagt gt

[0141] Protein Sequence Defining the Full Length Light Chain Sequence (Kappa Chain Variable Region and Constant Region) of 2G10 (SEQ ID NO: 71)

1 divmtqsqkf mstsvgdrvs vtckasqnvg tnvawyqqkp gqspkvliys asyrysgvpd 61 rftgsgsgtd ftltianvqs edlaeyfcqq ydsyprtfgg vtkleikrad aaptvsifpp 121 sseqltsgga svvcflnnfy pkdinvkwki dgserqngvl nswtdqdskd stysmsstlt 181 ltkdeyerhn sytceathkt stspivksfn rnec

[0142] Nucleic Acid Sequence Encoding the Full Length Heavy Chain Sequence (Heavy Chain Variable Region and IgGl Constant Region) of 2E6 (SEQ ID NO: 72)

1 gaggttcagc tccagcagtc tggggctgag ctggcaagac ctggggcttc agtgaagatg 61 tcctgcaagg cttctggcta cacctttacc agctactgga tgcactgggt aaaacagagg 121 cctggacagg gtctggaatg gattggcgct gtttatccta gaaacaatga tactacttac 181 aatcagaagt tcaagggcaa ggccaagctg actgctgtca catccgccag cactgcctac 241 atggcactca gcagcctaac aaatgaggac tctgcggtct attactgtct ttattttaac 301 tacaactttg actactgggg ccaaggcacc actctcacag tctcctcagc caaaacgaca 361 cccccatctg tctatccact ggcccctgga tctgctgccc aaactaactc catggtgacc 421 ctgggatgcc tggtcaaggg ctatttccct gagccagtga cagtgacctg gaactctgga 481 tccctgtcca gcggtgtgca caccttccca gctgtcctgc agtctgacct ctacactctg 541 agcagctcag tgactgtccc ctccagcacc tggcccagcg agaccgtcac ctgcaacgtt 601 gcccacccgg ccagcagcac caaggtggac aagaaaattg tgcccaggga ttgtggttgt 661 aagccttgca tatgtacagt cccagaagta tcatctgtct tcatcttccc cccaaagccc 721 aaggatgtgc tcaccattac tctgactcct aaggtcacgt gtgttgtggt agacatcagc 781 aaggatgatc ccgaggtcca gttcagctgg tttgtagatg atgtggaggt gcacacagct 841 cagacgcaac cccgggagga gcagttcaac agcactttcc gctcagtcag tgaacttccc

901 atcatgcacc aggactggct caatggcaag gagttcaaat gcagggtcaa cagtgcagct

961 ttccctgccc ccatcgagaa aaccatctcc aaaaccaaag gcagaccgaa ggctccacag

1021 gtgtacacca ttccacctcc caaggagcag atggccaagg ataaagtcag tctgacctgc

1081 atgataacag acttcttccc tgaagacatt actgtggagt ggcagtggaa tgggcagcca

1141 gcggagaact acaagaacac tcagcccatc atggacacag atggctctta cttcgtctac

1201 agcaagctca atgtgcagaa gagcaactgg gaggcaggaa atactttcac ctgctctgtg

1261 ttacatgagg gcctgcacaa ccaccatact gagaagagcc tctcccactc tcctggtaaa

[0143] Protein Sequence Defining the Full Length Heavy Chain Sequence (Heavy Chain Variable Region and IgGl Constant Region) of 2E6 (SEQ ID NO: 73)

1 evqlqqsgae larpgasvkm sckasgytft sywmhwvkqr pgqglewiga vyprnndtty 61 nqkfkgkakl tavtsastay malssltned savyyclyfn ynfdywgqgt tltvssaktt 121 ppsvyplapg saaqtnsmvt Igclvkgyfp epvtvtwnsg slssgvhtfp avlqsdlytl 181 sssvtvpsst wpsetvtcnv ahpasstkvd kkivprdcgc kpcictvpev ssvfifppkp 241 kdvltitltp kvtcvvvdis kddpevqfsw fvddvevhta qtqpreeqfn stfrsvselp 301 imhqdwlngk efkcrvnsaa fpapiektis ktkgrpkapq vytipppkeq makdkvsltc 361 mitdffpedi tvewqwngqp aenykntqpi mdtdgsyfvy sklnvqksnw eagntftcsv 421 lheglhnhht ekslshspgk

[0144] Nucleic Acid Sequence Encoding the Full Length Light Chain Sequence (Kappa

Chain Variable Region and Constant Region) of 2E6 (SEQ ID NO: 74)

1 caaattgttc tcacccagtc tccagcaatc atgtctgctt ctccagggga gaaggtcacc 61 atgacctgca gtgccagctc aagtgtaagt tacatgcact ggtaccagca gaagccagga 121 tcctccccca gactcctgat ttatgacaca tccaacctgg cttctggagt ccctgtgcac 181 ttcagtggca gtgggtctgg gacctcttac tctctcacaa tcatccgaat ggaggctgaa 241 gatgctgcca cttattactg ccagcagtgg agtagttacc cgtacacgtt cggagggggg 301 accaagctgg aaataaaacg ggctgatgct gcaccaactg tatccatctt cccaccatcc 361 agtgagcagt taacatctgg aggtgcctca gtcgtgtgct tcttgaacaa cttctacccc 421 aaagacatca atgtcaagtg gaagattgat ggcagtgaac gacaaaatgg cgtcctgaac 481 agttggactg atcaggacag caaagacagc acctacagca tgagcagcac cctcacgttg 541 accaaggacg agtatgaacg acataacagc tatacctgtg aggccactca caagacatca 601 acttcaccca ttgtcaagag cttcaacagg aatgagtgt

[0145] Protein Sequence Defining the Full Length Light Chain Sequence (Kappa Chain Variable Region and Constant Region) of 2E6 (SEQ ID NO: 75)

1 qivltqspai msaspgekvt mtcsasssvs ymhwyqqkpg ssprlliydt snlasgvpvh 61 fsgsgsgtsy sltiirmeae daatyycqqw ssypytfggg tkleikrada aptvsifpps 121 seqltsggas vvcflnnfyp kdinvkwkid gserqngvln swtdqdskds tysmsstltl 181 tkdeyerhns ytceathkts tspivksfnr nec

[0146] Nucleic Acid Sequence Encoding the Full Length Heavy Chain Sequence (Heavy Chain Variable Region and IgG2b Constant Region) of 2A11 (SEQ ID NO: 76)

1 caggttcagc tgcagcagtc tggacctgag ctggtgaagc ctggggcctc agtgaagatt 61 tcctgcaagg cttctggcta tgcattcagt agctcctgga tgaactgggt gaagcagagg 121 cctggaaagg gtcttgagtg gattggacgg atttatcctg gagatggaga tactaactac 181 aatgggaaat tcaagggcaa ggccacactg actgcagaca aatcctccag cacagcctac 241 atgcaactca gcagcctgac atctgaggac tctgcggtct acttctgtgc aagatcgggc 301 tccatctact atggtaacca cggggactac tttgactact ggggccaagg caccactctc

361 acagtctcct cagccaaaac aacaccccca tcagtctatc cactggcccc tgggtgtgga

421 gatacaactg gttcctccgt gacctctggg tgcctggtca aggggtactt ccctgagcca

481 gtgactgtga cttggaactc tggatccctg tccagcagtg tgcacacctt cccagctctc

541 ctgcagtctg gactctacac tatgagcagc tcagtgactg tcccctccag cacctggcca

601 agtcagaccg tcacctgcag cgttgctcac ccagccagca gcaccacggt ggacaaaaaa

661 cttgagccca gcgggcccat ttcaacaatc aacccctgtc ctccatgcaa ggagtgtcac

721 aaatgcccag ctcctaacct cgagggtgga ccatccgtct tcatcttccc tccaaatatc

781 aaggatgtac tcatgatctc cctgacaccc aaggtcacgt gtgtggtggt ggatgtgagc

841 gaggatgacc cagacgtcca gatcagctgg tttgtgaaca acgtggaagt acacacagct

901 cagacacaaa cccatagaga ggattacaac agtactatcc gggtggtcag caccctcccc

961 atccagcacc aggactggat gagtggcaag gagttcaaat gcaaggtgaa caacaaagac

1021 ctcccatcac ccatcgagag aaccatctca aaaattaaag ggctagtcag agctccacaa

1081 gtatacactt tgccgccacc agcagagcag ttgtccagga aagatgtcag tctcacttgc

1141 ctggtcgtgg gcttcaaccc tggagacatc agtgtggagt ggaccagcaa tgggcataca

1201 gaggagaact acaaggacac cgcaccagtt cttgactctg acggttctta cttcatatat

1261 agcaagctca atatgaaaac aagcaagtgg gagaaaacag attccttctc atgcaacgtg

1321 agacacgagg gtctgaaaaa ttactacctg aagaagacca tctcccggtc tccgggtaaa

[0147] Protein Sequence Defining the Full Length Heavy Chain Sequence (Heavy Chain Variable Region and IgG2b Constant Region) of 2A11 (SEQ ID NO: 77)

1 qvqlqqsgpe lvkpgasvki sckasgyafs sswmnwvkqr pgkglewigr iypgdgdtny 61 ngkfkgkatl tadkssstay mqlssltsed savyfcarsg siyygnhgdy fdywgqgttl 121 tvssakttpp svyplapgcg dttgssvtsg clvkgyfpep vtvtwnsgsl sssvhtfpal 181 lqsglytmss svtvpsstwp sqtvtcsvah passttvdkk lepsgpisti npcppckech 241 kcpapnlegg psvfifppni kdvlmisltp kvtcvvvdvs eddpdvqisw fvnnvevhta 301 qtqthredyn stirvvstlp iqhqdwmsgk efkckvnnkd lpspiertis kikglvrapq 361 vytlpppaeq lsrkdvsltc lvvgfnpgdi svewtsnght eenykdtapv ldsdgsyfiy 421 sklnmktskw ektdsfscnv rheglknyyl kktisrspgk

[0148] Nucleic Acid Sequence Encoding the Full Length Light Chain Sequence (Kappa Chain Variable Region and Constant Region) of 2A11 (SEQ ID NO: 78)

1 caaattgttc tcacccagtc tccagcaatc atgtctgctt ctccagggga gaaggtcacc 61 atgacctgca gtgccagctc aagtgtaagt tacatgcact ggtaccagca gaagccagga 121 tcctccccca gactcctgat ttatgacaca tccaacctgg cttctggagt ccctgtgcac 181 ttcagtggca gtgggtctgg gacctcttac tctctcacaa tcatccgaat ggaggctgaa 241 gatgctgcca cttattactg ccagcagtgg agtagttacc cgtacacgtt cggagggggg 301 accaagctgg aaataaaacg ggctgatgct gcaccaactg tatccatctt cccaccatcc 361 agtgagcagt taacatctgg aggtgcctca gtcgtgtgct tcttgaacaa cttctacccc 421 aaagacatca atgtcaagtg gaagattgat ggcagtgaac gacaaaatgg cgtcctgaac 481 agttggactg atcaggacag caaagacagc acctacagca tgagcagcac cctcacgttg 541 accaaggacg agtatgaacg acataacagc tatacctgtg aggccactca caagacatca 601 acttcaccca ttgtcaagag cttcaacagg aatgagtgt

[0149] Protein Sequence Defining the Full Length Light Chain Sequence (Kappa Chain Variable Region and Constant Region) of 2A11 (SEQ ID NO: 79)

1 qivltqspai msaspgekvt mtcsasssvs ymhwyqqkpg ssprlliydt snlasgvpvh 61 fsgsgsgtsy sltiirmeae daatyycqqw ssypytfggg tkleikrada aptvsifpps 121 seqltsggas vvcflnnfyp kdinvkwkid gserqngvln swtdqdskds tysmsstltl 181 tkdeyerhns ytceathkts tspivksfnr nec [0150] Nucleic Acid Sequence Encoding the Full Length Heavy Chain Sequence (Heavy Chain Variable Region and IgGl Constant Region) of 2D11 (SEQ ID NO: 80)

1 gaggttcagc tccagcagtc tggggctgag ctggcaagac ctggggcttc agtgaagatg 61 tcctgcaagg cttctggcta cacctttacc aggtactgga tgcactgggt aaaacagagg 121 cctggacagg gtctggaatg gattggcgct atttatcctg gaaatagtga tactacctac 181 aatcagaagt tcaagggcaa ggccaaactg actgcagtca catccgccag cactgcctac 241 atggagctca gcagcctaac aaatgaggac tctgcggtct attactgtat atacccctat 301 gattaccttg actactgggg ccaaggcacc actctcacag tctcctcagc caaaacgaca 361 cccccatctg tctatccact ggcccctgga tctgctgccc aaactaactc catggtgacc 421 ctgggatgcc tggtcaaggg ctatttccct gagccagtga cagtgacctg gaactctgga 481 tccctgtcca gcggtgtgca caccttccca gctgtcctgc agtctgacct ctacactctg 541 agcagctcag tgactgtccc ctccagcacc tggcccagcg agaccgtcac ctgcaacgtt 601 gcccacccgg ccagcagcac caaggtggac aagaaaattg tgcccaggga ttgtggttgt 661 aagccttgca tatgtacagt cccagaagta tcatctgtct tcatcttccc cccaaagccc 721 aaggatgtgc tcaccattac tctgactcct aaggtcacgt gtgttgtggt agacatcagc 781 aaggatgatc ccgaggtcca gttcagctgg tttgtagatg atgtggaggt gcacacagct 841 cagacgcaac cccgggagga gcagttcaac agcactttcc gctcagtcag tgaacttccc 901 atcatgcacc aggactggct caatggcaag gagttcaaat gcagggtcaa cagtgcagct 961 ttccctgccc ccatcgagaa aaccatctcc aaaaccaaag gcagaccgaa ggctccacag 1021 gtgtacacca ttccacctcc caaggagcag atggccaagg ataaagtcag tctgacctgc 1081 atgataacag acttcttccc tgaagacatt actgtggagt ggcagtggaa tgggcagcca 1141 gcggagaact acaagaacac tcagcccatc atggacacag atggctctta cttcgtctac 1201 agcaagctca atgtgcagaa gagcaactgg gaggcaggaa atactttcac ctgctctgtg 1261 ttacatgagg gcctgcacaa ccaccatact gagaagagcc tctcccactc tcctggtaaa

[0151] Protein Sequence Defining the Full Length Heavy Chain Sequence (Heavy Chain Variable Region and IgGl Constant Region) of 2D11 (SEQ ID NO: 81)

1 evqlqqsgae larpgasvkm sckasgytft rywmhwvkqr pgqglewiga iypgnsdtty 61 nqkfkgkakl tavtsastay melssltned savyyciypy dyldywgqgt tltvssaktt 121 ppsvyplapg saaqtnsmvt Igclvkgyfp epvtvtwnsg slssgvhtfp avlqsdlytl 181 sssvtvpsst wpsetvtcnv ahpasstkvd kkivprdcgc kpcictvpev ssvfifppkp 241 kdvltitltp kvtcvvvdis kddpevqfsw fvddvevhta qtqpreeqfn stfrsvselp 301 imhqdwlngk efkcrvnsaa fpapiektis ktkgrpkapq vytipppkeq makdkvsltc 361 mitdffpedi tvewqwngqp aenykntqpi mdtdgsyfvy sklnvqksnw eagntftcsv 421 lheglhnhht ekslshspgk

[0152] Nucleic Acid Sequence Encoding the Full Length Light Chain Sequence (Kappa Chain Variable Region and Constant Region) of 2D11 (SEQ ID NO: 82)

1 caaattgttc tcacccagtc tccagcaatc atgtctgcat ctccagggga gaaggtcacc

61 atgacctgca gtgccagctc aagtttaagt tacatgcact ggtaccagca gaagccaggc

121 acctccccca aaagatgggt ttatgacaca tccaaactgg cttctggagt ccctgctcgc

181 ttcagtggca gtgggtctgg gacctcttat tctctcacaa tcagcagcat ggaggctgaa

241 gatgctgcca cttattactg ccatcagcgg agtagttacc cgtacacgtt cggagggggg

301 accaagctgg aaataaaacg ggctgatgct gcaccaactg tatccatctt cccaccatcc

361 agtgagcagt taacatctgg aggtgcctca gtcgtgtgct tcttgaacaa cttctacccc

421 aaagacatca atgtcaagtg gaagattgat ggcagtgaac gacaaaatgg cgtcctgaac

481 agttggactg atcaggacag caaagacagc acctacagca tgagcagcac cctcacgttg

541 accaaggacg agtatgaacg acataacagc tatacctgtg aggccactca caagacatca

601 acttcaccca ttgtcaagag cttcaacagg aatgagtgt [0153] Protein Sequence Defining the Full Length Light Chain Sequence (Kappa Chain Variable Region and Constant Region) of 2D11 (SEQ ID NO: 83)

1 qivltqspai msaspgekvt mtcsasssls ymhwyqqkpg tspkrwvydt sklasgvpar 61 fsgsgsgtsy sltissmeae daatyychqr ssypytfggg tkleikrada aptvsifpps

121 seqltsggas vcflnnfyp kdinvkwkid gserqngvln swtdqdskds tysmsstltl

181 tkdeyerhns ytceathkts tspivksfnr nec

[0154] Table 4 shows the correspondence between the full length sequences of the antibodies discussed in this Example with those presented in the Sequence Listing.

Table 4

Example 3: Binding Affinities

[0155] The binding affinities and kinetics of interaction of monoclonal antibodies 2G10, 2E6, 2A11, and 2D11 to recombinant human Notch 1/Fc fusion protein (rhNotchl-Fc) were measured by surface plasmon resonance using a Biacore ^® T100 instrument (GE Healthcare, Piscataway, NJ).

[0156] Rabbit anti-mouse IgGs (Biacore, Cat. No. BR- 1008-38) were immobilized on carboxymethylated dextran CM4 sensor chips by amine coupling (GE Healthcare) using a standard coupling protocol according to vendor's instructions. The analyses were performed at 25°C, using PBS (Invitrogen, Cat. No. 14040-133) containing 0.05% surfactant P20 (GE Healthcare) as running buffer.

[0157] The antibodies were captured in individual flow cells, at a flow rate of 10 μΐ/min. Injection time was varied for each antibody to yield an Rmax between 30 and 60 RU. Buffer or rhNotchl-Fc diluted in running buffer was injected sequentially over a reference surface (no antibody captured) and the active surface (antibody to be tested) for 300 seconds, at 60 μΐ/min. The dissociation phase was monitored for up to 3600 seconds. The surface was then regenerated with two 60-second injections of 10 mM Glycine-HCl, pH 1.7, at a flow rate of 60 μΐ/min. The rhNotchl-Fc concentration range tested was 6.25 nM to 100 nM. Kinetic parameters were determined using the kinetic function of the BIAevalutation software (GE

Healthcare) with double reference subtraction. Kinetic values of the monoclonal antibodies on rhNotchl-Fc at 25°C are summarized in Table 5.

Table 5

[0158] The results in Table 5 demonstrate that antibodies 2E6, 2A11, and 2D11 bind rhNotchl-Fc with a K _D of about 10 nM or less, 7.5 nM or less, 5 nM or less, 4 nM or less.

Example 4: Binding Specificity

[0159] Antibodies 2G10, 2E6, 2A11 and 2G11 were tested for binding to human Notchl, human Notch2, or human Notch3, protein. Binding measurements were made by bio-layer interferometry (BLI), using a ForteBio Octet ^® QK instrument (ForteBio, Menlo Park, CA). Anti-human-Fc sensors were soaked in PBS containing 1 mg/ml BSA for 5 minutes prior to binding of antibodies. Then the following proteins (400 nM, in PBS containing 1 mg/ml BSA) were allowed to bind to the sensors: rhNotchl-Fc (R&D Systems, Minneapolis, MN; Cat. No. 3647-TK-050), rhNotch2-Fc (R&D Cat. No. 3735-NT-050), rhNotch3-Fc (R&D Cat. No. 1559-NT-050), or rmNotchl-Fc (R&D Cat. No. 5267-TK-050). Notch protein bound sensors were immersed in antibody solution (50 μg/ml) to allow binding of antibody to the Notch protein. Binding was detected by shifts in the interference pattern. These results demonstrated that the antibodies bind specifically to human Notch 1 protein, but do not bind to human Notch2 or human Notch3 protein.

[0160] To determine specificity of binding to cell surface Notch proteins, the antibodies were tested for binding to human Notch 1, human Notch2, human Notch3 and human Notch4 expressed on the surface of CHO cells using electrochemiluminescence (Meso Scale

Discovery). CHO Flpln™ cells (Invitrogen, Cat. No. R758-07) expressing each of the four human Notch proteins were produced, according to the vendor's instructions. A CHO line lacking any human Notch protein was also produced for use as a negative control. Cells were grown under standard conditions (37°C, DMEM/F12 + 10% FBS). For binding studies, cells were washed in PBS containing calcium and magnesium, removed from the plate, and disaggregated by treatment with dissociation buffer (GIBCO Cat. No. 13151014) for 10 minutes at 37 °C.

[0161] Cells were seeded at a density of 30,000 cells per well, in hybridoma media, in a standard 96-well binding plates (Meso Scale Discovery, Cat. No. L15XA-6). Cells were incubated for one hour at 37°C. Antibodies or control IgG were added at 5 μg/ml, in 50 μΐ hybridoma media, and incubated for 1 hour at 37°C. The plates were washed twice with PBS containing 3% BSA. Binding of the antibodies to cell surface was detected using 2 μg/ml of MSD anti-mouse IgG secondary antibody (Meso Scale Discovery, Cat. No. R32AC-1) for 1 hour at 4°C. Plates were washed twice with PBS containing 3% BSA, and 150 μΐ of read buffer (Meso Scale Discovery Cat. No. R92TC-1) was added. The plates were analyzed on a Sector Imager 2400 instrument (Meso Scale Discovery). This analysis showed that antibodies 2G10, 2E6, 2A11 and 2D11 bind to human Notchl displayed on the surfaces of cells, but do not bind to human Notch2, Notch3 (FIG. 6) or Notch4 (data not shown), displayed on the surfaces of cells. The antibodies also do not bind CHO-EV (empty vector) cells that express endogenous hamster Notch proteins. These results indicated that the antibodies bind specifically to human Notchl protein in vitro, and when the Notchl protein is displayed on a cell surface.

Examples 5: Inhibition of Notchl-Ligand Binding

[0162] Antibodies 2E6, 2A11 and 2D11 were tested for their ability to inhibit the binding of rhNotchl to human Jagl, Jag2, DLLl and DLL4. Binding measurements were made by bio- layer interferometry (BLI), using a ForteBio Octet QK instrument (ForteBio, Menlo Park, CA). The ligands tested were rhJagl-Fc (R&D Cat. No. 1277-JG-050 ), rhJag2-Fc (R&D Cat. No. 1726-JG-050), rhDLLl-Fc (R&D Cat. No. 5026-DL-050), and His tagged rhDLL4 (R&D Cat. No. 1506-D4-050).

[0163] To determine the degree of inhibition of Notch 1-ligand binding by each antibody, the Octet sensors were loaded with recombinant human Notch 1, and each antibody was allowed to bind, as described in Example 4 (above). Subsequently sensors were immersed in 500 μg/ml human IgG, to block non-specific binding. Ligands were prepared at 400 nM, in PBS containing 3% BSA, and were allowed to bind. The on-rate and off-rate for ligand binding were detected using the Octet ^® QK instrument and software. Antibodies 2E6, 2A11, and 2D11 blocked binding of all four ligands to rhNotchl-Fc.

Example 6: Inhibition of Notchl Activation

[0164] The effect of the antibodies on signaling by Notchl was tested as follows. Blocking of ligand-induced activation of Notchl and subsequent proteolytic cleavages that release the intracellular domain of Notchl, were analyzed by using immunoblots to detect activated Notchl -intracellular domain (Nl-ICD).

[0165] To activate Notchl signaling in cell lines, various Notchl -expressing cells were plated in 96- well plates on wells coated with Notch ligands. The wells were prepared by diluting oc-human Fc (Jackson ImmunoResearch, West Grove, PA) to 10 μg/ml in sterile- filtered carbonate -bicarbonate coating buffer, pH 9.4 (Pierce 28382). Then 1 μg of the diluted antibody was added to each well of a 96-well Maxisorp™ plate and incubated overnight at 4°C. The next day, wells were washed three times with PBS before adding 1 μg soluble ligand Fc fusion protein or human IgG Fc (Jackson Immunolabs) diluted in PBS/0.5%BSA. After incubating for two hours at room temperature on an orbital shaker, the wells were washed three times with PBS to remove unbound ligand. Karpas45 or Notchl 293 Flpln™ cells were counted and resuspended in fresh growth media at 0.75 x 10 ⁶ cells/ml or 0.3 x 10 ⁶ cells/ml, respectively. Cells were pre-incubated with 10 μg/ml blocking antibody for one hour at 37°C, before seeding 100 μΐ of the suspension into 96-well plates coated with ligand or hFc. Cells were incubated for four hours at 37°C before being dislodged from the well by pipetting and harvested. Wells were washed with PBS and pooled with harvested cells to ensure complete collection. Cells were sedimented in a refrigerated microcentrifuge, washed with 100 μΐ PBS, and lysed by resuspending cell pellet in 30 μΐ of RIPA buffer containing protease inhibitors. Lysates were clarified by centrifugation in a refrigerated microcentrifuge. Supernatants were boiled with 5X SDS sample buffer before SDS PAGE and for Western blotting. Blots were probed with a a- Notchl antibody specific for the cleaved intracellular domain (Cell Signaling, #2421). Notchl activation by all four ligands was inhibited by Notchl antibodies 2G10, 2E6, 2A11 and 2D11, at concentrations ranging from 0.1 μg/ml tolO μg/ml.

Example 7: Inhibition of Notchl-Dependent Transcription

[0166] Reporter cell lines dependent upon Notchl, Notch2, or Notch3 were produced by lentiviral introduction of a RBP-jK-dependent luciferase reporter gene (SABiosciences,

Frederick, MD) into 293-FlpIn™ Notchl cells, Karpas45 cells, and DU4475 cells. To activate Notchl -dependent signaling and transcription, cells were plated on ligand-coated wells prepared, as described in Example 6 (above). Cells were pre-incubated with a 3-fold dilution series of Notchl antibody concentrations ranging from 0-300 μg/ml, for one hour at 37°C, before seeding 100 μΐ of the suspension into 96-well plates coated with ligand or hFc. Cells were incubated in ligand-coated or human-Fc-coated wells for four or 24 hours at 37°C, in 5% C0 ₂ . Next, 100 μΐ of Promega Bright Glo™ (Promega, Madison, WI) was added to each well. The reaction was allowed to proceed for five minutes in the dark, and then the entire 200 μΐ volume was transferred to white walled plates and read using a luminometer. Polyclonal antibodies against Notchl (AF1057, R&D Systems), Notch2 (AF1190, R&D Systems) or Notch3 (AF1559, R&D Systems) were used as controls to confirm that ligand-stimulated reporter activity in each cell line was specifically dependent upon the introduced Notch receptor. The Notchl antibodies specifically inhibited Notchl -dependent transcription (FIG. 7A), and did not inhibit Notch2-dependent (FIG. 7B) or Notch3-dependent transcription (FIG. 7C).

[0167] Activation of Notchl -dependent transcription by each of the ligands Jagl, Jag2, DLLl and DLL4 was inhibited by the Notchl antibodies 2E6 (FIG. 8A), 2A11 (FIG. 8B) and 2D11 (FIG. 8C). The data in Table 6 show that antibody 2E6 inhibited activation of Notch 1- dependent transcription by Jagl, Jag2, DLLl and DLL4 in the Notchl -dependent reporter cell lines. Table 6

[0168] To determine the effect of Notch 1 antibodies on transcription of endogenous Notch target genes, Notchl signaling was activated by Jagl, as described above. The effect on expression of endogenous Notch targets, as a result of treatment of the Jagl -stimulated

Karpas45 cells with IgG control, Notchl antibodies, or DBZ (gamma secretase inhibitor) was assessed by quantitative RT-PCR. Karpas45 cells were seeded into 6-well plates, in 2 ml of media. Replicate wells of cells were treated with antibody 2E6, IgG control, 1 μΜ DBZ, or vehicle control (DMSO), immediately after seeding. Cells were incubated at 37°C, 5% C0 ₂ for 20 hours after treatment, collected, and rinsed with PBS. Cell pellets were frozen on dry ice and stored -80°C. RNA was prepared using Qiagen RNeasy™ miniprep columns (Qiagen GR8RNA). Quantitative RT-PCR was performed to analyze Notch target gene expression, using a commercial kit according to the kit vendor's instructions (Quantitect SYBR GREEN RT-PCR Kit; Qiagen). Results were analyzed using the comparative Ct method. Beta actin was used as an internal standard, and Stratagene Universal Human Reference RNA (Stratagene 740000) was used as an external standard for measurement of expression levels of the genes investigated. These results showed that antibody 2E6 inhibits transcription of endogenous Notch target genes, including Heyl.

Example 8: Inhibition of Human Cancer Cell Line Proliferation

[0169] Antibody 2E6 was tested for inhibition of ligand-dependent and ligand-independent proliferation of human cancer cells that express Notchl. The T-ALL cell line Karpas45 expresses elevated levels of Notchl. To screen for antagonistic Notchl antibodies, cells were grown in 96- well plates in wells coated with either human Fc or rhJagl-Fc. Growth was measured in the presence of various concentrations of antibodies (0 - 300 μg/ml in 100 μΐ final volume) by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays conducted two days after plating cells on ligand or human Fc control. These results showed that antibody 2E6 inhibits proliferation of Karpas45 cells. In addition, quantitative RT-PCR analysis, as described in Example 7 (above) demonstrated that antibody 2E6 inhibited expression of the Notch target gene Heyl in Karpas45 cells.

Example 9: Vascular Branching Morphogenesis

[0170] Antibodies 2G10, 2E6, 2A11 and 2D11 were shown to bind to Notchl on the surface of human umbilical vein endothelial cells (HUVEC) (ATCC Cat. No. CRL-1730), as determined by FACS analysis. To determine whether inhibition of Notchl signaling affects angiogenesis, these four antibodies were tested for promotion of endothelial cell branching morphogenesis. Matrigel™ was prepared in 24- well plates by adding 250 μΐ of growth factor reduced Matrigel (GFR; BD Bioscience Cat. No. 356231) to each well, and incubated for one hour at 37 °C. In parallel, HUVECs were washed in PBS, and resuspended in EGM-2 growth media (Lonza Cat. No. CC-3156) plus 2% FBS. Antibodies, human IgG, or positive controls were added to the media containing cells, and 40,000 cells per well were plated on polymerized GFR matrix. Branching morphogenesis was assessed at various time points, by image capture and analysis, using ImageJ public domain image processing software. All four antibodies promoted vascular branching morphogenesis resulting in increased branching of vessels and increased overall vascular area. In addition, quantitative RT-PCR analysis, as described in Example 7 (above), demonstrated that the antibodies inhibited Notchl signaling in endothelial cells as indicated by the down-regulated expression of Notch target genes, including Heyl and Hey2. Example 10: Inhibition of T-cell Fate Specification In Vivo

[0171] Antibodies 2G10, 2E6, 2A11 and 2D11 did not bind with high affinity to mouse Notchl. Therefore, to determine the effect of these Notchl antibodies on Notchl function in mice in vivo, the mouse Notchl gene was engineered to express a Notchl protein containing the human amino acid sequence from amino acid 413 to 488. No phenotypic difference was observed in these "humanized" Notchl mice. Importantly, the number and distribution of the thymocyte population in these animals was indistinguishable from wild-type mice. This indicated that the engineered Notchl protein was fully functional in the humanized mice.

[0172] Inhibition of thymocyte development and T-cell fate specification can be used as an indication that an anti-Notch 1 antibody is actually inhibiting Notchl function in vivo.

Therefore, antibody 2E6 was tested for inhibition of thymocyte development and T-cell fate specification in humanized Notchl mice. Mice (C57bl/6; 129Sv/Ev mixed background) homozygous for humanized Notch 1 gene were treated with the antibodies or IgG control at 40 mg/kg twice weekly, or the gamma secretase inhibitor DBZ at 10 μΜ/kg once daily, and monitored daily. After 18 days, the mice were sacrificed, thymus glands were removed, thymocytes were dissociated, and the total number of thymocytes was counted. FACS profiling was performed using antibodies against CD4 and CD8 (Beckton-Dickinson Cat. Nos. 553730 and 553031, respectively). The effect of the antibodies (or controls) on thymocyte number, and the distribution of CD4/CD8 double positive, double negative or single positive cells, were determined. Antibody 2E6 reduced the total number of thymocytes, and decreased the percent of CD4/CD8 double positive cells, while increasing the percentage of CD4 single positive, CD8 single positive, and CD4/CD8 double negative cells. These effects of antibody 2E6 were comparable to the effects observed when animals were treated with 10 μΜ/kg of the gamma-secretase inhibitor DBZ. This indicated that antibody 2E6 was inhibiting Notch 1 in vivo function in thymocyte development.

Example 11: Loss of Hair Pigmentation

[0173] Loss of hair pigmentation can be used as an indication that an anti-Notch 1 antibody is inhibiting Notch 1 function in vivo. Therefore, mice (C57bl/6; 129Sv/Ev mixed background) homozygous for humanized Notch 1 gene were treated with antibody 2E6, IgG control, or DBZ, as described in Example 10. Daily monitoring revealed loss of hair pigmentation in the antibody-treated mice within 2 weeks. No such loss of pigmentation was observed in IgG control treated mice.

Example 12: Lack of Toxicity

[0174] Mice were treated with antibody 2E6, IgG control, or DBZ, as described in Example 10 (FIG. 9A). Over time, DBZ treated animals exhibited loss of body weight, while animals treated with up to 150 mg/kg Notch 1 antibody three times per week exhibited normal weight gain (FIG. 9B). After 18 days, animals were sacrificed, small intestines were collected, fixed and embedded in paraffin. To observe goblet cells in the small intestine, sections of small intestine from antibody-treated, IgG-treated, and DBZ-treated animals were stained with Alcian Blue (Diagnostic Biosystems, Cat. No. KT 003). Mice treated with 40, 100 or 150 mg/kg of Notch 1 antibody 2E6 showed no increase in goblet cell numbers compared to control animals. By contrast, small intestines from animals treated with DBZ (10 μΜ/kg) showed extensive alcian blue staining. These results indicated that antibody 2E6 did not lead to goblet cell metaplasia, and had little or no intestinal toxicity in treated mice.

[0175] The results in FIG. 9A also demonstrate that antibody 2E6 at doses of 40, 100 or 150 mg/kg inhibit thymocyte development as effectively as 10 μπιοΐ/kg of DBZ. Therefore, as shown in FIGS. 9A-9B, and by alcian blue staining of the small intestine, antibody 2E6 does not have toxic effects (as measured by body weight loss and goblet cell conversion) at doses significantly higher than the dose required to inhibit thymocyte development.

Example 13: Inhibition of Angiogenesis In Vivo

[0176] The effect of antibody 2E6 on functional angiogenesis was determined using an in vivo matrigel plug assay. In this test, 400 ng/ml bFGF (R&D Systems) was prepared in 0.5 ml matrigel (Becton Dickinson). Mice were anaesthetized, and one or two matrigel plugs per animal were injected subcutaneously on either side on the ventral midline. Angiogenesis was allowed to proceed for 7 days, and the mice were treated intraperitoneally with 2-40 mg/kg Notch 1 antibody, or IgG control on day 0, and every 3 days thereafter. No significant loss of body weight was observed during these experiments. This indicated a lack of toxicity associated with treatment of the animals with 2-40 mg/kg of 2E6. Animals were sacrificed after 7 days. At this time, the plugs were removed, minced in water, and incubated overnight at 4°C. The following day, a standard curve of hemoglobin concentration was prepared using 180 mg/ml, 120 mg/ml, 60 mg/ml, 30 mg/ml, 15 mg/ml, 7.5 mg/ml and 0 mg/ml of hemoglobin in a 1: 1 mixture of water and Drabkin's reagent (Sigma- Aldrich, St. Louis, MO). Test samples were centrifuged to pellet matrigel and cells. A 225 μΐ sample of supernatant was removed, mixed with an equal volume of Drabkin's reagent, and incubated for 15 minutes at room temperature. Absorbance at 540 nm was read, and hemoglobin concentration was determined by comparison to a standard curve. Hemoglobin concentration was normalized for plug weight for each sample. The results of these experiments indicated that antibody 2E6 inhibited bFGF- induced angiogenesis in humanized (Notch l ^{hl l2/hl 12} knock-in) mice (FIG. 10). Dose response studies indicated that inhibition of bFGF-induced angiogenesis was inhibited at concentrations as low as 2 mg/kg.

[0177] To investigate the effect of antibody 2E6 on angiogenesis induced by human cancer cell lines in vivo, 0.5 - 1 x 10 ⁶ human cancer cells were prepared in 0.5 ml matrigel. Mice were anaesthetized, and one or two matrigel plugs per animal were injected subcutaneously on either side on the ventral midline. Angiogenesis was allowed to proceed for 7-12 days, and animals were treated intraperitoneally with 2-40 mg/kg antibody 2E6, or IgG control on day 0, and every 3 days thereafter. No significant loss of body weight was observed during these experiments. This indicated a lack of toxicity associated with treatment of the animals with 2- 40 mg/kg of 2E6. Animals were sacrificed after 10-12 days, depending on the cell line. Plugs were removed and processed (as described above), to determine hemoglobin concentration. The results of this experiment indicated that antibody 2E6 inhibited angiogenesis induced by human lung cancer (Calu-6), breast cancer (MDA-MB-231) or pancreatic cancer (SW1990) cell lines (FIG. 11).

[0178] The second matrigel plug from each mouse (as described in the preceding paragraph) was processed for histologic analysis, in parallel with the hemoglobin

measurements. Plugs were removed, fixed over night in 10% buffered formalin at room temperature, embedded in paraffin, and 10-20 um sections were prepared for

immunohistochemistry. To detect blood vessels present in the matrigel plug, thin sections were stained for CD31, using an anti-mouse-CD31 antibody (Biocare Medical, Cat. Nos. CM303 and RT517SK) according to the vendor's instructions. CD31 staining of the matrigel plugs demonstrated increased vessel branching and smaller vessels after treatment with antibody 2E6, by comparison to control IgG. These data demonstrated that antibody 2E6 promoted vascular branching in the treated mice. However, despite the increase in vascular branching, the decrease in blood content (as measured by hemoglobin content) suggests that antibody 2E6 decreased the function of the vessels that were present, and thus inhibited functional angiogenesis. This apparent decrease in functional angiogenesis caused by antibody 2E6 is consistent with increased branching and decreased vascular function resulting from genetic loss of function of the Notch pathway in endothelial tissue.

Example 14: Humanization of Anti-human Notchl Antibodies

A. Construction of Humanized and Chimeric Anti-Human Notchl Antibodies

[0179] This Example describes the humanization of the murine antibody designated 2E6, and the characterization of the resulting humanized antibodies. The humanized anti-Notch 1 antibodies were designed using methods well-known in the art. Two different humanized versions were made for each chain and a predicted N-linked glycosylation site in 2E6 heavy CDR2 was mutated to prevent any possible glycosylation. The designed amino acid sequences were converted to codon-optimized DNA sequences and synthesized by DNA2.0, Inc. to include (in the following order): 5' Hindlll restriction site, Kozak consensus sequence, amino terminal signal sequence, humanized variable region, human IgGl or Kappa constant region, stop codon, and a 3' EcoRI restriction site.

[0180] The anti-Notch 1 humanized antibody chains are designated with the prefix "Hu2E6_Hv" or "Hu2E6_Kv", referring to humanized 2E6 heavy or kappa light, respectively, and the designations are then followed by a numeric suffix (e.g., Hu2E6_Hvl, Hu2E6_Hv2, Hu2E6_Kvl, or Hu2E6_Kv2). In some cases, the designation is also followed by an amino acid substitution abbreviation (e.g., Hu2E6_Hvl T57A or Hu2E6_Hv2 T57A). Combinations of humanized heavy light chains are designated with the prefix "Hu2E6" and a numeric suffix.

[0181] Chimeric (murine variable region and human constant region) 2E6 heavy (human IgGl) and light (human Kappa) chains were also constructed. The murine variable regions were fused to the human constant region using overlap extension PCR, including (in the following order): 5' Hindlll restriction site, Kozak consensus sequence, amino terminal signal sequence, mouse variable region, human IgGl or Kappa constant region, stop codon, and 3' EcoRI restriction site.

[0182] The humanized and chimeric heavy chains were subcloned into pEE6.4 (Lonza, Basel, Switzerland) via Hindlll and EcoRI sites using In-Fusion™ PCR cloning (Clontech, Mountain View, CA). The humanized and chimeric Kappa light chains were subcloned into pEE14.4 (Lonza) via Hindlll and EcoRI sites using In-Fusion™ PCR cloning.

[0183] Humanized antibody chains or chimeric antibody chains were transiently transfected into 293T cells to produce antibody. Antibody was either purified or used in cell culture media supernatant for subsequent in vitro analysis. Binding of the chimeric and humanized antibodies to human Notch 1 was measured as described below. The results are summarized in Table 13.

[0184] Additionally, some humanized antibody heavy and light chain combinations were stably expressed in CHOK1SV cells using the GS System™ (Lonza Biologies) in order to produce large quantities of purified humanized antibody. A single expression vector was constructed by combining pEE6.4 and pEE14.4 based vectors. First, pEE6.4 containing full length humanized heavy chain cDNA was digested with Notl and Sail to isolate the hCMV- MIE promoter + full length humanized heavy chain cDNA + SV40 poly A fragment. This fragment was inserted into the pEE14.4 vector already containing full length humanized light chain cDNA via Notl/Sall sites, thus creating an expression vector that simultaneously expresses heavy and light chains. The combined heavy and light chain vector was linearized and transfected into CHOK1SV cells. Stable clones were selected in the presence of methionine sulfoximine.

[0185] Each of the possible combinations of the humanized immunoglobulin heavy chain and immunoglobulin light chain variable regions are set forth below in Table 7.

Table 7

I .idit Chai n Variable K ni on H aw Chain V ariable Region

Hu2E6_Kvl (SEQ ID NO 111) Hu2E6_Hvl (SEQ ID NO: 103)

Hu2E6_Kvl (SEQ ID NO 111) Hu2E6_Hvl T57A (SEQ ID NO: 105)

Hu2E6_Kvl (SEQ ID NO 111) Hu2E6_Hv2 (SEQ ID NO: 107)

Hu2E6_Kvl (SEQ ID NO 111) Hu2E6_Hv2 T57A (SEQ ID NO: 109)

Hu2E6_Kv2 (SEQ ID NO 113) Hu2E6_Hvl (SEQ ID NO: 103)

Hu2E6_Kv2 (SEQ ID NO 113) Hu2E6_Hvl T57A (SEQ ID NO: 105)

Hu2E6_Kv2 (SEQ ID NO 113) Hu2E6_Hv2 (SEQ ID NO: 107)

Hu2E6_Kv2 (SEQ ID NO 113) Hu2E6_Hv2 T57A (SEQ ID NO: 109)

[0186] The nucleic acid sequences encoding and the protein sequences defining variable regions of the humanized 2E6 antibodies are summarized below (amino terminal signal peptide sequences are not shown). CDR sequences (Kabat definition) are shown in bold and are underlined in the amino acid sequences.

[0187] Nucleic Acid Sequence Encoding the Hu2E6 Hyl Heavy Chain Variable Region (SEQ ID NO: 102)

1 gaagtgcagt tggtacaaag tggggccgaa gttgeaaage caggggcctc agtgaagatg

61 tettgeaagg ettceggata cacattcact tcatattgga tgcactgggt gaagcaagct 121 cccggccagg gtctggagtg gateggegea gtctacccta gaaacaacga taccacctat 181 aaccagaaat tcaagggcaa ggccaccctc accgctgaca ctagcacatc cacagcatac 241 atggagctgc gctctcttcg gagegacgat acagccgtct attactgtct gtatttcaat 301 tacaatttcg actactgggg acagggtact ctcctgaccg ttagttcc

[0188] Protein Sequence Defining the Hu2E6 Hyl Heavy Chain Variable Region (SEQ ID

NO: 103)

1 evqlvqsgae vakpgasvkm sckasgytft sywmhwvkqa pgqglewiga vyprnndtty 61 nqkfkgkatl tadtststay melrslrsdd tavyyclyfn ynfdywgqgt lltvss [0189] Nucleic Acid Sequence Encoding the Hu2E6 Hyl T57A Heavy Chain Variable Region (SEQ ID NO: 104)

1 gaagtgcagt tggtacaaag tggggccgaa gttgcaaagc caggggcctc agtgaagatg 61 tcttgcaagg cttccggata cacattcact tcatattgga tgcactgggt gaagcaagct 121 cccggccagg gtctggagtg gatcggcgca gtctacccta gaaacaacga tgccacctat 181 aaccagaaat tcaagggcaa ggccaccctc accgctgaca ctagcacatc cacagcatac 241 atggagctgc gctctcttcg gagcgacgat acagccgtct attactgtct gtatttcaat 301 tacaatttcg actactgggg acagggtact ctcctgaccg ttagttcc

[0190] Protein Sequence Defining the Hu2E6 Hyl T57A Heavy Chain Variable Region (SEQ ID NO: 105)

1 evqlvqsgae vakpgasvkm sckasgytft sywmhwvkqa pgqglewiga vyprnndaty 61 nqkfkgkatl tadtststay melrslrsdd tavyyclyfn ynfdywgqgt lltvss

[0191] Nucleic Acid Sequence Encoding the Hu2E6 Hv2 Heavy Chain Variable Region (SEQ ID NO: 106)

1 caggtgcagt tggtacaaag tggggccgaa gttaagaagc caggggcctc agtgaagatg

61 tcttgcaagg cttccggata cacattcact tcatattgga tgcactgggt gaggcaagct

121 cccggccagg gtctggagtg gatcggcgca gtctacccta gaaacaacga taccacctat

181 aaccagaaat tccagggcag ggccaccctc accgctgaca ctagcacatc cacagcatac

241 atggagctgc gctctcttcg gagcgacgat acagccgtct attactgtct gtatttcaat

301 tacaatttcg actactgggg acagggtact ctcctgaccg ttagttcc

[0192] Protein Sequence Defining the Hu2E6 Hv2 Heavy Chain Variable Region (SEQ ID NO: 107)

1 qvqlvqsgae vkkpgasvkm sckasgytft sywmhwvrqa pgqglewiga vyprnndtty 61 nqkfqgratl tadtststay melrslrsdd tavyyclyfn ynfdywgqgt lltvss

[0193] Nucleic Acid Sequence Encoding the Hu2E6 Hv2 T57A Heavy Chain Variable Region (SEQ ID NO: 108)

1 caggtgcagt tggtacaaag tggggccgaa gttaagaagc caggggcctc agtgaagatg 61 tcttgcaagg cttccggata cacattcact tcatattgga tgcactgggt gaggcaagct 121 cccggccagg gtctggagtg gatcggcgca gtctacccta gaaacaacga tgccacctat 181 aaccagaaat tccagggcag ggccaccctc accgctgaca ctagcacatc cacagcatac 241 atggagctgc gctctcttcg gagcgacgat acagccgtct attactgtct gtatttcaat 301 tacaatttcg actactgggg acagggtact ctcctgaccg ttagttcc

[0194] Protein Sequence Defining the Hu2E6 Hv2 T57A Heavy Chain Variable Region (SEQ ID NO: 109)

1 qvqlvqsgae vkkpgasvkm sckasgytft sywmhwvrqa pgqglewiga vyprnndaty 61 nqkfqgratl tadtststay melrslrsdd tavyyclyfn ynfdywgqgt lltvss [0195] Nucleic Acid Sequence Encoding the Hu2E6 Kyl Kappa Chain Variable Region (SEQ ID NO: 110)

1 gaaattgtcc tgacacagtc acccgcaaca atgtctgcct ctccaggcga gagagtcacc

61 atgtcttgca gggcttcctc ctctgtgagc tacatgcatt ggtaccagca aaagccaggt 121 cagtcccctc ggctgcttat ctatgacacc tccaaccgag cctctggagt tcccgcccac

181 ttcagcggca gcgggagtgg gacagattac actctgacca taagttcaat ggagcctgag

241 gactttgcaa cctattactg ccagcaatgg agcagttatc cctatacttt cggccaggga 301 accaaactcg aaatcaag [0196] Protein Sequence Defining the Hu2E6 Kyl Kappa Chain Variable Region (SEQ ID NO: 111)

1 eivltqspat msaspgervt mscrasssvs ymhwyqqkpg qsprlliydt snrasgvpah 61 fsgsgsgtdy tltissmepe dfatyycqqw ssypyt fgqg tkleik [0197] Nucleic Acid Sequence Encoding the Hu2E6 Kv2 Kappa Chain Variable Region (SEQ ID NO: 112)

1 gaaattgtcc tgacacagtc acccgcaaca ttgtctgcct ctccaggcga gagagtcacc

61 atgtcttgca gggcttcctc ctctgtgagc tacatgcatt ggtaccagca aaagccaggt

121 caggctcctc ggctgcttat ctatgacacc tccaaccgag ccactggagt tcccgccagg 181 ttcagcggca gcgggagtgg gacagattac actctgacca taagttcaat ggagcctgag

241 gactttgcaa cctattactg ccagcaatgg agcagttatc cctatacttt cggccaggga 301 accaaactcg aaatcaag

[0198] Protein Sequence Defining the Hu2E6 Kv2 Kappa Chain Variable Region (SEQ ID NO: 113)

1 eivltqspat lsaspgervt mscrasssvs ymhwyqqkpg qaprlliydt snratgvpar 61 fsgsgsgtdy tltissmepe dfatyycqqw ssypyt fgqg tkleik

[0199] The amino acid sequences defining the immunoglobulin heavy chain variable regions for the antibodies produced in Example 14 are aligned in FIG. 12. Amino terminal signal peptide sequences (for proper expression/secretion) are not shown. CDR _l5 CDR ₂ , and CDR ₃ (Kabat definition) are identified by boxes. FIG. 13 shows an alignment of the separate CDRi, CDR ₂ , and CDR ₃ sequences for each of the variable region sequences shown in FIG. 12.

[0200] The amino acid sequences defining the immunoglobulin light chain variable regions for the antibodies in Example 14 are aligned in FIG. 14. Amino terminal signal peptide sequences (for proper expression/secretion) are not shown. CDR _l5 CDR ₂ and CDR ₃ are identified by boxes. FIG. 15 shows an alignment of the separate CDR _l5 CDR ₂ , and CDR ₃ sequences for each of the variable region sequences shown in FIG. 14.

[0201] Table 8 is a concordance chart showing the SEQ ID NO. of each sequence discussed in this Example.

Table 8

u _ v g t appa an ₃

[0202] Humanized monoclonal antibody heavy chain CDR sequences (Kabat, Chothia, and IMGT definitions) are shown in Table 9. Table 9

[0203] Humanized monoclonal antibody Kappa light chain CDR sequences (Kabat, Chothia, and IMGT definitions) are shown in Table 10. Table 10

[0204] To create the complete chimeric and humanized heavy or kappa chain antibody sequences, each variable sequence above is combined with its respective human constant region. For example, a complete heavy chain comprises a heavy variable sequence followed by a human IgGl heavy chain constant sequence. A complete kappa chain comprises a kappa variable sequence followed by the human kappa light chain constant sequence.

[0205] Nucleic Acid Sequence Encoding the Human IgGl Heavy Chain Constant Region (SEQ ID NO: 114)

1 gcctcaacaa aaggaccaag tgtgttccca ctcgccccta gcagcaagag tacatccggg 61 ggcactgcag cactcggctg cctcgtcaag gattattttc cagagccagt aaccgtgagc 121 tggaacagtg gagcactcac ttctggtgtc catacttttc ctgctgtcct gcaaagctct 181 ggcctgtact cactcagctc cgtcgtgacc gtgccatctt catctctggg cactcagacc 241 tacatctgta atgtaaacca caagcctagc aatactaagg tcgataagcg ggtggaaccc 301 aagagctgcg acaagactca cacttgtccc ccatgccctg cccctgaact tctgggcggt 361 cccagcgtct ttttgttccc accaaagcct aaagatactc tgatgataag tagaacaccc 421 gaggtgacat gtgttgttgt agacgtttcc cacgaggacc cagaggttaa gttcaactgg 481 tacgttgatg gagtcgaagt acataatgct aagaccaagc ctagagagga gcagtataat 541 agtacatacc gtgtagtcag tgttctcaca gtgctgcacc aagactggct caacggcaaa 601 gaatacaaat gcaaagtgtc caacaaagca ctcccagccc ctatcgagaa gactattagt 661 aaggcaaagg ggcagcctcg tgaaccacag gtgtacactc tgccacccag tagagaggaa 721 atgacaaaga accaagtctc attgacctgc ctggtgaaag gcttctaccc cagcgacatc 781 gccgttgagt gggagagtaa cggtcagcct gagaacaatt acaagacaac ccccccagtg 841 ctggatagtg acgggtcttt ctttctgtac agtaagctga ctgtggacaa gtcccgctgg 901 cagcagggta acgtcttcag ctgttccgtg atgcacgagg cattgcacaa ccactacacc 961 cagaagtcac tgagcctgag cccagggaag [0206] Protein Sequence Defining the Human IgGl Heavy Chain Constant Region (SEQ ID NO: 115)

1 astkgpsvfp lapsskstsg gtaalgclvk dyfpepvtvs wnsgaltsgv htfpavlqss

61 glyslss vt vpssslgtqt yicnvnhkps ntkvdkrvep kscdkthtcp pcpapellgg 121 psvflfppkp kdtlmisrtp evtcv vdvs hedpevkfnw yvdgvevhna ktkpreeqyn

181 styr vsvlt vlhqdwlngk eykckvsnka lpapiektis kakgqprepq vytlppsree

241 mtknqvsltc lvkgfypsdi avewesngqp ennykttppv ldsdgsffly skltvdksrw 301 qqgnvfscsv mhealhnhyt qkslslspgk [0207] Nucleic Acid Sequence Encoding the Human Kappa Light Chain Constant Region (used for chimeric antibodies) (SEQ ID NO: 116)

1 cgcacagtcg ccgctccctc cgtgttcatc tttccaccaa gtgatgagca actgaagtct 61 ggtactgctt cagtcgtgtg tctgctgaac aatttctacc ctcgagaagc caaagtccaa 121 tggaaggtag acaacgcact gcagtccggc aatagccaag aatcagttac cgaacaggat 181 tcaaaggaca gtacatattc cctgagcagc actctgaccc tgtcaaaggc cgattacgag

241 aaacacaagg tctatgcttg cgaagtgaca catcagggac tgtccagccc agtgacaaaa 301 tcttttaacc gtggggagtg t

[0208] Nucleic Acid Sequence Encoding the Human Kappa Light Chain Constant Region (used for humanized antibodies) (SEQ ID NO: 117)

1 cgcacagttg ctgcccccag cgtgttcatt ttcccaccta gcgatgagca gctgaaaagc 61 ggtactgcct ctgtcgtatg cttgctcaac aacttttacc cacgtgaggc taaggtgcag 121 tggaaagtgg ataatgcact tcaatctgga aacagtcaag agtccgtgac agaacaggac 181 agcaaagact caacttattc actctcttcc accctgactc tgtccaaggc agactatgaa 241 aaacacaagg tatacgcctg cgaggttaca caccagggtt tgtctagtcc tgtcaccaag

301 tccttcaata ggggcgaatg t

[0209] Protein Sequence Defining the Human Kappa Light Chain Constant Region (used for chimeric and humanized antibodies) (SEQ ID NO: 118)

1 rtvaapsvfi fppsdeqlks gtas vclln nfypreakvq wkvdnalqsg nsqesvteqd

61 skdstyslss tltlskadye khkvyacevt hqglsspvtk sfnrgec

[0210] The following sequences represent the actual or contemplated full length heavy and light chain sequence (i.e., containing both the variable and constant regions sequences) for each antibody described in this Example. Signal sequences for proper secretion of the antibodies (e.g., signal sequences at the 5' end of the DNA sequences or the amino terminal end of the protein sequences) are not shown in the full length heavy and light chain sequences disclosed herein and are not included in the final secreted protein. Also not shown are stop codons for termination of translation required at the 3' end of the DNA sequences. It is within ordinary skill in the art to select a signal sequence and/or a stop codon for expression of the disclosed full length IgG heavy chain and light chain sequences. It is also contemplated that the variable region sequences can be ligated to other constant region sequences to produce active full length IgG heavy and light chains.

[0211] Nucleic Acid Sequence Encoding the Full Length Chimeric 2E6 Heavy Chain (Mouse Heavy Chain Variable Region and Human IgGl Constant Region) (SEQ ID NO: 119)

1 gaggttcagc tccagcagtc tggggctgag ctggcaagac ctggggcttc agtgaagatg 61 tcctgcaagg cttctggcta cacctttacc agctactgga tgcactgggt aaaacagagg 121 cctggacagg gtctggaatg gattggcgct gtttatccta gaaacaatga tactacttac 181 aatcagaagt tcaagggcaa ggccaagctg actgctgtca catccgccag cactgcctac 241 atggcactca gcagcctaac aaatgaggac tctgcggtct attactgtct ttattttaac 301 tacaactttg actactgggg ccaaggcacc actctcacag tctcctcagc ctcaacaaaa 361 ggaccaagtg tgttcccact cgcccctagc agcaagagta catccggggg cactgcagca 421 ctcggctgcc tcgtcaagga ttattttcca gagccagtaa ccgtgagctg gaacagtgga 481 gcactcactt ctggtgtcca tacttttcct gctgtcctgc aaagctctgg cctgtactca 541 ctcagctccg tcgtgaccgt gccatcttca tctctgggca ctcagaccta catctgtaat 601 gtaaaccaca agcctagcaa tactaaggtc gataagcggg tggaacccaa gagctgcgac 661 aagactcaca cttgtccccc atgccctgcc cctgaacttc tgggcggtcc cagcgtcttt 721 ttgttcccac caaagcctaa agatactctg atgataagta gaacacccga ggtgacatgt 781 gttgttgtag acgtttccca cgaggaccca gaggttaagt tcaactggta cgttgatgga 841 gtcgaagtac ataatgctaa gaccaagcct agagaggagc agtataatag tacataccgt 901 gtagtcagtg ttctcacagt gctgcaccaa gactggctca acggcaaaga atacaaatgc 961 aaagtgtcca acaaagcact cccagcccct atcgagaaga ctattagtaa ggcaaagggg 1021 cagcctcgtg aaccacaggt gtacactctg ccacccagta gagaggaaat gacaaagaac 1081 caagtctcat tgacctgcct ggtgaaaggc ttctacccca gcgacatcgc cgttgagtgg 1141 gagagtaacg gtcagcctga gaacaattac aagacaaccc ccccagtgct ggatagtgac 1201 gggtctttct ttctgtacag taagctgact gtggacaagt cccgctggca gcagggtaac 1261 gtcttcagct gttccgtgat gcacgaggca ttgcacaacc actacaccca gaagtcactg 1321 agcctgagcc cagggaag

[0212] Protein Sequence Defining the Full Length Chimeric 2E6 Heavy Chain (Mouse

Heavy Chain Variable Region and Human IgGl Constant Region) (SEQ ID NO: 120)

1 evqlqqsgae larpgasvkm sckasgytft sywmhwvkqr pgqglewiga vyprnndtty

61 nqkfkgkakl tavtsastay malssltned savyyclyfn ynfdywgqgt tltvssastk

121 gpsvfplaps skstsggtaa lgclvkdyfp epvtvswnsg altsgvhtfp avlqssglys

181 lssvvtvpss slgtqtyicn vnhkpsntkv dkrvepkscd kthtcppcpa pellggpsvf

241 lfppkpkdtl misrtpevtc vvdvshedp evkfnwyvdg vevhnaktkp reeqynstyr

301 vsvltvlhq dwlngkeykc kvsnkalpap iektiskakg qprepqvytl ppsreemtkn

361 qvsltclvkg fypsdiavew esngqpenny kttppvldsd gsfflysklt vdksrwqqgn

421 vfscsvmhea lhnhytqksl slspgk

[0213] Nucleic Acid Sequence Encoding the Full Length Chimeric 2E6 Light Chain (Mouse Kappa Chain Variable Region and Human Kappa Constant Region) (SEQ ID NO: 121)

1 caaattgttc tcacccagtc tccagcaatc atgtctgctt ctccagggga gaaggtcacc

61 atgacctgca gtgccagctc aagtgtaagt tacatgcact ggtaccagca gaagccagga

121 tcctccccca gactcctgat ttatgacaca tccaacctgg cttctggagt ccctgtgcac

181 ttcagtggca gtgggtctgg gacctcttac tctctcacaa tcatccgaat ggaggctgaa

241 gatgctgcca cttattactg ccagcagtgg agtagttacc cgtacacgtt cggagggggg

301 accaagctgg aaataaaacg cacagtcgcc gctccctccg tgttcatctt tccaccaagt

361 gatgagcaac tgaagtctgg tactgcttca gtcgtgtgtc tgctgaacaa tttctaccct

421 cgagaagcca aagtccaatg gaaggtagac aacgcactgc agtccggcaa tagccaagaa 481 tcagttaccg aacaggattc aaaggacagt acatattccc tgagcagcac tctgaccctg 541 tcaaaggccg attacgagaa acacaaggtc tatgcttgcg aagtgacaca tcagggactg 601 tccagcccag tgacaaaatc ttttaaccgt ggggagtgt

[0214] Protein Sequence Defining the Full Length Chimeric 2E6 Light Chain (Mouse

Kappa Chain Variable Region and Human Kappa Constant Region) (SEQ ID NO: 122)

1 qivltqspai msaspgekvt mtcsasssvs ymhwyqqkpg ssprlliydt snlasgvpvh 61 fsgsgsgtsy sltiirmeae daatyycqqw ssypytfggg tkleikrtva apsvfifpps 121 deqlksgtas vcllnnfyp reakvqwkvd nalqsgnsqe svteqdskds tyslsstltl 181 skadyekhkv yacevthqgl sspvtksfnr gee

[0215] Nucleic Acid Sequence Encoding the Full Length Humanized Hu2E6 Hyl Heavy Chain (Humanized Heavy Chain Variable Region and Human IgGl Constant Region) (SEQ ID NO: 123)

1 gaagtgcagt tggtacaaag tggggccgaa gttgeaaage caggggcctc agtgaagatg 61 tettgeaagg ettceggata cacattcact tcatattgga tgcactgggt gaagcaagct 121 cccggccagg gtctggagtg gateggegea gtctacccta gaaacaacga taccacctat 181 aaccagaaat tcaagggcaa ggccaccctc accgctgaca ctagcacatc cacagcatac 241 atggagctgc gctctcttcg gagegacgat acagccgtct attactgtct gtatttcaat 301 tacaatttcg actactgggg acagggtact ctcctgaccg ttagttccgc ctcaacaaaa 361 ggaccaagtg tgttcccact cgcccctagc agcaagagta catceggggg cactgcagca 421 ctcggctgcc tegtcaagga ttattttcca gagecagtaa ccgtgagctg gaacagtgga 481 gcactcactt ctggtgtcca tacttttcct gctgtcctgc aaagctctgg cctgtactca 541 ctcagctccg tcgtgaccgt gccatcttca tctctgggca ctcagaccta catctgtaat 601 gtaaaccaca agectagcaa tactaaggtc gataageggg tggaacccaa gagctgegae 661 aagactcaca cttgtccccc atgccctgcc cctgaacttc tgggeggtec cagegtcttt 721 ttgttcccac caaagectaa agatactctg atgataagta gaacacccga ggtgacatgt 781 gttgttgtag acgtttccca cgaggaccca gaggttaagt tcaactggta cgttgatgga 841 gtcgaagtac ataatgetaa gaccaagcct agagaggagc agtataatag tacataccgt 901 gtagtcagtg ttctcacagt gctgcaccaa gactggctca aeggcaaaga atacaaatgc 961 aaagtgtcca acaaagcact cccagcccct atcgagaaga ctattagtaa ggcaaagggg 1021 cagcctcgtg aaccacaggt gtacactctg ccacccagta gagaggaaat gacaaagaac 1081 caagtctcat tgacctgcct ggtgaaaggc ttctacccca gcgacatcgc cgttgagtgg 1141 gagagtaacg gtcagcctga gaacaattac aagacaaccc ccccagtgct ggatagtgac 1201 gggtctttct ttctgtacag taagctgact gtggacaagt cccgctggca gcagggtaac 1261 gtcttcagct gttccgtgat gcacgaggca ttgcacaacc actacaccca gaagtcactg 1321 agcctgagcc cagggaag

[0216] Protein Sequence Defining the Full Length Humanized Hu2E6 Hyl Heavy Chain (Humanized Heavy Chain Variable Region and Human IgGl Constant Region) (SEQ ID NO: 124)

1 evqlvqsgae vakpgasvkm sckasgytft sywmhwvkqa pgqglewiga vyprnndtty 61 nqkfkgkatl tadtststay melrslrsdd tavyyclyfn ynfdywgqgt lltvssastk 121 gpsvfplaps skstsggtaa lgclvkdyfp epvtvswnsg altsgvhtfp avlqssglys 181 lssvvtvpss slgtqtyicn vnhkpsntkv dkrvepkscd kthtcppcpa pellggpsvf 241 lfppkpkdtl misrtpevtc vvdvshedp evkfnwyvdg vevhnaktkp reeqynstyr 301 vsvltvlhq dwlngkeykc kvsnkalpap iektiskakg qprepqvytl ppsreemtkn 361 qvsltclvkg fypsdiavew esngqpenny kttppvldsd gsfflysklt vdksrwqqgn 421 vfscsvmhea lhnhytqksl slspgk [0217] Nucleic Acid Sequence Encoding the Full Length Humanized Hu2E6 Hyl T57A Heavy Chain (Humanized Heavy Chain Variable Region and Human IgGl Constant Region) (SEQ ID NO: 125)

1 gaagtgcagt tggtacaaag tggggccgaa gttgcaaagc caggggcctc agtgaagatg 61 tcttgcaagg cttccggata cacattcact tcatattgga tgcactgggt gaagcaagct 121 cccggccagg gtctggagtg gatcggcgca gtctacccta gaaacaacga tgccacctat 181 aaccagaaat tcaagggcaa ggccaccctc accgctgaca ctagcacatc cacagcatac 241 atggagctgc gctctcttcg gagcgacgat acagccgtct attactgtct gtatttcaat 301 tacaatttcg actactgggg acagggtact ctcctgaccg ttagttccgc ctcaacaaaa 361 ggaccaagtg tgttcccact cgcccctagc agcaagagta catccggggg cactgcagca 421 ctcggctgcc tcgtcaagga ttattttcca gagccagtaa ccgtgagctg gaacagtgga 481 gcactcactt ctggtgtcca tacttttcct gctgtcctgc aaagctctgg cctgtactca 541 ctcagctccg tcgtgaccgt gccatcttca tctctgggca ctcagaccta catctgtaat 601 gtaaaccaca agcctagcaa tactaaggtc gataagcggg tggaacccaa gagctgcgac 661 aagactcaca cttgtccccc atgccctgcc cctgaacttc tgggcggtcc cagcgtcttt 721 ttgttcccac caaagcctaa agatactctg atgataagta gaacacccga ggtgacatgt 781 gttgttgtag acgtttccca cgaggaccca gaggttaagt tcaactggta cgttgatgga 841 gtcgaagtac ataatgctaa gaccaagcct agagaggagc agtataatag tacataccgt 901 gtagtcagtg ttctcacagt gctgcaccaa gactggctca acggcaaaga atacaaatgc 961 aaagtgtcca acaaagcact cccagcccct atcgagaaga ctattagtaa ggcaaagggg 1021 cagcctcgtg aaccacaggt gtacactctg ccacccagta gagaggaaat gacaaagaac 1081 caagtctcat tgacctgcct ggtgaaaggc ttctacccca gcgacatcgc cgttgagtgg 1141 gagagtaacg gtcagcctga gaacaattac aagacaaccc ccccagtgct ggatagtgac 1201 gggtctttct ttctgtacag taagctgact gtggacaagt cccgctggca gcagggtaac 1261 gtcttcagct gttccgtgat gcacgaggca ttgcacaacc actacaccca gaagtcactg 1321 agcctgagcc cagggaag

[0218] Protein Sequence Defining the Full Length Humanized Hu2E6 Hyl T57A Heavy Chain (Humanized Heavy Chain Variable Region and Human IgGl Constant Region) (SEQ ID NO: 126)

1 evqlvqsgae vakpgasvkm sckasgytft sywmhwvkqa pgqglewiga vyprnndaty 61 nqkfkgkatl tadtststay melrslrsdd tavyyclyfn ynfdywgqgt lltvssastk 121 gpsvfplaps skstsggtaa lgclvkdyfp epvtvswnsg altsgvhtfp avlqssglys 181 lssvvtvpss slgtqtyicn vnhkpsntkv dkrvepkscd kthtcppcpa pellggpsvf 241 lfppkpkdtl misrtpevtc vvdvshedp evkfnwyvdg vevhnaktkp reeqynstyr 301 vsvltvlhq dwlngkeykc kvsnkalpap iektiskakg qprepqvytl ppsreemtkn 361 qvsltclvkg fypsdiavew esngqpenny kttppvldsd gsfflysklt vdksrwqqgn 421 vfscsvmhea lhnhytqksl slspgk

[0219] Nucleic Acid Sequence Encoding the Full Length Humanized Hu2E6 Hv2 Heavy Chain (Humanized Heavy Chain Variable Region and Human IgGl Constant Region) (SEQ ID NO: 127)

1 caggtgcagt tggtacaaag tggggccgaa gttaagaagc caggggcctc agtgaagatg

61 tcttgcaagg cttccggata cacattcact tcatattgga tgcactgggt gaggcaagct

121 cccggccagg gtctggagtg gatcggcgca gtctacccta gaaacaacga taccacctat

181 aaccagaaat tccagggcag ggccaccctc accgctgaca ctagcacatc cacagcatac

241 atggagctgc gctctcttcg gagcgacgat acagccgtct attactgtct gtatttcaat 301 tacaatttcg actactgggg acagggtact ctcctgaccg ttagttccgc ctcaacaaaa 361 ggaccaagtg tgttcccact cgcccctagc agcaagagta catccggggg cactgcagca 421 ctcggctgcc tcgtcaagga ttattttcca gagccagtaa ccgtgagctg gaacagtgga 481 gcactcactt ctggtgtcca tacttttcct gctgtcctgc aaagctctgg cctgtactca 541 ctcagctccg tcgtgaccgt gccatcttca tctctgggca ctcagaccta catctgtaat 601 gtaaaccaca agcctagcaa tactaaggtc gataagcggg tggaacccaa gagctgcgac 661 aagactcaca cttgtccccc atgccctgcc cctgaacttc tgggcggtcc cagcgtcttt 721 ttgttcccac caaagcctaa agatactctg atgataagta gaacacccga ggtgacatgt 781 gttgttgtag acgtttccca cgaggaccca gaggttaagt tcaactggta cgttgatgga 841 gtcgaagtac ataatgctaa gaccaagcct agagaggagc agtataatag tacataccgt 901 gtagtcagtg ttctcacagt gctgcaccaa gactggctca acggcaaaga atacaaatgc 961 aaagtgtcca acaaagcact cccagcccct atcgagaaga ctattagtaa ggcaaagggg 1021 cagcctcgtg aaccacaggt gtacactctg ccacccagta gagaggaaat gacaaagaac 1081 caagtctcat tgacctgcct ggtgaaaggc ttctacccca gcgacatcgc cgttgagtgg 1141 gagagtaacg gtcagcctga gaacaattac aagacaaccc ccccagtgct ggatagtgac 1201 gggtctttct ttctgtacag taagctgact gtggacaagt cccgctggca gcagggtaac 1261 gtcttcagct gttccgtgat gcacgaggca ttgcacaacc actacaccca gaagtcactg 1321 agcctgagcc cagggaag

[0220] Protein Sequence Defining the Full Length Humanized Hu2E6 Hv2 Heavy Chain (Humanized Heavy Chain Variable Region and Human IgGl Constant Region) (SEQ ID NO: 128)

1 qvqlvqsgae vkkpgasvkm sckasgytft sywmhwvrqa pgqglewiga vyprnndtty 61 nqkfqgratl tadtststay melrslrsdd tavyyclyfn ynfdywgqgt lltvssastk 121 gpsvfplaps skstsggtaa lgclvkdyfp epvtvswnsg altsgvhtfp avlqssglys 181 lssvvtvpss slgtqtyicn vnhkpsntkv dkrvepkscd kthtcppcpa pellggpsvf 241 lfppkpkdtl misrtpevtc vvdvshedp evkfnwyvdg vevhnaktkp reeqynstyr 301 vsvltvlhq dwlngkeykc kvsnkalpap iektiskakg qprepqvytl ppsreemtkn 361 qvsltclvkg fypsdiavew esngqpenny kttppvldsd gsfflysklt vdksrwqqgn 421 vfscsvmhea lhnhytqksl slspgk

[0221] Nucleic Acid Sequence Encoding the Full Length Humanized Hu2E6 Hv2 T57A Heavy Chain (Humanized Heavy Chain Variable Region and Human IgGl Constant Region) (SEQ ID NO: 129)

1 caggtgcagt tggtacaaag tggggccgaa gttaagaagc caggggcctc agtgaagatg 61 tcttgcaagg cttccggata cacattcact tcatattgga tgcactgggt gaggcaagct 121 cccggccagg gtctggagtg gatcggcgca gtctacccta gaaacaacga tgccacctat 181 aaccagaaat tccagggcag ggccaccctc accgctgaca ctagcacatc cacagcatac 241 atggagctgc gctctcttcg gagcgacgat acagccgtct attactgtct gtatttcaat 301 tacaatttcg actactgggg acagggtact ctcctgaccg ttagttccgc ctcaacaaaa 361 ggaccaagtg tgttcccact cgcccctagc agcaagagta catccggggg cactgcagca 421 ctcggctgcc tcgtcaagga ttattttcca gagccagtaa ccgtgagctg gaacagtgga 481 gcactcactt ctggtgtcca tacttttcct gctgtcctgc aaagctctgg cctgtactca 541 ctcagctccg tcgtgaccgt gccatcttca tctctgggca ctcagaccta catctgtaat 601 gtaaaccaca agcctagcaa tactaaggtc gataagcggg tggaacccaa gagctgcgac 661 aagactcaca cttgtccccc atgccctgcc cctgaacttc tgggcggtcc cagcgtcttt 721 ttgttcccac caaagcctaa agatactctg atgataagta gaacacccga ggtgacatgt 781 gttgttgtag acgtttccca cgaggaccca gaggttaagt tcaactggta cgttgatgga 841 gtcgaagtac ataatgctaa gaccaagcct agagaggagc agtataatag tacataccgt 901 gtagtcagtg ttctcacagt gctgcaccaa gactggctca acggcaaaga atacaaatgc 961 aaagtgtcca acaaagcact cccagcccct atcgagaaga ctattagtaa ggcaaagggg 1021 cagcctcgtg aaccacaggt gtacactctg ccacccagta gagaggaaat gacaaagaac 1081 caagtctcat tgacctgcct ggtgaaaggc ttctacccca gcgacatcgc cgttgagtgg 1141 gagagtaacg gtcagcctga gaacaattac aagacaaccc ccccagtgct ggatagtgac 1201 gggtctttct ttctgtacag taagctgact gtggacaagt cccgctggca gcagggtaac 1261 gtcttcagct gttccgtgat gcacgaggca ttgcacaacc actacaccca gaagtcactg 1321 agcctgagcc cagggaag

[0222] Protein Sequence Defining the Full Length Humanized Hu2E6 Hv2 T57A Heavy Chain (Humanized Heavy Chain Variable Region and Human IgGl Constant Region) (SEQ ID NO: 130)

1 qvqlvqsgae vkkpgasvkm sckasgytft sywmhwvrqa pgqglewiga vyprnndaty

61 nqkfqgratl tadtststay melrslrsdd tavyyclyfn ynfdywgqgt lltvssastk

121 gpsvfplaps skstsggtaa lgclvkdyfp epvtvswnsg altsgvhtfp avlqssglys

181 lssvvtvpss slgtqtyicn vnhkpsntkv dkrvepkscd kthtcppcpa pellggpsvf

241 lfppkpkdtl misrtpevtc vvdvshedp evkfnwyvdg vevhnaktkp reeqynstyr

301 vsvltvlhq dwlngkeykc kvsnkalpap iektiskakg qprepqvytl ppsreemtkn

361 qvsltclvkg fypsdiavew esngqpenny kttppvldsd gsfflysklt vdksrwqqgn

421 vfscsvmhea lhnhytqksl slspgk

[0223] Nucleic Acid Sequence Encoding the Full Length Humanized Hu2E6 Kyl Light Chain (Humanized Kappa Chain Variable Region and Human Constant Region) (SEQ ID NO:

131)

1 gaaattgtcc tgacacagtc acccgcaaca atgtctgcct ctccaggcga gagagtcacc

61 atgtcttgca gggcttcctc ctctgtgagc tacatgcatt ggtaccagca aaagccaggt

121 cagtcccctc ggctgcttat ctatgacacc tccaaccgag cctctggagt tcccgcccac

181 ttcagcggca gcgggagtgg gacagattac actctgacca taagttcaat ggagcctgag

241 gactttgcaa cctattactg ccagcaatgg agcagttatc cctatacttt cggccaggga

301 accaaactcg aaatcaagcg cacagttgct gcccccagcg tgttcatttt cccacctagc

361 gatgagcagc tgaaaagcgg tactgcctct gtcgtatgct tgctcaacaa cttttaccca

421 cgtgaggcta aggtgcagtg gaaagtggat aatgcacttc aatctggaaa cagtcaagag

481 tccgtgacag aacaggacag caaagactca acttattcac tctcttccac cctgactctg

541 tccaaggcag actatgaaaa acacaaggta tacgcctgcg aggttacaca ccagggtttg

601 tctagtcctg tcaccaagtc cttcaatagg ggcgaatgt

[0224] Protein Sequence Defining the Full Length Humanized Hu2E6 Kyl Light Chain (Humanized Kappa Chain Variable Region and Human Constant Region) (SEQ ID NO: 132)

1 eivltqspat msaspgervt mscrasssvs ymhwyqqkpg qsprlliydt snrasgvpah 61 fsgsgsgtdy tltissmepe dfatyycqqw ssypytfgqg tkleikrtva apsvfifpps 121 deqlksgtas vcllnnfyp reakvqwkvd nalqsgnsqe svteqdskds tyslsstltl 181 skadyekhkv yacevthqgl sspvtksfnr gee

[0225] Nucleic Acid Sequence Encoding the Full Length Humanized Hu2E6 Kv2 Light Chain (Humanized Kappa Chain Variable Region and Human Constant Region) (SEQ ID NO: 133)

1 gaaattgtcc tgacacagtc acccgcaaca ttgtctgcct ctccaggcga gagagtcacc 61 atgtcttgca gggcttcctc ctctgtgagc tacatgcatt ggtaccagca aaagccaggt

121 caggctcctc ggctgcttat ctatgacacc tccaaccgag ccactggagt tcccgccagg

181 ttcagcggca gcgggagtgg gacagattac actctgacca taagttcaat ggagcctgag

241 gactttgcaa cctattactg ccagcaatgg agcagttatc cctatacttt cggccaggga

301 accaaactcg aaatcaagcg cacagttgct gcccccagcg tgttcatttt cccacctagc

361 gatgagcagc tgaaaagcgg tactgcctct gtcgtatgct tgctcaacaa cttttaccca

421 cgtgaggcta aggtgcagtg gaaagtggat aatgcacttc aatctggaaa cagtcaagag

481 tccgtgacag aacaggacag caaagactca acttattcac tctcttccac cctgactctg

541 tccaaggcag actatgaaaa acacaaggta tacgcctgcg aggttacaca ccagggtttg

601 tctagtcctg tcaccaagtc cttcaatagg ggcgaatgt

[0226] Protein Sequence Defining the Full Length Humanized Hu2E6 Kv2 Light Chain (Humanized Kappa Chain Variable Region and Human Constant Region) (SEQ ID NO: 134)

1 eivltqspat lsaspgervt mscrasssvs ymhwyqqkpg qaprlliydt snratgvpar 61 fsgsgsgtdy tltissmepe dfatyycqqw ssypytfgqg tkleikrtva apsvfifpps

121 deqlksgtas vcllnnfyp reakvqwkvd nalqsgnsqe svteqdskds tyslsstltl 181 skadyekhkv yacevthqgl sspvtksfnr gee

[0227] For convenience, Table 11 provides a concordance chart showing the SEQ ID NO. of each sequence discussed in this Example.

Table 11

uman ze J u _ v uman ar a e + uman appa constant- -prote n

[0228] Table 12 below shows antibodies containing chimeric immunoglobulin heavy and light chains and each of the possible combinations of the full-length humanized

immunoglobulin heavy and light chains.

Table 12

[0229] The antibody construct containing the full length chimeric heavy and light chains is designated below:

Chimeric 2E6 (also referred to as Hu2E6-l) = Full Length Chimeric 2E6 Heavy Chain (Mouse Variable Region and Human IgGl Constant Region) (SEQ ID NO: 120) plus Full Length Chimeric 2E6 Light Chain (Mouse Variable Region and Human Kappa Constant Region) (SEQ ID NO: 122) [0230] Two of the possible antibody constructs containing the full length immunoglobulin heavy and light chains containing humanized variable regions are designated below:

Hu2E6-62 = Humanized Hu2E6_Hvl T57A Heavy Chain Variable Region and Human IgGl Constant Region (SEQ ID NO: 126) plus Hu2E6_Kvl Light Chain Variable Region and Human Kappa Constant Region (SEQ ID NO: 132)

Hu2E6-74 = Humanized Hu2E6_Hvl T57A Heavy Chain Variable Region and Human IgGl Constant Region (SEQ ID NO: 126) plus Hu2E6_Kv2 Light Chain Variable Region and Human Kappa Constant Region (SEQ ID NO: 134)

B, Binding Affinities of Humanized and Chimeric Anti-NOTCHl Monoclonal

Antibodies

[0231] The binding affinities and kinetics of interaction of monoclonal antibodies produced in Example 14 against recombinant human Notch 1 (monomeric protein containing EGF-Like repeats 1-13) were measured by surface plasmon resonance using a Biacore T100 (Biacore (GE Healthcare), Piscataway, NJ) instrument.

[0232] Goat anti-human IgG Fc (Jackson ImmunoResearch, Catalog No. 109-005-098) was immobilized on carboxymethylated dextran CM4 sensor chips (Biacore, Catalog No. BR- 1005-34) by amine coupling (Biacore, Catalog No. BR- 1000-50) using a standard coupling protocol according to the vendor's instructions. The analyses were performed at 37°C using PBS (Invitrogen, Catalog No. 14040-133) containing 0.05% surfactant P20 (Biacore, Catalog No. BR- 1000-54) as running buffer.

[0233] The antibodies were captured in individual flow cells at a flow rate of 10 μΐ/minute. Injection time was varied for each antibody to yield an R _max between 30 and 60 RU. Buffer or recombinant human Notch 1 monomer diluted in running buffer was injected sequentially over a reference surface (no antibody captured) and the active surface (antibody to be tested) for 240 sec at 60 μΐ/minute. The dissociation phase was monitored for up to 1200 sec. The surface was then regenerated with two 60 second injections of Glycine pH 2.25 (made from Glycine pH 2.0 (Biacore, Catalog No. BR- 1003-55) and pH 2.5 (Biacore, Catalog No. BR- 1003-56) at 30 μΐ/minute. Concentrations of recombinant human Notchl tested were between 30 nM and 3.75 nM (a 2-fold serial dilution) (results are summarized in Table 13). [0234] Kinetic parameters were determined using the kinetic function of the BIAevaluation software (Biacore) with double reference subtraction. Kinetic parameters for each antibody, k _a (association rate constant), k _d (dissociation rate constant) and K _D (equilibrium dissociation constant) were determined. The kinetic values of the monoclonal antibodies on recombinant human Notch 1 at 25 °C are summarized in Tables 13.

Table 13

[0235] The results in Table 13 demonstrate that the purified antibodies have affinities ranging from about 2.7 nM to about 8.0 nM when test at 25°C.

[0236] The kinetic values of the monoclonal antibodies on recombinant human Notch 1 at 37°C are summarized in Table 14.

Table 14

[0237] The results in Table 14 demonstrate that the purified antibodies have affinities ranging from about 6.7 nM to about 12 nM when test at 37°C.

[0238] The results in Table 13 and 14 demonstrate that the chimeric and each of the humanized 2E6 antibodies have fast association rates (k _a ), very slow disassociation rates (k _d ) and very high affinities (K _D ). The affinity of humanized variants (e.g., Hu2E6-56, Hu2E6-57, Hu2E6-58, Hu2E6-62, and Hu2E6-74) for monomeric Notch 1 is consistently better than the affinity of chimeric 2E6 (Hu2E6-l). Overall, these results show that the K _D of the humanized antibodies (e.g., Hu2E6-56, Hu2E6-57, Hu2E6-58, Hu2E6-62, and Hu2E6-74) was smaller (i.e., higher affinity) than the K _D for Hu2E6-l (chimeric 2E6).

C Comparison with Another Notch 1 Antibody

[0239] A2-NRR1, as disclosed in Wu et al, (2010) NATURE 464: 1052-57, is an antibody known to inhibit the function of human Notch 1. The binding specificities of antibodies mu2E6, Hu2E6-62, and A2-NRR1 against human Notch 1 protein expressed on the surface of the T- ALL cell line Karpas45s were measured as described above (See Example 4). Results are summarized in Table 15.

Table 15

[0240] The results in Table 15 demonstrate that Hu2E6-62 and A2-NRR1 have similar binding specificities for human Notch 1 protein. Further, both antibodies exhibited higher specificities than mu2E6.

Example 15: Inhibition of Notchl-Ligand Binding

[0241] Antibodies mu2E6 and Hu2E6-62 were tested for their ability to inhibit the binding of rhNotchl-Fc to human Jagl, Jag2, DLLl and DLL4. Binding measurements were made by bio-layer interferometry (BLI), using a ForteBio Octet ^® QK instrument as described in

Example 5. The ligands tested were rhJagl-Fc (R&D Cat. No. 1277-JG-050), rhJag2-Fc (R&D Cat. No. 1726-JG-050), rhDLLl-Fc (R&D Cat. No. 5026-DL-050), and His tagged rhDLL4 (R&D Cat. No. 1506-D4-050). The inhibitory activities of the antibodies on Notch 1-ligand binding are summarized in Table 16. Table 16

[0242] As shown in Table 16, Hu2E6-62 blocked binding of all four ligands to rhNotchl- Fc. Example 16: Inhibition of Notchl-Dependent Signaling and Transcription

[0243] Antibodies mu2E6, Hu2E6-62, and A2-NRR1 were tested for their ability to inhibit Notch 1 -dependent signaling and transcription in the presence of DLL4 as described in Example 7. Results are shown in Fig. 16 and demonstrate that Hu2E6-62 is approximately three times more potent than mu2E6 in inhibiting Notch 1 -dependent transcription. Further, antibodies Hu2E6-62 and A2-NRR1 are equally effective in their inhibitory activities.

[0244] Notch 1 antibodies mu2E6, Hu2E6-62, and A2-NRR1 were tested for their ability to inhibit Notch 1 -dependent transcription by each of the ligands Jagl, Jag2, DLLl and DLL4 as described in Example 7. The inhibitory activities of antibodies mu2E6, Hu2E6-62, and A2- NRR1 on Notch 1 -dependent transcription are summarized in Table 17. Table 17

iiiu2l 6 I l u21 h- l Λ2-ΝΚΚ Ι

Max Max Max

EC50 Inhibitio EC50 Inhibitio EC50 Inhibitio

Inhibition n n n of Notch 1 Jag 1 0.4 nM 100% 0.9 nM 95% 0.1 nM 92%

Signaling Jag 2 0.1 nM 85% 0.5 nM 97% NA NA

DLI A 0.2 nM 90% 0.1 nM 96% NA NA

DLI A 0.1 nM 93% 0.02 nM 100% 0.06 nM 100%

[0245] The data in Table 17 shows that Hu2E6-62 inhibits activation of transcription of Notch 1 -dependent reporter gene by Jagl, Jag2, DLLl or DLL4. The mu2E6 and Hu2E6-62 antibodies appeared to show equivalent inhibition of Jagl -dependent Notch 1 signaling. Further, the mu2E6 and Hu2E6-62 antibodies showed equivalent inhibition of Jagl or DLL4- dependent Notch 1 signaling when compared to the A2-NRR1 antibody.

[0246] A reporter cell line dependent upon Notch 1 was produced by lenti viral introduction of a RBP-jK-dependent luciferase reporter gene (SABiosciences, Frederick, MD) into DU4475 cells. To activate Notch 1 -dependent signaling and transcription, cells were plated on ligand- coated wells prepared, as described in Example 6 (above). Cells were pre-incubated with a 3- fold dilution series of antibody Hu2E6-62 concentrations ranging from 0-300 μg/ml, for one hour at 37°C, before seeding 100 μΐ of the suspension into 96-well plates coated with ligand or hFc. Cells were incubated in ligand-coated or human-Fc-coated wells for four or 24 hours at 37°C, in 5% C0 ₂ . Next, 100 μΐ of Promega Bright Glo™ (Promega, Madison, WI) was added to each well. The reaction was allowed to proceed for five minutes in the dark, and then the entire 200 μΐ volume was transferred to white walled plates and read using a luminometer. Polyclonal antibody against Notch 1 (AF1057, R&D Systems) was used as controls to confirm that ligand-stimulated reporter activity in each cell line was specifically dependent upon the introduced Notch receptor. Results demonstrate that antibody Hu2E6-62 specifically inhibited Notch 1 -dependent transcription (FIG. 17A).

[0247] To determine the effect of antibody Hu2E6-62 on transcription of endogenous Notch 1 target genes, Notch 1 signaling was activated by Jagl in DU4475 cells, as described above (See Example 7). The effect on expression of endogenous Notchl targets, as a result of treatment with IgG control or antibody Hu2E6-62 was assessed by quantitative RT-PCR.

DU4475 cells were seeded into 6-well plates, in 2 ml of media. Replicate wells of cells were treated with antibody Hu2E6-62, IgG control, or vehicle control (DMSO), immediately after seeding. Cells were incubated at 37°C, 5% C0 ₂ for 20 hours after treatment, collected, and rinsed with PBS. Cell pellets were frozen on dry ice and stored at -80°C. RNA was prepared using Qiagen RNeasy™ miniprep columns (Qiagen GR8RNA). Quantitative RT-PCR was performed to analyze Notch target gene expression, using a commercial kit according to the kit vendor's instructions (Quantitect SYBR GREEN RT-PCR Kit; Qiagen). Results were analyzed using the comparative Ct method. Beta actin was used as an internal standard, and Stratagene Universal Human Reference RNA (Stratagene 740000) was used as an external standard for measurement of expression levels of the genes investigated. Results as shown in FIG. 17B showed that antibody Hu2E6-62 inhibited transcription of endogenous Notch target genes, including Heyl, Hey 2, HeyL, and Hes5.

Example 17: Inhibition of T-cell Fate Specification In Vivo

[0248] The antibodies Hu2E6-62 and Nrrl were tested for inhibition of thymocyte development and T-cell fate specification in humanized Notch 1 mice as described above (See Example 10).

[0249] As shown in Fig. 18, antibody Hu2E6-62 reduced the total number of thymocytes by greater than 95%. Similar levels of thymocyte depletion were observed with A2-NRR1. The results indicate that antibodies Hu2E6-62 and A2-NRR1 inhibited the in vivo function of Notch 1 in thymocyte development to equivalent extents.

Example 18: Lack of Toxicity

[0250] To determine if the antibodies were associated with toxicity, mice were treated with 20 mg/kg of antibodies Hu2E6-62, or a IgG control three times per week, or 5 mg/kg of A2- NRR1 twice per week, as described in Example 10. As shown in Fig. 19A, the Hu2E6-62 treated animals exhibited normal weight gain indicating a lack of toxicity of these antibodies. However, the A2-NRR1 treated mice exhibited significant weight loss over the period of treatment, accompanied by diarrhea.

[0251] After 18 days, animals were sacrificed, small intestines were collected, fixed and embedded in paraffin. To observe goblet cells in the small intestine, sections of small intestine from antibody-treated and IgG-treated animals were stained with Alcian Blue (Diagnostic

Biosystems, Cat. No. KT 003). As shown in FIG. 19B, mice treated with antibody Hu2E6-62 showed no increase in goblet cell numbers compared to control animals treated with IgG. By contrast, small intestines from animals treated with A2-NRR1 showed extensive Alcian Blue staining. These results indicate that antibody Hu2E6-62 did not lead to goblet cell metaplasia, and had little or no intestinal toxicity in treated mice. By contrast, A2-NRR1 treatment led to dramatic goblet cell hyperplasia indicative of severe intestinal toxicity. A2-NRR1 treated animals also exhibited diarrhea, significant weight loss (FIG. 19A), and approximately 30% of the animals died within 18 days of treatment. Upon necropsy the animals that died during treatment with A2-NRRlwere found to have bloated intestines, consistent with goblet cell hyperplasia, and similar to the gross morphological phenotype observed in intestines of mice treated with gamma- secretase inhibitors (Example 12 and Fig 9B).

Example 19: Inhibition of Angiogenesis in vivo

[0252] The effect of antibody Hu2E6-62 on functional angiogenesis induced by bFGF was determined using an in vivo matrigel plug assay as described in Example 13. Briefly, mice were treated intraperitoneally with 20 mg/kg of antibody Hu2E6-62 or a IgG control on day 0, and every 3 days thereafter. No significant loss of body weight was observed during these experiments. Animals were sacrificed after 7 days. Plugs were removed and processed (as described above in Example 13), to determine hemoglobin concentration. The results as shown in FIG. 20 indicated that Hu2E6-62 inhibited bFGF-induced angiogenesis in humanized (Notch l ^{hl l2/hl 12} knock-in) mice.

[0253] The effect of antibody Hu2E6-62 on functional angiogenesis induced by human lung cancer cells (Calu-6) was determined, using an in vivo matrigel plug assay as described in Example 13. Briefly, 129Sv/Ev or immunocompromised SCID mice were treated

intraperitoneally with 20 mg/kg of antibody Hu2E6-62, or a IgG control on day 0, and every 3 days thereafter. No significant loss of body weight was observed during these experiments. Animals were sacrificed after 7 days. Plugs were removed and processed (as described above in Example 13), to determine hemoglobin concentration. The results of this experiment indicated that antibody Hu2E6-62 inhibited angiogenesis induced by the human lung cancer Calu-6 cells (FIG. 21).

[0254] A second matrigel plug was obtained from each bFGF treated mouse and processed for histologic analysis, in parallel with the hemoglobin measurements. Plugs were removed, fixed over night in 10% buffered formalin at room temperature, embedded in paraffin, and 10- 20 um sections were prepared for immunohistochemistry. To detect blood vessels present in the matrigel plug, thin sections were stained for CD31, using an anti-mouse-CD31 antibody (Biocare Medical, Cat. Nos. CM303 and RT517SK) according to the vendor's instructions. CD31 staining of the matrigel plugs demonstrated increased vessel branching and smaller vessels after treatment with antibody Hu2E6-62, when compared to mice treated with IgG control. (Data not shown). These data indicated that antibody Hu2E6-62 promoted vascular branching in the treated mice. However, despite the increase in vascular branching, the decrease in blood content (as measured by hemoglobin content) suggested that antibody Hu2E6-62 decreased the function of the vessels that were present, and thus inhibited functional angiogenesis. This apparent decrease in functional angiogenesis caused by antibody Hu2E6-62 is consistent with the increased branching and decreased vascular function associated with a genetic loss of function of the Notch pathway in endothelial tissue.

INCORPORATION BY REFERENCE

[0255] The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.

EQUIVALENTS

[0256] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and the range of equivalency of the claims are intended to be embraced therein.

[0257] WHAT IS CLAIMED IS :