WO2012080891A1 - Anti-notch-1 antibodies - Google Patents

Abstract

The present disclosure is directed toward monoclonal antibodies and antigen-binding portions thereof that bind to Notch1. The present disclosure also relates to methods of making anti-Notch1 antibodies, compositions comprising these antibodies and methods of using the antibodies and compositions.

Description

ANTI-NOTCH-1 ANTIBODIES

Cross Reference To Related Applications

This application claims the benefit of U.S. Provisional Application No. 61/423333, filed December 15, 2010.

Field

The present disclosure relates to antibodies that antagonize the activity of Notch- 1 , methods of producing such antibodies, methods of assaying such antibodies and methods of using such antibodies in the treatment of cancer. Background

Notch proteins are transmembrane receptor proteins. There are four such Notch receptors in mammals. During receptor maturation, the ectodomains of mammalian Notch receptors are cleaved at a S1 site by a furin-like protease, yielding an

extracellular subunit and a transmembrane subunit that are held together by a heterodimerization (HD) domain. The part of the HD domain associated with the extracellular subunit is referred to as HD-N, and the other part of HD (the extracellular moiety of the transmembrane subunit) is referred to as HD-C. The extracellular subunit contains a large epidermal growth factor (EGF)-like repeat region and three Lin 12 repeats. Ligand binding of the EGF-repeat region induces a proteolytic cleavage by an ADAM-type metalloprotease at the S2 site within the HD-C domain, which triggers subsequent cleavage by γ-secretase at the S3 site and releases the intracellular part of Notch from the membrane, allowing it to move into the nucleus and regulate gene transcription. (Gordon, W.R., et.al, Nature Structural & Molecular Biology (2007) 14:295-300).

Before ligand induced activation, Notch is maintained in a resting

metalloprotease-resistant confirmation by a conserved negative regulation region (NRR), which consists of the three Lin12 repeats and the HD domain. (Vardar et al., Biochemistry 2003, 41 : 7061 -7067; Sanchez-lrizarry et al., Mol. Cell. Biol. 2004, 24: 9265-9273; Gordon, W.R., et.al, Nature Structural & Molecular Biology (2007)

14:295-300). The NRR of the Notch proteins is also sometimes defined as only consisting of the Lin12 repeats and the N terminal HD domain (HD-N) after proteolytic cleavage at the S1 site. (Weng, A.P., et. al, Science (2004) 9265-9273). The NRR domain prevents the ligand-independent proteolysis of the notch pathway.

The Notch pathway functions during diverse developmental and physiological process including those affecting neurogenesis in flies and vertebrates. In general, Notch signaling is involved in lateral inhibition, lineage decisions, and the establishment of boundaries between groups of cells. (Bray, S.J., Nature Reviews, 2006, 678-688). However, Notch activities are also associated with a variety of human diseases, including cancer. For example, mutations of Notch-1 were detected in more than 50% of T-cell acute lymphoblastic leukemia. (Radtke, F, Nature Review, Cancer, 2003, 756- 767). Accordingly, there is a need for identifying therapeutic agents that regulate the Notch-1 signaling pathway for the use of treating cancer.

Summary

In one embodiment, the present disclosure provides an isolated antibody that specifically binds to human Notch-1 , wherein the antibody binds to at least a first epitope and a second epitope, wherein the first epitope resides within the Lin-A domain of Notch-1 , and the second epitope resides within the HD-C domain of Notch-1. In one aspect of this embodiment, the first epitope is a major epitope. For example, a non- conservative substitution of any of the amino acid residues of the first epitope can result in the loss of more than 60%, more than 80%, or more than 90% of the antibody's binding affinity to human Notch-1.

In another aspect, the second epitope is a major epitope. For example, a non-conservative substitution of any of the amino acid residues of the second epitope results in the loss of more than 60%, more than 80%, or more than 90% of the antibody's binding affinity to human Notch-1 .

In another aspect, both the first epitope and the second epitope are major epitopes. For example, a non-conservative substitution of any of the amino acid residues of the first epitope and the second epitope results in the loss of more than 60%, more than 80%, or more than 90% of the antibody's binding affinity to human Notch-1. In another aspect of this embodiment, the antibody binds to an additional 1 to 4 epitopes, wherein each of said additional epitopes resides within either the Lin-A domain or HD-C domain of human Notch-1 .

In another aspect of this embodiment, the only major epitopes that the antibody binds to are the first epitope and the second epitope. More specifically, the first epitope is a major epitope comprising 1 to 4 amino acid residues selected from 1463V, 1465S, 1466L and 1467Q of the LinA domain of human Notch-1. For example, the first epitope is a major epitope consisting of the four amino acid residues 1463V, 1465S, 1466L and 1467Q of the LinA domain of human Notch-1. Even more specifically, the second epitope is a major epitope comprising 1 to 5 amino acid residues selected from 1705G, 1706A, 1707L, 1709S and 1710L of the HD-C domain of human Notch-1. For example, the second epitope is a major epitope consisting of the 5 amino acid residues 1705G, 1706A, 1707L, 1709S and 1710L of the HD-C domain of human Notch-1. Even more specifically, a non-conservative substitution of any of the amino acid residues of the first epitope or the second epitope results in a loss of more than 70%, more than 80%, more than 90% or more than 95% of the antibody's binding affinity to human Notch-1.

In another aspect of this embodiment, the antibody is humanized, human, or chimeric. In another aspect of this embodiment, the antibody is a mouse antibody.

In another aspect of this embodiment, the antibody binds to human Notch-1 with a K_D of 1x 10^"5 M or less. For example, the antibody binds to human Notch-1 with a K_D of 1X10^"6 M or less, 5x10^"7M or less, 2x10^"7 M or less, 1X10^"7 M or less, 5x10^"8 M or less, 2x10^"8 M or less or 1X10^"8 M or less. For example, in one embodiment, KD is determined by surface plasmon resonsonance, such as by Biacore.

In another embodiment, the present disclosure provides an antibody that specifically binds to human Notch-1 , comprising: (i) an L-CDR1 amino acid sequence as set forth in SEQ ID NO: 19, or a variant thereof in which 1 to 5 residues of SEQ ID NO:19 are modified; (ii) an L-CDR2 amino acid sequence as set forth in SEQ ID NO:20; and (iii) an L-CDR3 amino acid sequence as set forth in SEQ I D NO:21 , or a variant thereof in which 1 residue of SEQ ID NO:21 is modified. As will be apparent, SEQ ID NO:19 is both the Kabat and Chothia L-CDR1 sequence that is present at amino acid positions 23-36 of the the V_L sequence as set forth in SEQ ID NO: 17. For example, 1 , 2, 3, 4 or 5 residues of SEQ ID NO:19 can be modified. In one embodiment, such modifications are conservative substitutions. In a further embodiment, such

modifications are selected from R23S, R23W, L24S, L24F, S25G, S25H, S25L, S25A, S25T, T26L, T26F, T26V, G27D, A28L, T31 S, S32I, and S32R, where the amino acid sequence numbering is provided with respect to the V_L sequence as set forth in SEQ ID NO:17. In a further example, 1 residue of SEQ ID NO:21 is modified. As will be apparent, SEQ ID NO:21 is both the Kabat and Chothia L-CDR3 sequence that is present at amino acid positions 91-99 of the the V_L sequence as set forth in SEQ ID NO:17. In one embodiment, such modification is a conservative substitution. In a further embodiment, such modification is selected from V94Y, V94N, V94Q, and V94S, where the amino acid sequence numbering is provided with respect to the V_L sequence as set forth in SEQ ID NO:17.

In a further embodiment, the disclosure provides an antibody that specifically binds to human Notch-1 , comprising: (i) an H-CDR1 amino acid sequence as set forth in SEQ ID NO:22, SEQ ID NO:23, or SEQ I D NO:24; (ii) an H-CDR2 amino acid sequence as set forth in SEQ ID NO:25, or SEQ ID NO:26, or a variant thereof in which 1 to 2 residues of SEQ ID NO:25 or SEQ ID NO: 26 is modified; and (iii) an H-CDR3 amino acid sequence as set forth in SEQ ID NO:27, or a variant thereof in which 1 to 5 residues of SEQ ID NO:27 are modified. In one embodiment, 1 or 2 residues of SEQ ID NO:25 or SEQ ID NO:26 are modified. As will be apparent, SEQ ID NO:25 is the Kabat H-CDR2 sequence that is present at amino acid positions 50-66 of the the V_H sequence as set forth in SEQ ID NO: 18, and SEQ ID NO:26 is the Chothia H-CDR2 sequence that is present at amino acid positions 52 to 57 of the V_H sequence as set forth in SEQ ID NO:18. In one embodiment, such modifications are conservative substitutions. In a further embodiment, such modifications are selected from P56H, P56N, P56G, and F57R, where the amino acid sequence numbering is provided with respect to the V_H sequence as set forth in SEQ ID NO: 18. In another embodiment, 1 to 5 residues of SEQ ID NO:27 are modified. As will be apparent, SEQ ID NO:27 is the Kabat and Chothia H-CDR3 sequence that is present at amino acid positions 99 to 1 10 of the V_H sequence as set forth in SEQ ID NO: 18. In one embodiment, such modifications are conservative substitutions. In another embodiment, such modifications are selected from S102K, A103P, Y104G, Y104W, A105Y, A105S, A105Q, R107T, and R107A, where the amino acid sequence numbering is provided with respect to the V_H sequence as set forth in SEQ ID NO: 18. Examples of such modifications and the effect on K_D are provided in Table 9.

In a further embodiment, the present disclosure provides an antibody that specifically binds to human Notch- 1 , comprising H-CDR1 , H-CDR2, H-CDR3, L-CDR1 , L-CDR2, and L-CDR3 sequences as described above, or modifications as described above.

In a further embodiment, the disclosure provides an antibody according to any one of antibodies described previously, comprising a V_H domain amino acid sequence that is at least 90% identical to SEQ ID NO:18. For example, the VH domain amino acid sequence is at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to SEQ ID NO:18. In a further embodiment, the antibody comprises a VH domain amino acid sequence as set forth in SEQ ID NO:18.

In a further embodiment, the disclosure provides an antibody according to any one of the antibodies described previously, comprising a V_L domain amino acid sequence that is at least 90% identical to SEQ ID NO: 17. For example, the VL domain amino acid sequence is at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to SEQ ID NO:17. In a further embodiment, the antibody comprises a VH domain amino acid sequence as set forth in SEQ ID NO:17.

In a further embodiment, the disclosure provides an antibody according to any one of the antibodies described previously, comprising a heavy chain amino acid sequence that is at least 90% identical to SEQ ID NO:37. For example, the heavy chain amino acid sequence is at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to SEQ I D NO:37. In a further embodiment, the antibody comprises a heavy chain amino acid sequence as set forth in SEQ ID NO:37.

In a further embodiment, the disclosure provides an antibody according to any one of the antibodies described previously, comprising a light chain amino acid sequence that is at least 90% identical to SEQ ID NO:36. For example, the light chain amino acid sequence is at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to SEQ I D NO:36. In a further embodiment, the antibody comprises a light chain amino acid sequence as set forth in SEQ ID NO:36.

In a further embodiment, the disclosure provides an antibody that specifically binds to human Notch-1 , comprising a V_H domain amino acid sequence that is at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to SEQ ID NO:18. In one specific embodiment, the disclosure provides an antibody comprising a VH domain amino acid sequence as set forth in SEQ ID NO:18.

In a further embodiment, the disclosure provides an antibody that specifically binds to human Notch-1 , comprising a V_L domain amino acid sequence that is at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to SEQ ID NO:17. In one specific embodiment, the disclosure provides an antibody comprising a V_L domain amino acid sequence as set forth in SEQ ID NO:17.

The present disclosure further provides an antibody that specifically binds to human Notch-1 , comprising: a V_H domain amino acid sequence that is at least 90% identical to SEQ ID NO: 18; and a VL domain amino acid sequence that is at least 90% identical to SEQ ID NO: 17. For example, in one embodiment, the V_H domain is at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to SEQ ID NO:18, and the V_L domain is at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to SEQ ID NO:17. In one specific embodiment, the antibody comprises a V_H domain as set forth in SEQ ID NO:18, and a V_L domain as set forth in SEQ ID NO:17.

The present disclosure further provides any of the antibodies described herein, wherein said antibody is of isotype IgA, IgD, IgE, IgG, or IgM. For example, in one embodiment, the isotype is IgG, and the subclass is IgG-i , lgG₂, lgG₃ or lgG₄, or is derived therefrom. For example, in one embodiment, the subclass is derived from lgG₂ and is lgG2AA-

The present disclosure further provides an antibody that specifically binds to human Notch-1 , comprising: a heavy chain amino acid sequence that is at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to SEQ ID NO:37; and a light chain amino acid sequence that is at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to SEQ ID NO:36. In a further embodiment, the heavy chain amino acid sequence comprises the sequence set forth in SEQ ID NO:37, wherein the C-terminal lysine of SEQ ID NO:37 is optionally not present. In an even further embodiment, the light chain amino acid sequence comprises the sequence set forth in SEQ ID NO:36. In a further embodiment, any of the antibodies described herein are isolated antibodies. In a further embodiment, any of the antibodies described herein are monoclonal antibodies.

In a further embodiment, antibodies consisting of the identical amino acid sequence of the mouse monoclonal antibody, 248A are excluded. For example, in one embodiment, excluded are antibodies comprising the identical CDR sequences and/or variable region sequences of the murine monoclonal antibody N248A, and nucleic acids that encode any such antibodies, as shown in Table 1 (SEQ ID NOs: 1- 16).

In a further embodiment, the present disclosure provides an isolated nucleic acid that encodes any of the antibodies described herein, or that encodes any of the heavy and/or light chains of antibodies described herein. For example, in one emobidment, the disclosure provides a nucleic acid comprising the sequence as set forth in SEQ ID NO:32. In a further embodiment, the disclosure provides an isolated nucleic acid comprising the sequence as set forth in SEQ ID NO:28.

In a further embodiment, the disclosure provides a host cell comprising any of the nucleic acids described herein. In a further embodiment, the disclosure provides a host cell that recombinantly produces any of the antibodies described herein. In one embodiment, any of the host cells decribed herein are isolated.

In a further embodiment, the present disclosure provides a pharmaceutical composition comprising any of the antibodies described herein and a pharmaceutically acceptable carrier.

In a further embodiment, the disclosure provides methods of treating abnormal cell growth, such as cancer, in a mammal in need thereof, comprising administering to said mammal any of the antibodies or pharmaceutical compositions described herein.

In a further embodiment, the disclosure provides any of the antibodies pharmaceutical compositions described herein, for use in treating abnormal cell growth in a mammal in need thereof. In another embodiment, the disclosure provides any of the antibodies disclosed herein for the use in treating cancer in a mammal, such as a human, in need thereof. In a further embodiment, such cancer is T-ALL or breast cancer. ln another embodiment, the disclosure provides the use of the antibodies disclosed herein, for the preparation of a medicament for the treatment of cancer.

Brief Description of the Figures/Drawings

Figure 1 illustrates the PCR synthesis of the cDNA of a human Notchl immunogen plasmid N I-NRR-TM(-), as described in Example 1.

Figure 2 illustrates the PCR synthesis of the cDNA of another human Notchl immunogen plasmid N 1-NRR-TM(+), also described in Example 1.

Figure 3 illustrates the Notch-1 dependent luciferase reporter assay results of two monoclonal antibodies: mAb N248A and mAb-C, as described in Example 3.

Figure 4 illustrates that the luciferase reporter assay indicates that mAb N248A inhibits Jagged-1 induced Notch-1 signaling. Hela/Jagged1 cells and N1 dP-c16 cells were co-cultured for the luciferase reporter assay. MlgG is a control mouse antibody. The y-axis numbers are luciferase reporter activity readings.

Figure 5 is a sequence alignment of the EFG, Lin-A, Lin-B, Lin-C, HD-N and

HD-C domains between human Notchl and human Notch2.

Figure 6A is a Western blot image illustrating that the level of NICD (Notchl intracellular domain) was reduced by mAb N248A in the HPB-ALL cells.

Figure 6B illustrates that growth of HPB-AII cells are inhibited by mAb N248A. Figure 7A and 7B illustrates that mAb N248A blocks the expression of Hes1 mRNA and Hes4 mRNA respectively.

Figure 8 illustrates the growth inhibition of HBP-ALL xenograft tumor by mAb N248A. Mice were dosed with mAb as indicated in the figure after tumor grew to 150- 300 mm³. Each group contains ten mice with randomized tumor size.

Figure 9 illustrates the change of plasma mAb N248A concentration after a single dose injection of 5 mg/kg in mice. Each data point was calculated based on three mice. T-i/2 is the half life of N248A in mouse sera.

Figure 10 illustrates the inhibition of NICD in HBP-ALL xenograft tumors after a single dose injection of 5 mg/kg of N248A in mice. The Western blot bands of the tumors treated with control antibody D16A were set as 100% intensity, which equals 0% inhibition. Figure 1 1 shows the results of a luciferase reporter assay (as described in Example 1 ) comparing the antibodies N248A and A12.2.

Figure 12 shows the results of domain swap and an ELISA assay to analyze the binding epitopes of A12.2 (as described in Examples 1 and 5) as compared to N248A.

Figure 13 shows the analysis of the ability of A12.2 to inhibit growth of HPB-ALL cells, using the methods described in Example 6.

Detailed Description

The present disclosure relates to isolated monoclonal antibodies, particularly human monoclonal antibodies and mouse monoclonal antibodies as well as

human/mouse chimeric antibodies that bind specifically to Notch-1 with high affinity. The disclosure provides isolated antibodies, methods of making such antibodies, immunoconjugates and bispecific molecules comprising such antibodies. The present disclosure further relates to pharmaceutical compositions containing the antibodies, immunconjugates or bispecific molecules of the disclosure. The disclosure also relates to methods of using the antibodies, such as to inhibit Notch-1 activation, as well as to treat diseases associated with over activation or over expression of Notch-1 , such as abnormal cell growth (e.g. cancer - such as breast cancer and T-cell acute

lymphoblastic leukemia (T-ALL)). Accordingly, the disclosure also provides methods of using the anti-Notch-1 antibodies to treat various types of abnormal cell growth, such as cancer.

General Techniques

The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.

Definitions

The terms "Notch-1 " or "Notchl " are used interchangeably, and include variants, isoforms and species homologs of human Notch-1 protein. Native human Notch-1 protein, for example, is made up of a leader peptide, a large epidermal growth factor (EGF)-like repeat region, three Lin 12 repeats, a N terminal heterodimerization domain (HD-N), a C terminal heterodimerization domain (HD-C), a transmembrane (TM) sequence and an intracellular domain (NICD). The NCBI/GenBank accession number of the full length human Notch-1 is NM_017617.2

The term "Notch-1 negative regulatory region", or "Notch-1 NRR" as used herein, unless otherwise indicted, refers to any native or synthetic polypeptide region of Notch-1 consisting of the three Lin12 domains and the amino acid sequence or sequences located between the three Lin12 domains and the transmebrane domain of Notch-1. In one embodiment, the "Notch-1 NRR" includes the three Lin12 domains and two heterodimerization domains HD-N, and HD-C, wherein the HD-N and HD-C domains of Notch-1 are covalently bonded and not yet cleaved by the furin-like protease (before S1 cleavage). In another embodiment, the "Notch-1 NRR" includes the three Lin12 domains and the two heterodimerization domains HD-N, and HD-C, wherein the HD-N and HD-C domains are non-covalently bonded (after S1 cleavage). In one aspect of this embodiment, the S2 site within the HD-C domain has not been cleaved by the ADAM- type metalloproteases. In another particular aspect of this embodiment, the S2 site within the HD-C domain is being cleaved or has already been cleaved by the ADAM- type metalloproteases. (Gordon, W.R., et.al, Nature Structural &Molecular Biology, 2007, volume 14, 295-300).

An "antibody" is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab')2, Fv), single chain (ScFv) and domain antibodies such as shark and camelid antibodies), and fusion proteins comprising an antibody portion (such as domain antibodies), and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site. An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class.

Depending on the antibody amino acid sequence of the constant domain of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes (isotypes) of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses, e.g., lgG1 , lgG2, lgG3, lgG4, lgA1 and lgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

An "isolated antibody", as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds Notch-1 is substantially free of antibodies that specifically bind antigens other than Notch-1 ). An isolated antibody that specifically binds Notch-1 may, however, have cross-reactivity to other antigens, such as Notch-1 molecules from other species. Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals.

The term "isolated polypeptide" or "isolated antibody" is a protein, polypeptide or antibody that by virtue of its origin or source of derivation (1 ) is not associated with naturally associated components that accompany it in its native state, (2) is free of other proteins from the same species, (3) is expressed by a cell from a different species, or (4) does not occur in nature. Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be "isolated" from its naturally associated components. A protein, polypeptide, or antibody may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art. Examples of isolated antibodies include a Notch-1 antibody that has been affinity purified using Notch-1 , and a Notch-1 antibody that has been synthesized by a cell line in vitro.

The term "isolated nucleic acid" as used herein means a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin the "isolated nucleic acid" (1 ) is not associated with all or a portion of

polynucleotides with which the "isolated polynucleotide" is found in nature, (2) is operably linked to a polynucleotide to which it is not linked in nature, or (3) does not occur in nature as part of a larger sequence.

As used herein, "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which typically include antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present disclosure may be made by the hybridoma method, or may be made by recombinant DNA methods, which are well known to those of skill in the art. The monoclonal antibodies may also be isolated from phage libraries generated using techniques that are well known to those of skill in the art.

As used herein, "humanized" antibody refers to forms of non-human (e.g. murine) antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. Preferably, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FW) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, the humanized antibody may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FW regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human

immunoglobulin. Other forms of humanized antibodies have one or more CDRs (L- CDR1 , L-CDR2, L-CDR3, H-CDR1 , H-CDR2, or H-CDR3) which are altered with respect to the original antibody, which are also termed one or more CDRs "derived from" one or more CDRs from the original antibody.

As used herein, the terms "human antibody" or "fully human antibody" are intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences.

Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies of the disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. This definition of a human antibody includes antibodies comprising at least one human heavy chain polypeptide or at least one human light chain polypeptide. One such example is an antibody comprising murine light chain and human heavy chain polypeptides. Human antibodies can be produced using various techniques known in the art. In some embodiments, the human antibody is selected from a phage library, where that phage library expresses human antibodies. Human antibodies can also be made by immunization of animals into which human immunoglobulin loci have been transgenically introduced in place of the endogenous loci, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625, 126; 5,633,425; and

5,661 ,016. Alternatively, the human antibody may be prepared by immortalizing human B lymphocytes that produce an antibody directed against a target antigen (such B lymphocytes may be recovered from an individual or may have been immunized in vitro).

The term "chimeric antibody" is intended to refer to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody. The term "recombinant antibody", as used herein, includes all antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further below), (b) antibodies isolated from a host cell transformed to express the antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of immunoglobulin gene sequences to other DNA sequences. Such recombinant antibodies have variable regions in which the framework and CDR regions are derived from germline immunoglobulin sequences. In certain embodiments, however, such recombinant antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to germline V_H and V_L sequences, may not naturally exist within the antibody germline repertoire in vivo.

The term "compete", as used herein with regard to an antibody, refers to when a first antibody, or an antigen-binding portion thereof, competes for binding with a second antibody, or an antigen-binding portion thereof, where binding of the first antibody with its cognate epitope is detectably decreased in the presence of the second antibody compared to the binding of the first antibody in the absence of the second antibody. The alternative, where the binding of the second antibody to its epitope is also detectably decreased in the presence of the first antibody, can, but need not be the case. That is, a first antibody can inhibit the binding of a second antibody to its epitope without that second antibody inhibiting the binding of the first antibody to its respective epitope. However, where each antibody detectably inhibits the binding of the other antibody with its cognate epitope or ligand, whether to the same, greater, or lesser extent, the antibodies are said to "cross-compete" with each other for binding of their respective epitope(s). For instance, cross-competing antibodies can bind to the epitope, or portion of the epitope, to which the antibodies as disclosed herein bind. Use of both competing and cross-competing antibodies is encompassed by the present disclosure. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope, or portion thereof, and the like), the skilled artisan would appreciate, based upon the teachings provided herein, that such competing and/or cross-competing antibodies are encompassed and can be useful for the methods disclosed herein.

As used herein, a "major epitope" refers to an epitope, wherein if any one of the amino acid residues of the epitope is replaced by an alanine or a non conservative substitution, the binding affinity of the antibody to the antigen which the epitope belongs to, is decreased by more than 50%.

As used herein, "isotype" or "class" of an antibody refers to the five major classes of immunoglobulins (e.g., IgA, IgD, IgE, IgG, and IgM) that are encoded by the heavy chain constant region genes. The constant domains of antibodies are not involved in binding to antigen, but exhibit various effector functions. Depending on the amino acid sequence of the heavy chain constant region, a given human antibody or

immunoglobulin can be assigned to one of five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM. The structures and three-dimensional configurations of different classes of immunoglobulins are well-known. Of the various human immunoglobulin classes, only human lgG1 , lgG2, lgG3, lgG4, and IgM are known to activate

complement. Human lgG1 and lgG3 are known to mediate ADCC in humans.

As used herein, "subclass" refers to the further specification within a class of the heavy chain constant region gene, such as, for example, the lgG1 , lgG2, lgG3, or lgG4 subclasses within the IgG isotype.

The phrases "an antibody recognizing an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody which binds specifically to an antigen."

As known in the art, the term "Fc region" is used to define a C-terminal region of an immunoglobulin heavy chain. The "Fc region" may be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl- terminus thereof. The numbering of the residues in the Fc region is that of the EU index as known in the art. The Fc region of an immunoglobulin generally comprises two constant domains, CH2 and CH3. A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. A "variant Fc region" comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification, yet retains at least one effector function of the native sequence Fc region. Preferably, the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide, e.g. from about one to about ten amino acid substitutions, and preferably, from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of the parent polypeptide. The variant Fc region herein will preferably possess at least about 80% sequence identity with a native sequence Fc region and/or with an Fc region of a parent polypeptide, and most preferably, at least about 90% sequence identity therewith, more preferably, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity therewith.

The terms "Fc receptor" or "FcR" are used to describe a receptor that binds to the

Fc region of an antibody. For example, the FcR can be a native sequence human FcR. Furthermore, the FcR can be one that binds an IgG antibody (a gamma receptor) and includes receptors of the FcyRI, FcyRII, FcyRIII, and FcyRIV subclasses, including allelic variants and alternatively spliced forms of these receptors. FcyRII receptors include FcyRIIA (an "activating receptor") and FcyRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. As will be appreciated by those of skill in the art, inhibiting receptor FcyRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. FcRs have been extensively reviewed and are well known to those of skill in the art. Other FcRs, including those to be identified in the future, are encompassed by the term "FcR" herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus.

As used herein, an antibody that "specifically binds to human Notch-1 ", or that binds to human Notch-1 with "high affinity", or "preferentially binds" is intended to refer to an antibody that binds to human Notch-1 with a K_D of 1 x 10^"6 M or less. The term "k_on", as used herein, is intended to refer to the on-rate, or association rate of a particular antibody-antigen interaction, whereas the term "koff," as used herein, is intended to refer to the off-rate, or dissociation rate of a particular antibody-antigen interaction. The term "K_D", as used herein, is intended to refer to the equilibrium dissociation constant, which is obtained from the ratio of k₀fr to k_on (i.e,. k₀ff/k_on) and is expressed as a molar concentration (M). K_D values for antibodies can be determined using methods well established in the art. One method for determining the K_D of an antibody is by using surface plasmon resonance, typically using a biosensor system such as a Biacore® system.

As used herein, the term "subject" includes any human or nonhuman animal.

The term "nonhuman animal" includes all vertebrates, e.g., mammals and non- mammals, such as nonhuman primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc.

The terms "polypeptide", "oligopeptide", "peptide" and "protein" are used interchangeably herein to refer to chains of amino acids of any length, preferably, relatively short (e.g., 10-100 amino acids). The chain may be linear or branched, it may comprise modified amino acids, and/or may be interrupted by non-amino acids. The terms also encompass an amino acid chain that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. It is understood that the polypeptides can occur as single chains or associated chains.

As known in the art, "polynucleotide," or "nucleic acid," as used interchangeably herein, refer to chains of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a chain by DNA or RNA polymerase. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the chain. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide(s). Further, any of the hydroxyl groups ordinarily present in the sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid supports. The 5' and 3' terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups. Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2'-0-methyl-, 2'-0-allyl, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, alpha- or beta-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(0)S("thioate"), P(S)S ("dithioate"), (0)NR₂ ("amidate"),

P(0)R, P(0)OR', CO or CH₂ ("formacetal"), in which each R or R' is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether (-0-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.

A "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. As known in the art, the variable regions of the heavy and light chain each consist of four framework regions (FW) connected by three complementarity

determining regions (CDRs) also known as hypervariable regions. The CDRs in each chain are held together in close proximity by the FWs and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies. There are at least two techniques for determining CDRs: (1 ) an approach based on cross-species sequence variability (referred to as Kabat CDRs); and (2) an approach based on crystallographic studies of antigen-antibody complexes (referred to as Chothia CDRs). As used herein, a CDR may refer to CDRs defined by either approach or by a combination of both approaches. The first, second, and third CDR regions in the light chain are referred to herein, respectively, as L-CDR1 , L-CDR2, and L-CDR3. The framework regions of the light chain are similarly denoted as L-FW1 , L-FW2, L-FW3, and L-FW4. Furthermore, the first, second, and third CDR regions in the heavy chain are referred to herein, respectively, as H-CDR1 , H-CDR2, and H-CDR3. The framework regions of the heavy chain are similarly denoted as H-FW1 , H-FW2, H-FW3, and H- FW4.

As known in the art a "constant region" of an antibody refers to the constant region of the antibody light chain or the constant region of the antibody heavy chain, either alone or in combination.

A "host cell" includes an individual cell or cell culture that can be or has been a recipient for vector(s) for incorporation of polynucleotide inserts. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a polynucleotide(s) of the present disclosure.

As used herein, "vector" means a construct, which is capable of delivering, and, preferably, expressing, one or more gene(s) or sequence(s) of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells. As used herein, "expression control sequence" means a nucleic acid sequence that directs transcription of a nucleic acid. An expression control sequence can be a promoter, such as a constitutive or an inducible promoter, or an enhancer. The expression control sequence is operably linked to the nucleic acid sequence to be transcribed.

As used herein, "pharmaceutically acceptable carrier" or "pharmaceutical acceptable excipient" includes any material which, when combined with an active ingredient, allows the ingredient to retain biological activity and is non-reactive with the subject's immune system. Examples include, but are not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solution, water, emulsions such as oil/water emulsion, and various types of wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate buffered saline (PBS) or normal (0.9%) saline. Compositions comprising such carriers are formulated by well known conventional methods.

An "individual" or a "subject" is a mammal, more preferably, a human. Mammals also include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice and rats.

Human Notch-1 receptor

Human Notch 1 cDNA encodes a protein of 2556 amino acid residues consisting of a leader peptide, 36 EGF-like repeats, negative regulatory region (NRR), a transmembrane (TM) sequence and an intracellular domain. The Notch-1 NRR starts from amino acid residue 1447 and ends at 1734. The Notch-1 NRR consists of LNR-A (also referred to herein as Lin-A - Notch-1 AA residues 1447-1483), LNR-B (also referred to herein as Lin-B - Notch-1 AA residues 1484-1525), LNR-C (also referred to herein as Lin-C - Notch-1 AA residues 1526-1565), N-terminal heterodimerization domain (HD-N, Notch-1 AA residues 1566-1665) and C-terminal heterodimerization domain (HD-C, Notch-1 AA residues 1666 to 1734).

The antibodies of the disclosure are characterized by particular functional features or properties of the antibodies. For example, the antibodies bind specifically to human Notch-1 with a K_D of 1 x 10^"5 M or less. For example, an antibody of the disclosure binds to Notch-1 with a K_D of 1 x 10^"6 M or less, with a K_D of 1x10^"7 M or less, or with a K_D or 1 x 10^"8 M or less.

Assays to evaluate the binding ability of the antibodies toward Notch-1 include, but are not limited to ELISAs, Western blots, RIAs, and flow cytometry analysis. The binding kinetics (e.g., binding affinity) of the antibodies also can be assessed by assays known in the art, such as by Biacore analysis.

Monoclonal Antibody mAb N248A

One illustrative antibody of the disclosure is the mouse monoclonal antibody N248A, generated, isolated, tested and structurally characterized as described in Examples 1-3 and 8. Table 1 lists the amino acid (a. a.) and nucleic acid (n.a.) sequences of various regions of mAb N248A.

Table 1 : N248A Sequence

Description Sequence SEQ ID NO

V_H (n.a.) CAGGTTCAGCTGCAGCAGTCTGGAGCTGAG 1

CTGATGAAGCCTGGGGCCTCAGTGAAGATAT

C CTG CAAGG CTACTG G CTACACATTCAGTAA

CTACTGGATGGAGTGGGTAAAGCAGAGGCC

TGGACATGGCCTTGAGTGGATTGGAGAGATT

TTACCTGGAAGGGGTAGAACTAACTACAATG

AG AACTTCAAG GG CAAG G CCACATTCACTGC

AGATACATCCTCCAACACAGTCTACATGCAA

CTCAACAGCCTGACATCTGAGGACTCTGCCG

TCTATTACTGTGCAAGATTCCACAGCTCGGC

CTATTACTATACTATGGACTACTGGGGTCAAA

GAACCTCGGTCACCGTCTCCTCA

V_H (a.a.) QVQLQQSGAELMKPGASVKISCKATGYTFSNY 2

WMEWVKQRPGHGLEWIGEILPGRGRTNYNEN FKGKATFTADTSSNTVYMQLNSLTSEDSAVYY CARFHSSAYYYTMDYWGQRTSVTVSS

V_L (n.a.) CAGGCTGTTGTGACTCAGGAATCTGCACTCA 3

C CACATCACCTG GTGAAACAGTCACACTCAC

TTGTCGCTCAAGTACTGGGGCTGTTACAACT

AGTAACTATGCCAACTGGGTCCAAGAAAAAC

CAGATCA I I I ATTCACTGGTCTAATAGGTGGT

ACCAACAACCGAGCTCCAGGTATTCCTGCCA

GATTCTCAGGCTCCCTGATTGGAGACAAGGC

TGCCCTCACCATCACAGGGGCACAGACTGA

GGATGAGGCAATATA I I I CTGTGCTCTATGG

TACAGCAACCACTGGGTGTTCGGTGGAGGA

ACCAAACTGACTGTCCTA Description Sequence SEQ ID NO

V_L (a.a.) QAVVTQESALTTSPGETVTLTCRSSTGAVTTS 4

NYANWVQEKPDHLFTGLIGGTNNRAPGIPARF SGSLIGDKAALTITGAQTEDEAIYFCALWYSNH WVFGGGTKLTVL

L-CDR1 (n.a.) CGCTCAAGTACTGGGGCTGTTACAACTAGTA 5

ACTATGCCAAC

L-CDR2 (n.a.) GGTACCAACAACCGAGCTCCA 6

L-CDR3 (n.a.) GCTCTATGGTACAGCAACCACTGGGTG 7

L-CDR1 (a.a.) RSSTGAVTTSNYAN 8

L-CDR2 (a.a.) GTNNRAP 9

L-CDR3 (a.a.) ALWYSNHWV 10

H-CDR1 (n.a.) AACTACTGGATGGAG 1 1

H-CDR2 (n.a.) GAGATTTTACCTGGAAGGGGTAGAACTAACT 12

ACAATGAGAACTTCAAGGGC

H-CDR3 (n.a.) TTCCACAGCTCGGCCTATTACTATACTATGG 13

ACTAC

H-CDR1 (a.a.) NYWME 14

H-CDR2 (a.a.) EILPGRGRTNYNENFKG 15

H-CDR3 (a.a.) FHSSAYYYTMDY 16

As shown in Example 4, mAb N248A has a KD of less than 0.33 nM. As shown in Example 5, it was shown that mAb N248A binds at least two distinguishable Notch-1 epitopes, one epitope is within the Lin-A domain and the other epitope is within the HD- C domain.

As shown in Example 6, mAb N248A inhibits both T-cell acute lymphoblastic leukemia (T-ALL) and breast cancer cell growth in cell culture. As shown in Example 7, mAb N248A also inhibits T-cell lymphoblastic leukemia in murine xenograft tumor model. Monoclonal Antibody A 12.2

A further illustrative antibody of the disclosure is the humanized monoclonal antibody A12.2, which is further described in Example 9. Table 2 provides the amino acid (a. a.) and nucleic acid (n.a.) sequences of various regions of mAb A12.2.

Table 2: A12.2 Sequence

Description Sequence SEQ ID NO

V_L (a.a.) QTWTQE PSFSVS PGGTVTLTCRLS TGAVTTSNYANWVQ 17

QTPGQAPRGL I GGTNNRAPGVPDRFSGS I LGNKAALT I T GAQADDESDYYCALWVSNHWVFGTGTKVTVL

V_H (a.a.) E VQ L VE S GGGL VQ PGG S L RL S C AAS GYTFSNYW E WVRQ 18

APGKGLEWIGE ILPGRPFTNYNENFKGRFTI SADNAKNS LYLQ NSLRAEDTAVYYCARFHSSAYAYR DYWGQGTTV TVSS

L-CDR1 (a.a.) RLSTGAVTTSNYAN 19 Kabat & Chothia

L-CDR2 (a.a.) GTNNRAP 20 Kabat & Chothia

L-CDR3 (a.a.) ALWVSNHWV 21 Kabat & Chothia

H-CDR1 (a.a.) NYW E 22 Kabat

H-CDR1 (a.a.) GYTFSNY 23 Chothia

H-CDR1 (a.a.) GYTFSNYWME 24 Kabat & Chothia

H-CDR2 (a.a.) E ILPGRPFTNYNENFKG 25 Kabat

H-CDR2 (a.a.) LPGRPF 26 Chothia

H-CDR3 (a.a.) FHS S AYAYRMD Y 27 Kabat & Chothia

V_L (n.a.) CAGACTGTGGTGACCCAGGAGCCTTCTTTCAGCGTGTCC 28

CCTGGGGGCACTGTGACTCTGACCTGCAGGCTGTCTACC Description Sequence SEQ ID NO

GGTGCTGTGACTACATCCAACTATGCTAATTGGGTCCAG CAGACCCCAGGGCAGGCTCCACGGGGCCTGATCGGTGGC ACCAACAACAGGGCTCCTGGAGTCCCCGACAGGTTCAGC GGTTCCATCCTGGGCAACAAGGCCGCCCTGACCATCACA GGCGCTCAGGCCGACGATGAATCTGATTACTATTGCGCC CTGTGGGTGTCCAACCATTGGGTGTTTGGCACCGGCACC AAGGTGACCGTGCTG

L-CDR1 (n.a.) AGGCTGTCTACCGGTGCTGTGACTACATCCAACTATGCT 29

AAT

L-CDR2 (n.a.) GGCACCAACAACAGGGCTCCT 30

L-CDR3 (n.a.) GCCCTGTGGGTGTCCAACCATTGGGTG 31

V_H (n.a.) GAGGTCCAGCTGGTCGAGAGTGGCGGTGGGCTGGTCCAG 32

CCAGGTGGAAGCCTGCGCCTCTCTTGTGCTGCTAGCGGG TACACATTTTCCAACTACTGGATGGAATGGGTCCGCCAG GCTCCCGGGAAGGGCCTCGAATGGATCGGTGAGATCCTG CCAGGACGACCCTTCACCAACTATAACGAAAATTTTAAG GGCCGGTTTACCATCAGTGCAGACAATGCCAAGAACTCT CTGTACCTGCAGATGAACAGCCTGAGAGCCGAAGATACC GCAGTGTATTACTGTGCCAGGTTCCATAGCAGCGCATAT GCTTACAGGATGGACTATTGGGGGCAGGGCACAACGGTC ACCGTCTCCTCA

H-CDR1 (n.a.) AACTACTGGATGGAA 33

H-CDR2 (n.a.) GAGATCCTGCCAGGACGACCCTTCACCAACTATAACGAA 34

AATTTTAAGGGC

H-CDR3 (n.a.) TTCCATAGCAGCGCATATGCTTACAGGATGGACTAT 35

Light Chain (a. a.) QTWTQEPSFSVSPGGTVTLTCRLSTGAVTTSNYANWVQ 36

QTPGQAPRGLIGGTNNRAPGVPDRFSGSILGNKAALTIT GAQADDESDYYCALWVSNHWVFGTGTKVTVLGQPKAAPS VTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSS PVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYS CQVTHEGSTVEKTVAPTECS

Heavy Chain EVQLVESGGGLVQPGGSLRLSCAASGYTFSNYW EWVRQ 37 (lgG2AA) (a.a.) APGKGLEWIGEILPGRPFTNYNENFKGRFTISADNAKNS

LYLQ NSLRAEDTAVYYCARFHSSAYAYR DYWGQGTTV TVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPE PVTVSWNSGALTSGWTFPAVLQSSGLYSLSSWTVPSS NFGTQTYTC VDHKPSNTKVDKTVERKCCVECPPCPAPP VAGPSVFLFPPKPKDTL ISRTPEVTCWVDVSHEDPEV QFNWYVDGVEVHNAKTKPREEQFNSTFRWSVLTWHQD Description Sequence SEQ ID NO

WLNGKEYKCKVSNKGLPS S I EKT I S KTKGQPRE PQVYTL PPSREE TKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPP LDSDGSFFLYSKLTVDKSRWQQGNVFSCSV H E ALHNHYTQKS LS LS PGK

As described further in Example 9, A12.2 has a a K_D of 0.315 nM, as determined at 25°C by Biacore analysis. As further described in Example 1 1 , both N248A1 and A12.2 bind to similar epitopes in the Notch-1 Lin-A domain and the Notch-1 HD-C domain.

Anti-Notch-1 antibodies that bind to the Lin-A domain and the HD-C domain

It is within the contemplation of the current disclosure that antibodies that bind to the Notch-1 Lin-A and HD-C domain with a high affinity will reduce Notch-1 signal transduction, and therefore may demonstrate biological activity in vitro and in vivo to inhibit cancer cell growth, in particular, T-ALL cancer cell growth. Such antibodies may be produced following general methods known to those of ordinary skill in the art. In one embodiment, such antibodies can be produced through immunization of a mouse with an immunogen comprising the Notch-1 LinA domain and the Notch-1 HD-C domain, as shown in Examples 1 and 2, followed by hybridoma cloning of the antibodies thus generated, and assaying the cloned antibodies by ELISA assay, as shown in Example 2. The Notch-1 binding affinity of the antibodies selected according to the ELISA assay can be measured on a surface plasma resonance Biacore 3000 instrument, as shown in Example 4.

The anti-Notch-1 antibodies of the current disclosure, wherein the antibodies that bind to the Notch-1 LinA domain and Notch-1 HD-C domain can be produced by any other methods known in the art other than described in the above paragraph. The route and schedule of immunization of the host animal are generally in keeping with established and conventional techniques for antibody stimulation and production, as further described herein. General techniques for production of human and mouse antibodies are known in the art and/or are described herein. Anti-Notch-1 antibodies generated by hybridoma technologies.

It is within the contemplation of the current disclosure that that any mammalian subject including humans or antibody producing cells therefrom can be manipulated to serve as the basis for production of mammalian, including human, hybridoma cell lines. Typically, the host animal is inoculated intraperitoneally, intramuscularly, orally, subcutaneously, intraplantar, and/or intradermal^ with an amount of immunogen, including as described herein.

Hybridomas can be prepared from the lymphocytes and immortalized myeloma cells using the general somatic cell hybridization technique of Kohler, B. and Milstein, C. (1975) Nature 256:495-497 or as modified by Buck, D. W., et al., In Vitro, 18:377-381 (1982). Available myeloma lines, including but not limited to X63-Ag8.653 and those from the Salk Institute, Cell Distribution Center, San Diego, Calif., USA, may be used in the hybridization. Generally, the technique involves fusing myeloma cells and lymphoid cells using a fusogen such as polyethylene glycol, or by electrical means well known to those skilled in the art. After the fusion, the cells are separated from the fusion medium and grown in a selective growth medium, such as hypoxanthine-aminopterin-thymidine (HAT) medium, to eliminate unhybridized parent cells. Any of the media described herein, supplemented with or without serum, can be used for culturing hybridomas that secrete monoclonal antibodies. As another alternative to the cell fusion technique, EBV immortalized B cells may be used to produce the Notch-1 monoclonal antibodies of the subject disclosure. The hybridomas are expanded and subcloned, if desired, and supernatants are assayed for anti-immunogen activity by conventional immunoassay procedures (e.g., radioimmunoassay, enzyme immunoassay, or fluorescence immunoassay).

Hybridomas that may be used as a source of antibodies encompass all derivatives, progeny cells of the parent hybridomas that produce monoclonal antibodies specific for Notch-1 , or a portion thereof.

Hybridomas that produce such antibodies may be grown in vitro or in vivo using known procedures. The monoclonal antibodies may be isolated from the culture media or body fluids, by conventional immunoglobulin purification procedures such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography, and ultrafiltration, if desired. Undesired activity, if present, can be removed, for example, by running the preparation over adsorbents made of the immunogen attached to a solid phase and eluting or releasing the desired antibodies off the immunogen. Immunization of a host animal with a human Notch-1 , or a fragment containing the target amino acid sequence conjugated to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example,

maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N- hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCI2, or R1 N=C=NR, where R and R1 are different alkyl groups, can yield a population of antibodies (e.g., monoclonal antibodies).

Humanization of anti-Notch-1 antibodies generated by immunization in a host animal.

It is within the contemplation of the current disclosure that anti-Notch-1 antibodies of the disclosure wherein the antibodies are generated by immunization in a host animal can be manipulated in many ways to increase their biological activity and

pharmaceutical properties. One way of such manipulation is humanization.

Methods of humanizing antibodies are well known to those of ordinary skill in the art. In general, there are four basic steps to humanize a monoclonal antibody. These are: (1 ) determining the nucleotide and predicted amino acid sequence of the starting antibody light and heavy variable domains (2) designing the humanized antibody, i.e., deciding which antibody framework region to use during the humanizing process (3) the actual humanizing methodologies/techniques and (4) the transfection and expression of the humanized antibody. See, for example, U.S. Patent Nos. 4,816,567; 5,807,715; 5,866,692; 6,331 ,415; 5,530, 101 ; 5,693,761 ; 5,693,762; 5,585,089; and 6, 180,370.

A number of "humanized" antibody molecules comprising an antigen-binding site derived from a non-human immunoglobulin have been described in the literature, including chimeric antibodies having rodent or modified rodent V regions and their associated CDRs fused to human constant domains. Other references describe rodent CDRs grafted into a human supporting framework region (FR) prior to fusion with an appropriate human antibody constant domain. Other references describe rodent CDRs supported by recombinantly engineered rodent framework regions. Such "humanized" molecules are designed to minimize unwanted immunological response toward rodent anti-human antibody molecules which limits the duration and effectiveness of therapeutic applications of those moieties in human recipients. For example, the antibody constant region can be engineered such that it is immunologically inert (e.g., does not trigger complement lysis).

Human anti-Notch-1 antibodies

It is within the contemplation of the current disclosure that fully human anti-Notch-1 antibodies may be obtained by using commercially available mice that have been engineered to express specific human immunoglobulin proteins. Transgenic animals that are designed to produce a more desirable (e.g., fully human antibody) or more robust immune response may also be used for generation of humanized or human antibodies. Examples of such technologies are Xenomouse™ from Abgenix, Inc. (Fremont, CA) and HuMAb-Mouse® and TC Mouse™ from Medarex, Inc. (Princeton, NJ).

It is also within the contemplation of the current disclosure that fully human anti-Notch-1 antibodies may be obtained recombinantly following general methods of phage display technology, as will be readily apparent to those of skill in the art.

Alternatively, the phage display technology can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors. According to this technique, antibody V domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties of the B cell. Several sources of V-gene segments can be used for phage display. A repertoire of V genes from unimmunized human donors can be constructed and antibodies to a diverse array of antigens (including self-antigens) can be isolated following general techniques that have been disclosed in the literature. In a natural immune response, antibody genes accumulate mutations at a high rate (somatic hypermutation). Some of the changes introduced will confer higher affinity, and B cells displaying high-affinity surface immunoglobulin are preferentially replicated and differentiated during subsequent antigen challenge. This natural process can be mimicked by employing the technique known as "chain shuffling." In this method, the affinity of "primary" human antibodies obtained by phage display can be improved by sequentially replacing the heavy and light chain V region genes with repertoires of naturally occurring variants (repertoires) of V domain genes obtained from unimmunized donors. This technique allows the production of antibodies and antibody fragments with affinities in the pM-nM range. Strategies for making very large phage antibody repertoires (also known as "the mother-of-all libraries") are known to those of skill in the art.

Gene shuffling can also be used to derive human antibodies from rodent antibodies, where the human antibody has similar affinities and specificities to the starting rodent antibody. According to this method, which is also referred to as "epitope imprinting", the heavy or light chain V domain gene of rodent antibodies obtained by phage display technique is replaced with a repertoire of human V domain genes, creating rodent-human chimeras. Selection on antigen results in isolation of human variable regions capable of restoring a functional antigen-binding site, i.e., the epitope governs (imprints) the choice of partner. When the process is repeated in order to replace the remaining rodent V domain, a human antibody is obtained. Unlike traditional humanization of rodent antibodies by CDR grafting, this technique provides completely human antibodies, which have no framework or CDR residues of rodent origin.

Although the above discussion pertains to humanized and human antibodies, the general principles discussed are applicable to customizing antibodies for use, for example, in dogs, cats, primate, equines and bovines. One or more aspects of humanizing an antibody described herein may be combined, e.g., CDR grafting, framework mutation and CDR mutation.

Engineered and modified anti-Notch-1 antibodies made recombinantly

In general, antibodies may be made recombinantly by placing the DNA

sequences of the desired antibody into expression vectors followed by transfection and expression in host cells, including but not limited to E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein. Other host cells, such as transgenic plant cells or transgenic milk cells may also be used.

An antibody may also be modified recombinantly. For example, the DNA of the human heavy and light chain constant regions may be used in place of the homologous murine sequences of the murine antibody DNA, or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non- immunoglobulin polypeptide. In similar manner, "chimeric" or "hybrid" antibodies can be prepared that have the binding specificity of an anti-Notch-1 monoclonal antibody herein.

Antibody variable regions can also be modified by CDR grafting. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies by constructing expression vectors that include CDR sequences from the specific naturally occurring antibody grafted onto framework sequences from a different antibody with different properties.

Accordingly, another aspect of the disclosure pertains to an isolated monoclonal antibody comprising a heavy chain variable region comprising CDR1 , CDR2, and CDR3 sequences as described herein, and a light chain variable region comprising CDR1 , CDR2, and CDR3 sequences as desribed herein. Thus, such antibodies contain the VH and V_L CDR sequences of the monoclonal antibodies described herein, yet may contain different framework sequences from these antibodies. Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences.

Another type of variable region modification is to mutate amino acid residues within the V_H and/or V_L CDR1 , CDR2 and/or CDR3 regions to thereby improve one or more binding properties (e.g., affinity) of the antibody of interest. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutation(s) and the effect on antibody binding, or other functional property of interest, can be evaluated using in vitro or in vivo assays as described herein. Typically, conservative modifications (as discussed below) are introduced. The mutations may be amino acid substitutions, additions or deletions. Moreover, typically no more than one, two, three, four or five residues within a CDR region are modified. Epitope Mapping

The binding epitopes of monoclonal antibodies on an antigen may be mapped by a number of methods depending on the type of antigen-antibody interactions.

If an antibody binds to a single epitope consisting of sequential amino acid residues in an antigen, whose binding usually is not affected by antigen conformational changes, the binding epitope is called a linear epitope. A peptide scanning method is commonly used to identify linear binding epitopes, which requires synthesizing a series of overlapping 10-15 mer peptides that cover the entire length of the antigen sequence. The peptides are arrayed on a protein-cross-linking membrane in duplicate dotted format. Antibody binding affinity to the peptide array is analyzed similar to an ELISA assay. The peptides-arrayed membrane is first incubated in 1 X PBST with 5% fetal calf serum to block nonspecific binding, then incubated with testing antibodies or nonspecific control antibody followed by incubation with HRP-labeled secondary antibody. The antibody binding strength is read out using a chemiluminescence imaging instrument.

Alternatively, a linear binding epitope may be identified using antigen protein domains displayed on yeast cell surface or using antigen protein fragments displayed on bacterial cell surface followed by flow-cytometric sorting or FACS.

Limited proteolysis of peptide antigen and antibody complex, combined with mass spectrometry, may provide another approach to locate linear binding epitopes. Antigen and antibody are mixed and incubated at appropriate conditions to form a binding complex, which is digested by protease under controlled temperature and time. The bound reaction mixture is then passed through a protein-A affinity column to retain the antibody bound with an antigen epitope fragment, which is analyzed by mass spectrometry after eluted from the column.

Mapping of conformational epitopes depends on the interaction of antibody to antigen in its native conformation. A number of techniques have been reported to be useful in determining conformational epitopes. One of the methods commonly used is amino acid mutagenesis. Individual amino acid residues in the antigen protein speculated to bind with the antibody are mutated, and the mutated antigen protein is then expressed and subjected to antibody binding analysis to determine if the binding affinity is impaired. However, systematic amino acid mutagenesis across the complete antigen protein sequence is laborious. To narrow down the regions of antigen protein that interact with antibody, substitution of an individual antigen domain by a closely related protein domain can be a useful method.

As will be recognized by those of skill in the art, shotgun mutagenesis mapping was developed to overcome the shortcomings of conventional amino acid mutagenesis. This method utilizes a comprehensive mutation library made from antigen cDNA, with each plasmid clone containing a unique point mutation and the entire mutation library covering every amino acid of the antigen coding region. The library of plasmid clones are transfected in HEK-293T or other human cells, and the cells are then arrayed in 384-well microplates. Antibody binding activity is assayed after fixation of cells on the microplate. If amino acid mutations caused a loss of reactivity, it is identified as an antibody binding epitope.

Cocrystalization of antigen-antibody complex, X-ray diffraction and structural analysis gives direction visualization of antigen-antibody interaction. When combined with amino acid mutagenesis, the technologies would provide powerful evidence and vivid picture for antibody binding epitopes. However, cocrystalization and structural analysis are technically challenging, requires large quantity of purified antigen and antibody, and can be a time-consuming trial and error process.

In order to make an anti-Notch-1 antibody that binds to an epitope or a specified set of epitopes, one can generate anti-Notch-1 antibodies, then determine the epitope or the set of epitopes that each of these antibodies binds to according to the above mapping methods generally known in the art. One can then select those anti-Notch-1 antibodies that bind to the specific epitope or the specific set of epitopes.

Variant Sequences

As disclosed herein, variants of the antibody sequences may be made by recombinant modifications, such as by conservative substitution of one or more of the amino acid residues of the antibody or by one or more deletions or additions of amino acids to that of the antibody.

Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue or the antibody fused to an epitope tag. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody of an enzyme or a polypeptide which increases the half-life of the antibody in the blood circulation.

Substitution variants have at least one amino acid residue in the antibody molecule removed and a different residue inserted in its place. The sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but FW alterations are also contemplated. In general, a conservative amino acid substitution should not substantially change the structural characteristics of the parent sequence; e.g., a replacement amino acid should not alter the anti-parallel β-sheet that makes up the immunoglobulin binding domain that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence. In general, glycine and proline would not be used in an anti-parallel β-sheet. Examples of conservative substitutions are shown in Table 3.

Table 3: Amino Acid Substitutions

Exemplary Conservative

Original Residue Substitutions

Ala (A) Val; Leu; lie

Arg (R) Lys; Gin; Asn

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

Asp (D) Glu; Asn

Cys (C) Ser; Ala

Gin (Q) Asn; Glu

Glu (E) Asp; Gin

Gly (G) Ala

His (H) Asn; Gin; Lys; Arg

Leu; Val; Met; Ala; Phe;

lie (I)

Norleucine

Norleucine; lie; Val; Met;

Leu (L)

Ala; Phe Exemplary Conservative

Original Residue Substitutions

Lys (K) Arg; Gin; Asn

Met (M) Leu; Phe; lie

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

Pro (P) Ala

Ser (S) Thr

Thr (T) Ser

Trp (W) Tyr; Phe

Tyr (Y) Trp; Phe; Thr; Ser

lie; Leu; Met; Phe; Ala;

Val (V)

Norleucine

Substantial modifications in the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side-chain properties:

(1 ) Non-polar: Norleucine, Met, Ala, Val, Leu, lie;

(2) Polar without charge: Cys, Ser, Thr, Asn, Gin;

(3) Acidic (negatively charged): Asp, Glu;

(4) Basic (positively charged): Lys, Arg;

(5) Residues that influence chain orientation: Gly, Pro; and

(6) Aromatic: Trp, Tyr, Phe, His.

Non-conservative substitutions are made by exchanging a member of one of these classes for another class.

Any cysteine residue not involved in maintaining the proper conformation of the antibody also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, cysteine bond(s) may be added to the antibody to improve its stability, particularly where the antibody is an antibody fragment such as an Fv fragment. Affinity matured anti-Notch-1 antibodies

The disclosure includes affinity matured embodiments. For example, affinity matured antibodies can be produced by procedures known in the art.

The following methods may be used for adjusting the affinity of an antibody and for characterizing a CDR. One way of characterizing a CDR of an antibody and/or altering (such as improving) the binding affinity of a polypeptide, such as an antibody, referred to "library scanning mutagenesis". Generally, library scanning mutagenesis works as follows. One or more amino acid positions in the CDR are replaced with two or more (such as 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20) amino acids using art recognized methods. This generates small libraries of clones (in some embodiments, one for every amino acid position that is analyzed), each with a complexity of two or more members (if two or more amino acids are substituted at every position). Generally, the library also includes a clone comprising the native

(unsubstituted) amino acid. A small number of clones, e.g., about 20-80 clones

(depending on the complexity of the library), from each library are screened for binding affinity to the target polypeptide (or other binding target), and candidates with increased, the same, decreased, or no binding are identified.

In some embodiments, every amino acid position in a CDR is replaced, in some embodiments, one at a time, with all 20 natural amino acids using art recognized mutagenesis methods. This generates small libraries of clones, in some embodiments, one for every amino acid position that is analyzed, each with a complexity of 20 members, if all 20 amino acids are substituted at every position.

In some embodiments, the library to be screened comprises substitutions in two or more positions, which may be in the same CDR or in two or more CDRs. Thus, the library may comprise substitutions in two or more positions in one CDR. The library may comprise substitutions in two or more positions in two or more CDRs. The library may comprise substitutions in 3, 4, 5, or more positions, said positions found in two, three, four, five or six CDRs. The substitution may be prepared using low redundancy codons. Each CDR may be a Kabat CDR, a Chothia CDR, or an extended CDR.

Candidates with improved binding may be sequenced, thereby identifying a CDR substitution mutant which results in improved affinity, which substitution is also referred to an "improved" substitution. Candidates that bind may also be sequenced, thereby identifying a CDR substitution which retains binding.

Multiple rounds of screening may be conducted. For example, candidates each comprising an amino acid substitution at one or more position of one or more CDR, with improved binding are also useful for the design of a second library containing at least the original and substituted amino acid at each improved CDR position (i.e., amino acid position in the CDR at which a substitution mutant showed improved binding).

Preparation, and screening or selection of this library is discussed further below.

Library scanning mutagenesis also provides a means for characterizing a CDR, in so far as the frequency of clones with improved binding, the same binding, decreased binding or no binding also provide information relating to the importance of each amino acid position for the stability of the antibody-antigen complex. For example, if a position of the CDR retains binding when changed to all 20 amino acids, that position is identified as a position that is unlikely to be required for antigen binding. Conversely, if a position of CDR retains binding in only a small percentage of substitutions, that position is identified as a position that is important to CDR function. Thus, the library scanning mutagenesis methods generate information regarding positions in the CDRs that can be changed to many different amino acids (including all 20 amino acids), and positions in the CDRs which cannot be changed or which can only be changed to a few amino acids.

Candidates with improved affinity may be combined in a second library, which includes the improved amino acid, the original amino acid at that position, and may further include additional substitutions at that position, depending on the complexity of the library that is desired, or permitted using the desired screening or selection method. In addition, if desired, adjacent amino acid position can be randomized to at least two or more amino acids. Randomization of adjacent amino acids may permit additional conformational flexibility in the mutant CDR, which may in turn, permit or facilitate the introduction of a larger number of improving mutations. The library may also comprise substitution at positions that did not show improved affinity in the first round of screening.

The second library is screened or selected for library members with improved and/or altered binding affinity using any method known in the art, including screening using Biacore surface plasmon resonance analysis, and selection using any method known in the art for selection, including phage display, yeast display, and ribosome display. Post translational modification of anti-Notch-1 antibodies

Antibodies can also be modified by post translational modifications, including, but not limited to glycosylation with different sugars, acetylation, and phosphorylation.

Antibodies are glycosylated at conserved positions in their constant regions. The oligosaccharide side chains of the immunoglobulins affect the protein's function and the intramolecular interaction between portions of the glycoprotein, which can affect the conformation and presented three-dimensional surface of the glycoprotein.

Oligosaccharides may also serve to target a given glycoprotein to certain molecules based upon specific recognition structures. Glycosylation of antibodies has also been reported to affect antibody-dependent cellular cytotoxicity (ADCC). In particular, CHO cells with tetracycline-regulated expression of 3(1 ,4)-N-acetylglucosaminyltransferase III (GnTIII), a glycosyltransferase catalyzing formation of bisecting GlcNAc, has been reported to have improved ADCC activity.

Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine, asparagine-X-threonine, and

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

Addition of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above- described tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites). The glycosylation pattern of antibodies may also be altered without altering the underlying nucleotide sequence. Glycosylation largely depends on the host cell used to express the antibody. Since the cell type used for expression of recombinant glycoproteins, e.g. antibodies, as potential therapeutics is rarely the native cell, variations in the glycosylation pattern of the antibodies can be expected.

In addition to the choice of host cells, factors that affect glycosylation during recombinant production of antibodies include growth mode, media formulation, culture density, oxygenation, pH, purification schemes and the like. Various methods have been proposed to alter the glycosylation pattern achieved in a particular host organism including introducing or overexpressing certain enzymes involved in oligosaccharide production. Glycosylation, or certain types of glycosylation, can be enzymatically removed from the glycoprotein, for example, using endoglycosidase H (Endo H), N- glycosidase F, endoglycosidase F1 , endoglycosidase F2, endoglycosidase F3. In addition, the recombinant host cell can be genetically engineered to be defective in processing certain types of polysaccharides. These and similar techniques are well known in the art.

Other methods of post translational modification include using coupling techniques known in the art, including, but not limited to, enzymatic means, oxidative substitution and chelation. Modifications can be used, for example, for attachment of labels for immunoassay.

It is likely that antibodies expressed by different cell lines or in transgenic animals will have different glycosylation from each other. However, all antibodies encoded by the nucleic acid molecules provided herein, or comprising the amino acid sequences provided herein are part of the present invention, regardless of the glycosylation of the antibodies.

Anti-Notch-1 antibodies with modified constant region

In some embodiments of the disclosure, the antibody comprises a modified constant region, such as a constant region that is immunologically inert or partially inert, e.g., does not trigger complement mediated lysis, does not stimulate antibody- dependent cell mediated cytotoxicity (ADCC), or does not activate microglia; or have reduced activities (compared to the unmodified antibody) in any one or more of the following: triggering complement mediated lysis, stimulating antibody-dependent cell mediated cytotoxicity (ADCC), or activating microglia. Different modifications of the constant region may be used to achieve optimal level and/or combination of effector functions. In some embodiments, the antibody comprises a human heavy chain lgG2 constant region comprising the following mutations: A330P331 to S330S331 (amino acid numbering with reference to the wild type lgG2 sequence). In still other

embodiments, the constant region is aglycosylated for N-linked glycosylation. In some embodiments, the constant region is aglycosylated for N-linked glycosylation by mutating the glycosylated amino acid residue or flanking residues that are part of the N-glycosylation recognition sequence in the constant region. For example,

N-glycosylation site N297 may be mutated to A, Q, K, or H. In some embodiments, the constant region is aglycosylated for N-linked glycosylation. The constant region may be aglycosylated for N-linked glycosylation enzymatically (such as removing carbohydrate by enzyme PNGase), or by expression in a glycosylation deficient host cell.

Modifications within the Fc region can typically be used to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity. Furthermore, an antibody of the disclosure may be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation pattern, again to alter one or more functional properties of the antibody. Each of these aspects is described in further detail below. The numbering of residues in the Fc region is that of the EU index of Kabat.

In one case, the hinge region of CH1 is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. The number of cysteine residues in the hinge region of CH1 is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.

In another case, the Fc hinge region of an antibody is mutated to decrease the biological half life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that the antibody has impaired Staphylococcyl protein A (SpA) binding relative to native Fc- hinge domain SpA binding. ln another case, the antibody is modified to increase its biological half life.

Various approaches are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F. Alternatively, to increase the biological half life, the antibody can be altered within the CH 1 or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG.

In yet other cases, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector function(s) of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand to which affinity is altered can be, for example, an Fc receptor or the C1 component of complement.

In another case, one or more amino acids selected from amino acid residues 329, 331 and 322 can be replaced with a different amino acid residue such that the antibody has altered C1 q binding and/or reduced or abolished complement dependent cytotoxicity (CDC).

In another example, one or more amino acid residues within amino acid positions 231 and 239 are altered to thereby alter the ability of the antibody to fix complement.

In yet another example, the Fc region is modified to increase the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for an Fey receptor by modifying one or more amino acids at the following positions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301 , 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331 , 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. Moreover, the binding sites on human lgG1 for FcyR1 , FcyRII, FcyRIII and FcRn have been mapped and variants with improved binding have been described in the literature and are well known to those of skill in the art. Specific mutations at positions 256, 290, 298, 333, 334 and 339 have been shown to improve binding to FcyRIII. Additionally, the following combination mutants have been shown to improve FcyRIII binding: T256A/S298A, S298A/E333A, S298A/K224A and

S298A/E333A/K334A.

In still another example, the glycosylation of an antibody is modified. For example, an aglycoslated antibody can be made (i.e., the antibody lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for antigen. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for antigen.

Additionally or alternatively, an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies of the disclosure to thereby produce an antibody with altered glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (alpha (1 ,6) fucosyltransferase), such that antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8-/- cell lines were created by the targeted disruption of the FUT8 gene in CHO/DG44 cells using two replacement vectors. A further example is a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation by reducing or eliminating the alpha 1 ,6 bond-related enzyme. Further examples are cell lines which have a low enzyme activity for adding fucose to the N- acetylglucosamine that binds to the Fc region of the antibody or does not have the enzyme activity, for example the rat myeloma cell line YB2/0 (ATCC CRL 1662). A further example is a variant CHO cell line, Led 3 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell. Further examples include cell lines engineered to express glycoprotein-modifying glycosyl transferases (e.g., beta(1 ,4)-N- acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures which results in increased ADCC activity of the antibodies. Alternatively, the fucose residues of the antibody may be cleaved off using a fucosidase enzyme. For example, the fucosidase alpha-L-fucosidase removes fucosyl residues from antibodies.

Another modification of the antibodies herein that is contemplated by the disclosure is pegylation. An antibody can be pegylated to, for example, increase the biological (e.g., serum) half life of the antibody. To pegylate an antibody, the antibody, or fragment thereof, typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment. Typically, the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (Ci to C-io) alkoxy- or aryloxy- polyethylene glycol or polyethylene glycol-maleimide. In certain cases, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies of the present disclosure.

Other antibody modifications include antibodies that comprise, in addition to a binding domain directed at the target molecule, an effector domain having an amino acid sequence substantially homologous to all or part of a constant domain of a human immunoglobulin heavy chain. These antibodies are capable of binding the target molecule without triggering significant complement dependent lysis, or cell-mediated destruction of the target. In some embodiments, the effector domain is capable of specifically binding FcRn and/or FcYRIIb. These are typically based on chimeric domains derived from two or more human immunoglobulin heavy chain CH2 domains. Antibodies modified in this manner are particularly suitable for use in chronic antibody therapy, to avoid inflammatory and other adverse reactions to conventional antibody therapy.

Fusion protein The disclosure also encompasses fusion proteins comprising one or more fragments or regions from the antibodies or polypeptides of this disclosure. In one embodiment, a fusion polypeptide is provided that comprises at least 10 contiguous amino acids of the variable light chain region and/or at least 10 amino acids of the variable heavy chain region of the antibodies of the current disclosure. In other embodiments, a fusion polypeptide is provided that comprises at least about 10, at least about 15, at least about 20, at least about 25, or at least about 30 contiguous amino acids of the variable light chain region and/or at least about 10, at least about 15, at least about 20, at least about 25, or at least about 30 contiguous amino acids of the variable heavy chain region. In another embodiment, the fusion polypeptide comprises a light chain variable region and/or a heavy chain variable region, of the antibodies of the current disclosure. In another embodiment, the fusion polypeptide comprises one or more CDR(s) of the antibodies of the current disclosure. For purposes of this disclosure, a fusion protein contains one or more antibodies and another amino acid sequence to which it is not attached in the native molecule, for example, a heterologous sequence or a homologous sequence from another region. Exemplary heterologous sequences include, but are not limited to a "tag" such as a FLAG tag or a 6His tag.

A fusion polypeptide can be created by methods known in the art, for example, synthetically or recombinantly.

Bispecific Molecules

An antibody of the disclosure, or antigen-binding portions thereof, can be derivatized or linked to another functional molecule, e.g., another peptide or protein (e.g., another antibody or ligand for a receptor) to generate a bispecific molecule that binds to at least two different binding sites or target molecules. The antibody of the disclosure may in fact be derivatized or linked to more than one other functional molecule to generate multispecific molecules that bind to more than two different binding sites and/or target molecules; such multispecific molecules are also intended to be encompassed by the term "bispecific molecule" as used herein. To create a bispecific molecule of the disclosure, an antibody of the disclosure can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic, such that a bispecific molecule results.

Polynucleotides encoding the anti-Notch-1 antibodies

The disclosure also provides isolated polynucleotides encoding the antibodies and peptides of the disclosure, and vectors and host cells comprising the

polynucleotide.

In one aspect, the disclosure provides compositions, such as a pharmaceutical composition, comprising any of the polynucleotides of the disclosure. In some embodiments, the composition comprises an expression vector comprising a polynucleotide encoding the antibody of the disclosure. In other embodiment, the composition comprises an expression vector comprising a polynucleotide encoding any of the antibodies or polypeptides of the disclosure.

In another aspect, the disclosure provides a method of making any of the polynucleotides described herein.

Polynucleotides complementary to any such sequences are also encompassed by the present disclosure. Polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules include HnRNA molecules, which contain introns and correspond to a DNA molecule in a one-to-one manner, and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide of the present disclosure, and a polynucleotide may, but need not, be linked to other molecules and/or support materials.

Polynucleotides may comprise a native sequence (i.e., an endogenous sequence that encodes an antibody or a portion thereof) or may comprise a variant of such a sequence. Polynucleotide variants contain one or more substitutions, additions, deletions and/or insertions such that the immunoreactivity of the encoded polypeptide is not diminished, relative to a native immunoreactive molecule. The effect on the immunoreactivity of the encoded polypeptide may generally be assessed as described herein. Variants preferably exhibit at least about 70% identity, more preferably, at least about 80% identity, yet more preferably, at least about 90% identity, and most preferably, at least about 95% identity to a polynucleotide sequence that encodes a native antibody or a portion thereof.

Two polynucleotide or polypeptide sequences are said to be "identical" if the sequence of nucleotides or amino acids in the two sequences is the same when aligned for maximum correspondence as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. A "comparison window" as used herein, refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, or 40 to about 50, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.

Preferably, the "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid bases or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e. the window size) and multiplying the results by 100 to yield the percentage of sequence identity.

Variants may also, or alternatively, be substantially homologous to a native gene, or a portion or complement thereof. Such polynucleotide variants are capable of hybridizing under moderately stringent conditions to a naturally occurring DNA sequence encoding a native antibody (or a complementary sequence).

Suitable "moderately stringent conditions" include prewashing in a solution of 5 X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50°C to 65°C, 5 X SSC, overnight; followed by washing twice at 65°C for 20 minutes with each of 2X, 0.5X and 0.2X SSC containing 0.1 % SDS.

As used herein, "highly stringent conditions" or "high stringency conditions" are those that: (1 ) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1 % sodium dodecyl sulfate at 50°C; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1 % bovine serum albumin/0.1 % Ficoll/0.1 %

polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCI, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 g/ml), 0.1 % SDS, and 10% dextran sulfate at 42°C, with washes at 42°C in 0.2 x SSC (sodium chloride/sodium citrate) and 50% formamide at 55°C, followed by a high-stringency wash consisting of 0.1 x SSC containing EDTA at 55°C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to

accommodate factors such as probe length and the like.

It will be appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide as described herein. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present disclosure. Further, alleles of the genes comprising the polynucleotide sequences provided herein are within the scope of the present disclosure. Alleles are endogenous genes that are altered as a result of one or more mutations, such as deletions, additions and/or substitutions of nucleotides. The resulting mRNA and protein may, but need not, have an altered structure or function. Alleles may be identified using standard techniques (such as hybridization, amplification and/or database sequence comparison).

The polynucleotides of this disclosure can be obtained using chemical synthesis, recombinant methods, or PCR.

For preparing polynucleotides using recombinant methods, a polynucleotide comprising a desired sequence can be inserted into a suitable vector, and the vector in turn can be introduced into a suitable host cell for replication and amplification, as further discussed herein. Polynucleotides may be inserted into host cells by any means known in the art. Cells are transformed by introducing an exogenous polynucleotide by direct uptake, endocytosis, transfection, F-mating or electroporation. Once introduced, the exogenous polynucleotide can be maintained within the cell as a non-integrated vector (such as a plasmid) or integrated into the host cell genome. The polynucleotide so amplified can be isolated from the host cell by methods well known within the art.

Alternatively, PCR, which is well known in the art, allows reproduction of DNA sequences.

RNA can be obtained by using the isolated DNA in an appropriate vector and inserting it into a suitable host cell. When the cell replicates and the DNA is transcribed into RNA, the RNA can then be isolated using methods well known to those of skill in the art.

Suitable cloning vectors may be constructed according to standard techniques, or may be selected from a large number of cloning vectors available in the art. While the cloning vector selected may vary according to the host cell intended to be used, useful cloning vectors will generally have the ability to self-replicate, may possess a single target for a particular restriction endonuclease, and/or may carry genes for a marker that can be used in selecting clones containing the vector. Suitable examples include plasmids and bacterial viruses, e.g., pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19, pBR322, pMB9, ColE1 , pCR1 , RP4, phage DNAs, and shuttle vectors such as pSA3 and pAT28. These and many other cloning vectors are available from commercial vendors such as BioRad, Strategene, and Invitrogen.

Expression vectors generally are replicable polynucleotide constructs that contain a polynucleotide according to the disclosure. It is implied that an expression vector must be replicable in the host cells either as episomes or as an integral part of the chromosomal DNA. Suitable expression vectors include but are not limited to plasmids, viral vectors, including adenoviruses, adeno-associated viruses, retroviruses, cosmids, and expression vector(s), all of which are well known to those of skill in the art. Vector components may generally include, but are not limited to, one or more of the following: a signal sequence; an origin of replication; one or more marker genes; suitable

transcriptional controlling elements (such as promoters, enhancers and terminator). For expression (i.e., translation), one or more translational controlling elements are also usually required, such as ribosome binding sites, translation initiation sites, and stop codons.

The vectors containing the polynucleotides of interest can be introduced into the host cell by any of a number of appropriate means, including electroporation, transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE- dextran, or other substances; microprojectile bombardment; lipofection; and infection (e.g., where the vector is an infectious agent such as vaccinia virus). The choice of introducing vectors or polynucleotides will often depend on features of the host cell.

The disclosure also provides host cells comprising any of the polynucleotides described herein. Any host cells capable of over-expressing heterologous DNAs can be used for the purpose of isolating the genes encoding the antibody, polypeptide or protein of interest. Non-limiting examples of mammalian host cells include but not limited to COS, HeLa, and CHO cells. Suitable non-mammalian host cells include prokaryotes (such as E. coli or B. subtillis) and yeast (such as S. cerevisae, S. pombe; or K. lactis). Preferably, the host cells express the cDNAs at a level of about 5 fold higher, more preferably, 10 fold higher, even more preferably, 20 fold higher than that of the corresponding endogenous antibody or protein of interest, if present, in the host cells. Screening the host cells for a specific binding to Notch-1 or a Notch-1 domain is effected by an immunoassay or FACS. A cell overexpressing the antibody or protein of interest can be identified.

Pharmaceutical Compositions

In another aspect, the present disclosure provides a composition, e.g., a pharmaceutical composition, containing one or a combination of monoclonal antibodies, or antigen-binding portion(s) thereof, of the present disclosure, formulated together with a pharmaceutically acceptable carrier. Such compositions may include one or a combination of (e.g., two or more different) antibodies, or immunoconjugates or bispecific molecules of the disclosure. For example, a pharmaceutical composition of the disclosure can comprise a combination of antibodies (or immunoconjugates or bispecifics) that bind to different epitopes on the target antigen or that have

complementary activities.

Pharmaceutical compositions of the disclosure also can be administered in combination therapy, i.e., combined with other agents. For example, the combination therapy can include an anti-Notchl antibody of the present disclosure combined with at least one other anti-inflammatory or immunosuppressant agent. Examples of therapeutic agents that can be used in combination therapy are described in greater detail below in the section on uses of the antibodies of the disclosure.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Typically, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., antibody, antigen-binding portion thereof, immunoconjuage, or bispecific molecule, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

In certain embodiments, the antibodies of the present disclosure may be present in a neutral form (including zwitter ionic forms) or as a positively or negatively-charged species. In some cases, the antibodies may be complexed with a counterion to form a pharmaceutically acceptable salt. Thus, the pharmaceutical compounds of the disclosure may include one or more pharmaceutically acceptable salts.

A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound (e.g. antibody) and does not impart undesired toxicological effects. For example, the term "pharmaceutically acceptable salt" includes a complex comprising one or more antibodies and one or more counterions, where the counterions are derived from pharmaceutically acceptable inorganic and organic acids and bases.

Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl- substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as Ν,Ν'-dibenzylethylenediamine, N- methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like. Furthermore, pharmaceutically acceptable inorganic bases include metallic ions. Metallic ions include, but are not limited to, appropriate alkali metal salts, alkaline earth metal salts and other physiological acceptable metal ions. Salts derived from inorganic bases include aluminum, ammonium, calcium, cobalt, nickel, molybdenum, vanadium, manganese, chromium, selenium, tin, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, rubidium, sodium, and zinc, and in their usual valences.

Pharmaceutically acceptable acid addition salts of the antibodies of the present disclosure can be prepared from the following acids, including, without limitation formic, acetic, acetamidobenzoic, adipic, ascorbic, boric, propionic, benzoic, camphoric, carbonic, cyclamic, dehydrocholic, malonic, edetic, ethylsulfuric, fendizoic,

metaphosphoric, succinic, glycolic, gluconic, lactic, malic, tartaric, tannic, citric, nitric, ascorbic, glucuronic, maleic, folic, fumaric, propionic, pyruvic, aspartic, glutamic, benzoic, hydrochloric, hydrobromic, hydroiodic, lysine, isocitric, trifluoroacetic, pamoic, propionic, anthranilic, mesylic, orotic, oxalic, oxalacetic, oleic, stearic, salicylic, aminosalicylic, silicate, p-hydroxybenzoic, nicotinic, phenylacetic, mandelic, embonic, sulfonic, methanesulfonic, phosphoric, phosphonic, ethanesulfonic, ethanedisulfonic, ammonium, benzenesulfonic, pantothenic, naphthalenesulfonic, toluenesulfonic, 2- hydroxyethanesulfonic, sulfanilic, sulfuric, nitric, nitrous, sulfuric acid monomethyl ester, cyclohexylaminosulfonic, β-hydroxybutyric, glycine, glycylglycine, glutamic, cacodylate, diaminohexanoic, camphorsulfonic, gluconic, thiocyanic, oxoglutaric, pyridoxal 5- phosphate, chlorophenoxyacetic, undecanoic, N-acetyl-L-aspartic, galactaric and galacturonic acids.

Pharmaceutically acceptable organic bases include trimethylamine, diethylamine, N, N'-dibenzylethylenediamine, chloroprocaine, choline, dibenzylamine, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), procaine, cyclic amines, quaternary ammonium cations, arginine, betaine, caffeine, clemizole, 2-ethylaminoethanol, 2- diethylaminoethanol, 2-dimethylaminoethanol, ethanediamine, butylamine,

ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, ethylglucamine, glucamine, glucosamine, histidine, hydrabamine, imidazole, isopropylamine,

methylglucamine, morpholine, piperazine, pyridine, pyridoxine, neodymium, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, tripropylamine, triethanolamine, tromethamine, methylamine, taurine, cholate, 6-amino-2-methyl-2- heptanol, 2-amino-2-methyl-1 ,3-propanediol, 2-amino-2-methyl-1-propanol, aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, strontium, tricine, hydrazine, phenylcydohexylamine, 2-(N-morpholino)ethanesulfonic acid, bis(2-hydroxyethyl)amino- tris(hydroxymethyl)methane, N-(2-acetamido)-2-aminoethanesulfonic acid, 1 ,4- piperazinediethanesulfonic acid, 3-morpholino-2-hydroxypropanesulfonic acid, 1 ,3- bis[tris(hydroxymethyl)methylamino]propane, 4-morpholinepropanesulfonic acid, 4-(2- hydroxyethyl)piperazine-1-ethanesulfonic acid, 2-[(2-hydroxy-1 , 1- bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid, N,N-bis(2-hydroxyethyl)-2- aminoethanesulfonic acid, 4-(N-morpholino)butanesulfonic acid, 3-(N,N-bis[2- hydroxyethyl]amino)-2-hydroxypropanesulfonic acid, 2-hydroxy-3- [tris(hydroxymethyl)methylamino]-1-propanesulfonic acid, 4-(2-hydroxyethyl)piperazine- 1-(2-hydroxypropanesulfonic acid), piperazine-1 ,4-bis(2-hydroxypropanesulfonic acid) dihydrate, 4-(2-hydroxyethyl)-1 -piperazinepropanesulfonic acid, N,N-bis(2- hydroxyethyl)glycine, N-(2-hydroxyethyl)piperazine-N'-(4-butanesulfonic acid), N- [tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid, N-tris(Hydroxymethyl)methyl- 4-aminobutanesulfonic acid, N-(1 , 1 -dimethyl-2-hydroxyethyl)-3-amino-2- hydroxypropanesulfonic acid, 2-(cyclohexylamino)ethanesulfonic acid, 3- (cyclohexylamino)-2-hydroxy-1 -propanesulfonic acid, 3-(cyclohexylamino)-1- propanesulfonic acid, N-(2-acetamido)iminodiacetic acid, 4-(cyclohexylamino)-1- butanesulfonic acid, N-[tris(hydroxymethyl)methyl]glycine, 2-amino-2-(hydroxymethyl)- 1 ,3-propanediol, and trometamol.

A pharmaceutical composition of the disclosure also may include a

pharmaceutically acceptable anti-oxidant. Examples of pharmaceutically acceptable antioxidants include: (1 ) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil- soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum

monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the disclosure is contemplated. Supplementary active compounds can also be incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.

Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation include, but are not limited to, vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.01 percent to about ninety-nine percent of active ingredient, preferably from about 0.1 percent to about 70 percent, most preferably from about 1 percent to about 30 percent of active ingredient in combination with a pharmaceutically acceptable carrier.

Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the disclosure are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

For administration of the antibody, the dosage ranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight. For example dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or within the range of 1 to 10 mg/kg. An exemplary treatment regime entails administration once per week, once every two weeks, once every three weeks, once every four weeks, once per month, once every 3 months or once every three to 6 months. Dosage regimens for an anti-Notch-1 antibody of the disclosure include, for example, 1 mg/kg body weight or 3 mg/kg body weight via intravenous administration, with the antibody being given using one of the following dosing schedules: (i) every four weeks for six dosages, then every three months; (ii) every three weeks; (iii) 3 mg/kg body weight once followed by 1 mg/kg body weight every three weeks.

In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered falls within the ranges indicated. Antibody is usually administered on multiple occasions. Intervals between single dosages can be, for example, weekly, monthly, every three months or yearly. Intervals can also be irregular as indicated by measuring blood levels of antibody to the target antigen in the patient. In some methods, dosage is adjusted to achieve a plasma antibody concentration of about 1 to 1000 μg/mL and in some methods about 25 to 300 μg/mL.

Alternatively, antibody can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody in the patient. In general, human antibodies show the longest half life, followed by humanized antibodies, chimeric antibodies, and nonhuman antibodies. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present disclosure may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present disclosure employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

A "therapeutically effective dosage" of an anti-Notch-1 antibody of the disclosure preferably results in a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. For example, for the treatment of Notch-1 - positive tumors, a "therapeutically effective dosage" preferably inhibits cell growth or tumor growth by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80% relative to untreated subjects. The ability of a compound to inhibit tumor growth can be evaluated in an animal model system predictive of efficacy in human tumors.

Alternatively, this property of a composition can be evaluated by examining the ability of the compound to inhibit, such inhibition in vitro by assays known to the skilled practitioner. A therapeutically effective amount of a therapeutic compound can decrease tumor size, or otherwise ameliorate symptoms in a subject. One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected. A composition of the present disclosure can be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. Routes of administration for antibodies of the disclosure include intravenous, intramuscular, intradermal, intraperitoneal,

subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Alternatively, an antibody or antigen biding portion thereof of the disclosure can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.

The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.

Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art.

Uses and Methods of the Disclosure

The antibodies, antibody compositions, and methods of the present disclosure have numerous in vitro and in vivo diagnostic and therapeutic utilities involving the diagnosis and treatment of Notch-1 mediated disorders. For example, these molecules can be administered to cells in culture, in vitro or ex vivo, or to human subjects, e.g., in vivo, to treat, prevent and to diagnose a variety of disorders. As used herein, the term "subject" is intended to include human and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles. Preferred subjects include human patients having disorders mediated by Notch-1 activity. The methods are particularly suitable for treating human patients having a disorder associated with aberrant Notch-1 expression or activation. When antibodies to Notch-1 are administered together with another agent, the two can be administered in either order or simultaneously.

Given the specific binding of the antibodies of the disclosure for Notch-1 , the antibodies of the disclosure can be used to specifically detect Notch-1 expression on the surface of cells and, moreover, can be used to purify Notch-1 via immunoaffinity purification.

Furthermore, the antibodies, antibody compositions and methods of the present disclosure can be used to treat a subject with abnormal cell growth, e.g., a cancer. In one particular embodiment, the cancer is T-ALL. In another particular embodiment, the cancer is breast cancer.

Other types of abnormal cell growth that may be treated by the antibodies of the disclosure include, for example, mesothelioma, hepatobilliary (hepatic and billiary duct), a primary or secondary CNS tumor, a primary or secondary brain tumor, lung cancer (NSCLC and SCLC), bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, ovarian cancer, colon cancer, rectal cancer, cancer of the anal region, stomach cancer, gastrointestinal (gastric, colorectal, and duodenal), breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, testicular cancer, chronic or acute leukemia, chronic myeloid leukemia, lymphocytic lymphomas, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, neoplasms of the central nervous system (CNS), primary CNS lymphoma, non hodgkins's lymphoma, spinal axis tumors, brain stem glioma, pituitary adenoma, adrenocortical cancer, gall bladder cancer, multiple myeloma, cholangiocarcinoma, fibrosarcoma, neuroblastoma, retinoblastoma, or a combination of one or more of the foregoing cancers. Suitable routes of administering the antibody compositions (e.g., humanized or human monoclonal antibodies, multispecific and bispecific molecules and

immunoconjugates) of the disclosure in vivo and in vitro are well known in the art and can be selected by those of ordinary skill. For example, the antibody compositions can be administered by injection (e.g., intravenous or subcutaneous). Suitable dosages of the molecules used will depend on the age and weight of the subject and the concentration and/or formulation of the antibody composition.

As previously described, humanized anti-Notch-1 antibodies of the disclosure can be co-administered with one or other more therapeutic agents, e.g., a cytotoxic agent, a radiotoxic agent or an immunosuppressive agent. The antibody can be linked to the agent (as an immunocomplex) or can be administered separate from the agent. In the latter case (separate administration), the antibody can be administered before, after or concurrently with the agent or can be co-administered with other known therapies, e.g., an anti-cancer therapy, e.g., radiation. Such therapeutic agents include, among others, anti-neoplastic agents such as doxorubicin (adriamycin), cisplatin bleomycin sulfate, carmustine, chlorambucil, and cyclophosphamide hydroxyurea which, by themselves, are only effective at levels which are toxic or subtoxic to a patient. Cisplatin can be intravenously administered as a 100 mg/dose once every four weeks and adriamycin is intravenously administered as a 60 to 75 mg/mL dose once every 21 days.

Co-administration of the human anti-Notch-1 antibodies of the present disclosure with chemotherapeutic agents provides two anti-cancer agents which operate via different mechanisms which yield a cytotoxic effect to human tumor cells. Such co-administration can solve problems due to development of resistance to drugs or a change in the antigenicity of the tumor cells which would render them unreactive with the antibody.

Kits

Also within the scope of the present disclosure are kits comprising the antibody compositions of the disclosure (e.g., human or humanized antibodies, bispecific or multispecific molecules, or immunoconjugates) and instructions for use. The kit can further contain one ore more additional reagents, such as an immunosuppressive reagent, a cytotoxic agent or a radiotoxic agent, or one or more additional antibodies of the disclosure (e.g., a human antibody having a complementary activity which binds to an epitope in the Notch-1 antigen distinct from the first human antibody).

Accordingly, patients treated with antibody compositions of the disclosure can be additionally administered (prior to, simultaneously with, or following administration of a human antibody of the disclosure) another therapeutic agent, such as a cytotoxic or radiotoxic agent, which enhances or augments the therapeutic effect of the human antibodies.

The present disclosure is further illustrated by the following examples which should not be construed as further limiting. The contents of all figures and all references, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.

Examples

Example 1 : Generation and expression of Notch 1 immunogen

The immunogen constructs were generated by multiplexing PCR (Figure 1 ) for monoclonal antibody (mAb) generation. As illustrated in Figure 1 , the Notchl

immunogen cDNA was synthesized by multiple overlapping PCR using the Notchl full- length cDNA clone as template (OriGene, Cat. No. TC308883, Rockville, MD) and High Fidelity PCR reagent system following the manufacturer's protocol (Roche, Indianapolis, IN). The recombinant Notchl immunogen cDNA, containing N-terminal leader peptide, EGF-like repeats 35 to 36, NRR, (including Lin A, B and C domains and the HD domain) and a small portion of intracellular sequence, was cloned in a Fc-fusion protein vector with Notchl immunogen fused to the N-terminus of Fc sequence. This Notchl immunogen plasmid is referred to as NI -NRR-TM(-).

A similar plasmid was constructed in parallel as shown in Figure 2, which contains the same sequence as N I-NRR-TM(-) except that the transmembrane (TM) sequence (the last 24 amino acid residues) was used to replace the intracellular sequence (the last 44 amino acid residues) of NI-NRR-TM(-). This PCR-amplified Notchl immunogen cDNA was cloned in pcDNA3.1 D/V5-His (Invitrogen). The plasmid is referred to as N1-NRR-TM(+). Expression and purification of Notch 1 immunogen protein

NI -NRR-TM(-) was expressed in FreestyleTM 293-F cells (Invitrogen, Inc., Calsbad, CA) by transient transfection using FreestyleTM Max Reagent (Invitrogen) and the manufacturer's protocol, verified by Western blot analysis. Briefly, 1 X 10⁷ cells were seeded in a tissue culture shaker flask containing 30 milliliters (ml.) of 293-F cell growth medium (Invitrogen). The secreted protein was analyzed by taking an aliquot of 0.5 ml_ conditioned medium every 24 hours from day 2 to day 7 after transfection. Twenty microliters (μΙ_) of conditioned medium and 2X protein sample loading buffer (BioRad, Hercules, CA) were combined, heated at 100°C for 5 minutes. The samples were separated through electrophoresis in a 4 to 12% gradient SDS-PAGE (Invitrogen). The proteins were transferred from gel to blotting membrane using a dry blotting device (Invitrogen), then the membrane was blocked in 5% non-fat dry milk in PBST (PBS with 0.05% tween-20) for one hour. Detection of N1-MRRHD-TM(-)/Fc fusion proteins was performed by incubation with human Fc-specific, HRP-conjugated antibody (Bethyl Lab. Inc. Montgomery, TX). The membrane was washed three times in PBST before developing with Supersignal Chemiluminescent Substrate (Pierce, Rockford, I L). The protein expression time course study showed that the conditioned medium of 5 to 6 day culture contains most secreted N1 -NRR-TM(-)/Fc fusion protein. Therefore, N1 -NRR- TM(-)/Fc protein expression was scaled up in 10 liters of culture volume, and the protein was purified through protein G affinity column (Invitrogen).

Establishing cell lines expressing Notchl immunogen

N1 -NRR-TM(+) was stably transfected in a mouse cell line, L-929 (ATCC, CCL-1 , Manassas, VA), expressed as cell surface membrane-anchored protein. The stable cell line was established by transfection using LipoFectamineTM 2000 (Invitrogen), and the cells were selected against 1 mg/mL of neomycin (G418) for about 9 to 15 days until individual colonies were visible by eye and picked up for clonal growth. The expression level of N1-NRR-TM(+)/V5 protein was assessed by Western blot using protein extract made from each stable transfection clone. More specifically, cells of each clone were removed from culture vessels, rinsed with phosphate buffered saline (PBS) and subjected to Western blot analysis described as above. The protein was detected by HRP-conjugated anti-V5 antibody (Invitrogen). The cell clones expressing highest level of N1 -NRR-TM(+) protein was selected for the use of immunization and cell-based antibody binding assay.

Example 2: Generation of Notchl mAb

Immunization and hybridoma cloning

Balb/c mice were immunized using human Notchl immunogen, NI-NRR-TM(-), and a long immunization protocol. The first immunization was given via subcutaneous (sc) injection with twenty micrograms ^g) of the antigen mixed in Complete Freunds Adjuvant (CFA) emulsion, followed by three biweekly sc injections with each delivering 20 μg of antigen mixed in Incomplete Freunds Adjuvant (IFA) emulsion. The serum was taken a week after fourth antigen injection to check the titer of antibodies by ELISA. The mouse with high response titer was euthanized, and the spleen was surgically removed for hybridoma cloning.

A single cell suspension of spleenocytes were prepared by forcing the spleen through a 100-micron stainless steel screen, then through a cell strainer, and wash twice in 30ml_ RPMI. The spleenocytes were mixed with Sp2/0-Ag14 cells (Sigma, St. Louis, MO) in three to one ratio, and cell fusion was facilitated by adding 50% PEG- 1500 and gentle stirring. The mixture of cells were precipitated by centrifugation, and gently washed with RPMI, followed by incubation in RPMI-1640 medium with 20% fetal calf serum (FCS) at 37°C for 30 minutes. The cells were suspended in RPMI-1640 containing 20% FCS, standard HAT (hypoxanthine, aminopterin and thymidine), 25% spleen-conditioned medium, 2 mM glutamate and 100ug/ml Pen-Strip, (Invitrogen; Calsbad, CA), dispensed in 96-well plates and cultured in 37°C/5%C02 incubator for 8 to 20 days to allow HAT-resistant hybridoma clones established. The conditioned media from each hybridoma clone were subjected to ELISA screening.

ELISA screening of monoclonal antibodies (mAb)

Enzyme-linked immunoabsorbent assay (ELISA) were performed using NuncTM MaxiSorp 96-well plates (ThermoFisher Scientific, Rochester, NY), which were prepared in two sets: the positive test plates coated overnight with 100 ng of N1-NRR-TM(-)/Fc protein in each well and the negative control plates coated with 100 ng of human Fc protein. Conditioned media from hybridoma clones were screened for their ability to bind N1 -NRR-TM(-)/Fc protein. One hundred microliters of each hybridoma supernatant were added to the coated plates, and incubated at room temperature for one hour. The wells were washed three times with PBST (1 X PBS with containing 0.05% Tween-20). Horse radish peroxidase (HRP) conjugated goat-anti-mouse Fc antibody was added to detect the mAbs bound to the antigen. Excessive HRP was washed off by three times of washes with PBST, 200 μΙ_ per well for each wash. ABTS (2,2'-azino-bis-[3- ethylbenzthiazoline-6-sulfonic acid]) solution was then added as substrate for HRP color development. The reaction was stopped and plates were scanned by a plate reader at 405 nm. Positive wells were re-screened with N1-NRR-TM(-)/Fc protein-coated plates and counter-screened with human Fc-coated plates in the same manner as described above. The hybridoma mAbs only binding to N1-NRR-TM(-)/Fc protein but not to human Fc were true Notchl-binding antibodies, which were selected to proceed for functional screening. Example 3: Identification and characterization of Notchl -antagonist mAb

Establishing luciferase reporter assay cell lines

Luciferase reporter assay was used to assess Notchl receptor-mediated signaling and transcriptional activity in a variety of settings. For assaying ligand-induced Notchl activation and mAb inhibition, the tool cell lines were developed to enhance Notch signaling. It was well established that the active form of Notch receptor consisting of intracellular domain translocates to the nucleus, and forms a complex with CSL

[named after CBF1 , Su(H) and LAG-1] binding factor 1 , which binds to the core sequence called CSL-binding motif in a gene promoter region, activating the

downstream gene transcription. Based on those discoveries, the Notchl -mediated luciferase reporter plasmid was generated. Briefly, a concatamers of eight CSL binding motifs were inserted in the multiple cloning site of pTA-Luc (BD Biosiences, Palo Alto, CA). A hygromycin selection marker (see next paragraph) was added to the down stream of luciferase gene. This yielded the luciferase reporter plasmid, CSLuc.

The full length Notchl expression construct was obtained from OriGene

(Rockville, MD) and verified by sequencing as identical to NM_017617.2

(NCBI/GenBank accession number). A hygromycin selection marker with SV40 promoter was PCR-synthesized from pcDNA3.1 /Hygromycin (Invitrogen), and connected to a growth hormone 3' poly-A signaling sequence from pcDNA5/RFT/V5-His (Invitrogen) by standard PCR joining method. The completed hygromycin marker was inserted in the Cla I site of the Notchl expression plasmids. This plasmid is renamed as Notch1/Hyg. To enhance Notchl activity, PEDT domain (Weng, A.P., et. al, Science, 2004, 9265-9273 et. al.) was deleted from Notch1/Hyg by site-directed mutagenesis (Genewiz, South Plainfield, NJ). The resulting plasmid was named as Notchl-dPEST. Human Jaggedl cDNA plasmid was obtained from Open Biosystems (Huntsville, AL). Jaggedl coding region was PCR-synthesized, and inserted into pcDNA3.3-TOPO expression vector (Invitrogen).

Notchl -dependent assay cell lines were generated by cotransfecting Notch 1/hyg and CSLuc plasmids into U2-OS (ATCC Number HTB-96, Manassas, VA) cells, or by cotransfecting Notchl -dPEST and CSLuc into 293T (ATCC Number CRL-1 1268, Manassas, VA) cells using LipoFectamine 2000 according to the manufacturer's protocol (Invitrogen). Stably-transfected cells were clonally selected against 200-800 μg/ml hygromycin in DMEM growth medium (Invitrogen), the cell clones were screened by Western blot analysis as described in Example 1 and by luciferase reporter assay described in following sections. A cell line with relatively high level of Notchl expression (based on Western blot) and Delta like-4 (DII4)-induced luciferase activity was selected for use in functional assay. Two such example cell lines are U2-OS/Notch1 -CSLuc (nick name: N1 CU3) and 293/Notch1-dPEST-CSLuc (nick name: N1 dP-c16). Through similar procedure, a cell line stably-expressing human Jaggedl was generated from a parental cell line, Hela (ATCC number CCL-2). The cell line was named as Hela/JAG1.

Luciferase reporter assay and identification of Notchl -antogonist hybridoma clones For identifying Notchl -inhibitory hybridoma clones, luciferase reporter assay was performed to assess DII4-induced Notchl activity in N1 CU3 cells. The 96-well tissue culture plates (BD Bioscience) were coated with 50 to 100 nanograms (ng) of recombinant DII4 (R&D Systems, Minneapolis, MN) per well. N1 CU3 cells were seeded at 50,000 cells per well in the DII4- or BSA-coated plates, 30 to 50 ul of conditioned media from hybridoma clones were added at same time, and cultured for 24 to 40 hours. At the end of the culture, cells were directly lysed in 1 X Passive Lysis Buffer (Promega, Madison, Wl) after removing all medium, and luciferase reporter activities were assayed using Bright-GloTM Luciferase Assay System following manufacturer's protocol (Promega, Madison, Wl) and MicroLumat Plus LB 96V luminometer (Berthhold Technologies, Bad Wildbad, Germany). Hybridoma supernatants with statistically significant inhibition to DII4-induced Notch reporter activity were subjected to affinity purification through Protein-G column (Pierce, Rockford, IL) following manufacturer's protocol. The purified mAb were further analyzed by luciferase reporter assays again to confirm the inhibitory function to Notchl -dependent signaling.

Characterization of the anti-Notchl mAb by luciferase reporter assays

Among the mAbs inhibiting Notchl -mediated signaling, one mAb (N248A) showed the most potent inhibitory activity, which was characterized in detail through several different luciferase reporter assays. Figure 3 shows that mAb N248A had much higher potency inhibiting DII4 ligand-induced Notchl signaling than that of companion mAb, mAb-C when DII4 was coated on the surface of culture plate to induce Notch signaling. The 293/Notch1-dPEST-CSLuc cells were used in the assay. The y-axis numbers are luciferase reporter activity readings.

To assess whether mAb N248A can inhibit other Notch ligand-induced signaling, Hela/Jagged1 cells and N1 dP-c16 cells were co-cultured and luciferase reporter assay as described above was performed. mAb N248A indeed completely inhibited Jaggedl - induced Notchl signaling (Figure 4).

Example 4: Determining Antibody Binding Affinity

The physical binding affinity of anti-Notchl mAb N248A, to Notchl antigen was measured on a surface plasmon resonance Biacore 3000 instrument equipped with a research-grade sensor chip (Chip type: CM5) using HBSP running buffer (Biacore AB, Uppsala, Sweden - now GE Healthcare) plus 1 mM CaC^. Protein A was amine- coupled at saturating levels onto the chip using a standard N- hydroxysuccinimide/ethyldimethylaminopropyl carbodiimide (NHS/EDC) chemistry. N 1- NRR-TM(-)Fc protein (described in Example 1 ) was captured to the chip surface by Protein A in all three flow cells at 40, 13, 4 μg/mL. Anti-Notchl mAb N248A was diluted in a 3-fold series, injected for 1 minute at 100 μί/Γηίηυίβ. Dissociation was monitored for 20 minutes. The chip was regenerated after the last injection of each titration with two 30 second pulses of 100 mM phosphoric acid. Buffer cycles provided blanks for double- referencing the data, which were then fit globally to a simple binding model using Biaevaluation software v.4.1. Affinities were deduced from the quotient of the kinetic rate constants (K_D = koff/kon). The data show that mAb N248A has K_on = 5.19e-5 (Ms), Koff <1.7e-4 (1/s) and KD < 0.33 nM. The tight K_D is contributed both by fast K_on and very slow K_off, which is slower than that resolvable by our assay (Refer to 5% rule, Biacore 3000 manual).

Example 5: Analysis of Notchl -antagonist mAb binding epitopes

Mapping mAb binding epitopes by domain swap with human Notch2

For understanding the mechanism of action by Notchl-antagonist mAb, binding epitopes of Notchl-antagonist mAbs were analyzed by domain swap and ELISA binding assays. The Notch 1-NRR-TM(-)/Fc protein was divided into six domains: EGF

(including EGF35-36), Lin12-A (or Lin-A), Lin-B, Lin-C, heterodimerization domain-N (or HD-N) and HD-C (Figure 5). Each of the domains were swapped for that corresponding to human Notch2 by PCR synthesis in a series of chimeric Notch 1 -NRR-TM(-)/Fc expression constructs. The domain-swap plasmids were transfected in FreestyleTM 293-F cells (Invitrogen, Inc., Calsbad, CA) using FreestyleTM Max Reagent (Invitrogen) as described above in Example 1 . After being cultured for three days, the conditioned media was subjected to Western blot analysis and an ELISA binding assay by Notchl mAbs. Western blot (method as in Example 1 ) showed that five of the six domain-swap constructs expressed well except that Lin-B/Notch2 swap was poorly expressed.

In ELISA assay, the 100 microliters of the conditioned medium from the above antigen plasmids-transfected cell culture were loaded in each well of the 96-well plate, and plates were incubated at room temperature for 4 hours or 40°C for overnight. The condition medium was then removed from the coated plates. For primary antibody binding, 300 ng of each mAb (Table 4) in 100 microliters of PBS was added to the coated well. The rest of the ELISA procedure is the same as described in Example 2. The mAb N99a, N326A and N440A were monoclonal antibodies that bind to Notch-1 , generated and isolated by the same procedures of that of mAb N248A. As shown in

Table 4, binding of mAb N248A to the chimeric antigens was completely abolished when the Lin-A or HD-C domain was swapped to corresponding Notch2 domain. On the other hand, swapping of EGF, Lin-C or HD-C domain did not affect its binding. The mAbs N326A and N440A were distinctly different from mAb N248A. These two mAbs require HD-N and HD-C domains for their binding activity. N99A is somewhat similar to mAb N248A in that its binding requires Lin-A and HD-C domains. However, swap of the HD-N domain also reduced N99A binding activity. These data supported the conclusion that mAb N248A has at least two distinguishable sets of binding epitopes, one in Lin-A domain and the other in HD-C domain. Whether there is another epitope in Lin-B domain was resolved in a separate experiment. Similarly, all the other three Notchl- antagonist mAbs have two identifiable sets of binding epitopes, one in the HD-C domain and the other in the domains of Notch 1 N-terminal subunit. Based on the recently published crystal structures of Notchl (Gordon, WR et al., Blood, 2009, Volume 1 13, 4381 -4390) and Notch2 (Gordon et al., Nature Structure Molecular Biology, 2007, Volume 14, 295-300) NRR regions, the three Lin12 domains are wrapped around the HD domains, blocking random cleavage and activation by ADAM protease, and therefore maintaining the receptor in non-active or silent status. The mAb N248A binds to Notchl at two distinct sets of epitopes, causing the Notchl to be locked down in the silent conformation, and thus preventing the receptor from being activated by its ligands.

Notchl gene mutations, mostly point mutations and some small deletions and insertions, have been reported in more than 50% of T-ALL. The mutations are clustered in two regions: one in the C-terminus of the intracellular moiety and the other in the HD- N domain. These findings support the notion that mAb N248A would have better therapeutic utility in T-ALL than the other three mAbs listed in Table 4 because the HD- N domain swap did not affect the binding of mAb N248A to Notchl while the binding of the other three was affected (see Table 4). Table 4: ELISA reading of Notchl mAb binding to chimeric antigens

N248A binds to human Notchl, but not mouse Notchl

To locate the binding epitopes of N248A1 , we generated murine Notchl-NRRHD expression plasmids containing mouse Notchl cDNA coding region from nucleotide 1 - 99 and nucleotide 4327 to 5169 (NCBI Accession #, NM_008714), and performed transient transfection and ELISA binding assay (methods described in previous sections, Example 1 and 5). The results showed that N248A1 does not bind to mouse Notchl , and only binds to human Notchl (Table 5). We further made domain-swap chimeric Notchl-NRRHD expression constructs using the human Notchl-NRRHD sequence (nucleotides 1-129 and 4338-5202, NCBI Accession # NM_017617) as frame work, systematically exchanged the human Lin-A, Lin-B or HD-C domains with the corresponding mouse domains. An ELISA binding assay using this human/mouse domain swap protein as bait demonstrated that the binding of N248A1 to human Notchl antigen is abolished when the Lin-A domain is exchanged to mouse sequence while the Lin-B or HD-C domain exchange did not affect the binding. In contrast, the other control mAb, 22F7, loses binding only when Lin-B is exchanged to the mouse sequence.

Therefore, the binding epitope that determines whether N248A1 only binds to human Notchl , not to mouse, is located in the Lin-A domain. Table 5: ELISA readings of Notchi mAb binding to human, mouse and chimeric antigens

Identify binding epitope of N248A 1 in Lin-A domain

To identify binding epitope of N248A1 in the Lin-A domain, we mutated the two amino acids which are different between the human and mouse Lin-A domains, i.e. 1457E/A and 1465S/N, (Table 6). ELISA results showed that mutation 1457E/A did not affect the binding, but mutation 1465S/N abolished the binding, indicating that amino acid Asn (N) in mouse Lin-A is the sole amino acid residue responsible for blocking N248A1 binding to mouse Notchi . Several amino acids surrounding 1465S were mutated to alanine sequentially (Table 6). Mutation of 1463V/A, 1466L/A or 1467Q/A also abolished the N248A1 binding. However, the control mAb A2 was not affected by the mutations 1463V/A or 1465S/N (Table 6). These experiments demonstrated that the binding epitope of N248A1 in Lin-A involves 1463V, 1465S, 1466L and 1467Q.

Table 6: Analysis of N248A1 point mutation and ELISA binding activity

Identify binding epitope of N248A 1 in HD-C domain

A serial of five sub-domain swap chimeric antigens (Table 7) were generated by sequentially swapping clusters of amino acids from the Notchi sequence to the Notch2 sequence. ELISA results showed that the sub-domain swap-1 significantly reduced N248A1 binding while the other four subdomain swap antigens does not affect N248A1 binding. On the other hand, the parallel control mAb 19H7 showed significant binding affinity reduction on sub-domain swap 1 , 3 and 5 (Table 7). All the subdomain swap antigen expression and secretion in conditioned media was confirmed by Western blot analysis (Methods in above). All subdomain swap antigens except subdomain swap 5 have equal or higher expression than the human Notchl-NRRHD (huN1 -NRRHD) protein. The subdomain swap 5 expressed at about 50% level of huN1 -NRRHD based on Western blot band intensity comparison (data not shown). These experiments showed that the binding epitope of N248A1 in HD-C includes five amino acids, 1705G, 1706A, 1707L, 1709S and 1710L (human Notchl coding cDNA sequence, NCBI accession # NM_017617), as highlighted in Table 7.

Table 7: ELISA readings of Notchl mAbs binding to human Notchl -NRRHD and chimeric antigens with subdomain swapped to Notch2 sequences

Example 6: Notchl mAb inhibits cancer cell growth in cell culture

Inhibition of HPB-ALL leukemia cells growth and reduction of NICD by mAb N248A

The T-cell acute lymphoblastic leukemia (T-ALL) cell line, HPB-ALL, was derived from a childhood T-ALL, and obtained from DSMZ (Braunschweig, Germany). This cell line harbors a Notchl mutation that leads to high level of Notchl intracellular domain (NICD), the active form of Notchl , as a result of enhanced gamma secretase cleavage. For growth inhibition assays, HPB-ALL cells were seeded in 96-well plates at 10,000 cell/well in RPMI1640 media supplemented with 10% FBS (Invitrogen). Serially diluted mAb N248A or D16A was added at beginning, and cells were cultured at 37°C for 7 day. At the end of the culture, phosphate buffered saline (PBS) containing 0.1 mg/mL of Resazurin (Sigma-Aldrich, St. Louis, MO) was added to the cells, and the plates were incubated at 37°C for 4 hours. Fluorescent signals were read through dual filters with excitation = 560 nm and emission = 590 nm. IC50 values were calculated using the sigmoidal dose-response (variable slope) in GraphPad Prism (GraphPad Software, Inc., La Jolla, CA).

For in vitro NICD analysis, HPB-ALL cells were seeded in 6-well plates at 2 x 10⁶ cells per well and cultured in RPMI 1640 with 10% FCS (Invitrogen). mAb N248A or control antibody D16A was added to the culture at variable concentrations as indicated in Figure 6. Cells were cultured in the presence of antibodies at 37°C for 24 hours. They were then collected and lysed in cold 1X Cell Lysis Buffer (Cell Signaling

Technologies, Boston MT). Proteins were extracted from cell lysate by centrifuging at 13,000 rpm for 10 minute at 4°C. Protein concentrations were determined using a BCA assay (Pierce, Rockford, IL). The level of NICD in each sample was determined by western blot analysis.

In each Western blot analysis, about 50 μg of lysate was resolved by

electrophoresis through a polyacrylamide gel (BioRad Laboratories, Hercules, CA), and transferred to nitrocellulose membrane, which was subjected to immunoblot analysis using rabbit anti-NICD antibody (Cell Signaling Technology, Inc., Danvers, MA) and mouse a actin antibody (Sigma-Aldrich, St. Louis, MO). IRDye 680 or 800 conjugated secondary antibodies (LI-COR Biosciences, Lincoln, NE) were used to visualize the Western blot bands. The images were analyzed using Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE).

The results demonstrated that inhibition of HPB-ALL cell growth by mAb N248A is correlated to the reduction of NICD levels. At 24 hours of treatment, mAb N248A reduced NICD levels in a dose dependent manner, the highest reduction was observed at 30 μg/mL. After 7 days of treatment, mAb N248A caused significant inhibition of cell growth. IC50 value for the cell growth inhibition is approximately 0.78 μg/mL or -5.2 nM.

Inhibition of breast cancer cell growth

It is well known in the literature that expression of Notchl is aberrantly increased in breast cancer which is known to be associated with poor survival rate. For example, in certain experiments involving transgenic mice expressing the activated form of Notchl in mammary tissue, nearly all the mice developed breast cancer by one year. To test the hypothesis that blocking Notchl-mediated signaling would inhibits breast cancer cell growth, several breast cancer cell lines were cultured in the presence of "I C^g/ml mAb N248A antibody, Herceptin (Genentech/Roche, South San Francisco, CA) or control mouse immunoglobulin G (mlgG). All cells were cultured in RPMI 1640 (Invitrogen) with 1 % FCS for two to three days. The viable cells was quantified by Cell Titer GlowTM (Promega), and scanned by MicroLumat Plus LB 96V luminometer

(Berthhold Technologies, Bad Wildbad, Germany). To the same panel of breast cancer cells, expression of Notchi and Jaggedl on cell surface was analyzed by FACS. The results demonstrated that the growth inhibition of the breast cancer cells by mAb N248A is roughly correlated to Notchi and Jaggedl expression level. mAb N248A exerts the strongest inhibition to MDA-MB-231 cells, which expresses relatively high level of Notchi and Jaggedl . Interestingly, BT475 cell-derived, Heceptin-resistant cell line, BT475HR, showed increased expression of Notchi and Jaggedl comparing to parental BT475 cell line. mAb N248A inhibited BT475HR cell growth, while Heceptin did not. The data indicated potential utility of mAb N248A in therapeutic treatment of breast cancer which has increased expression of Notchi or resistant to current drug, Heceptin.

Table 8: Tumor cell growth inhibition assay of anti-Notchl mAb, N248A.

The expression index in Table 8 represents fold increase of FACS geometric mean after the breast cancer cells were immuno-stained with anti-Notch 1 or anti- Jaggedl antibody. Relative cell proliferation index stands for percentage of control cell cultured in parallel without adding any agent. BT-474HR is a Herceptin-resistant cell line derived from BT-474 (ATCC).

To confirm that the growth inhibition of breast cancer cells by N248A is mediated by blocking Notch signaling, the expression of two well-known Notch down-stream target genes was assessed by quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR). MDA-MB-231 cells were cultured in the presence of N248A or control mAb for two days, and then harvested to isolate total RNA using RNAeasy reagent kit and protocol (Qiagen). The results demonstrated that mAb N248A indeed blocked HES1 and HES4 expression (Figure 7), confirming the mechanism of action by N248A.

Example 7: Notchl mAb inhibits T-cell acute lymphoblastic leukemia (T-ALL) in murine xenograft tumor model

T-ALL tumor growth inhibition by Notchl mAb

For establishing mouse model T-ALL xenograft model, immune-compromised athymic female Nude (Nu/Nu) mice (average at 20 grams, 6-8 weeks old), were obtained from Charles River Laboratories (Wilmington, MA) and housed in specific pathogen-free conditions following the guidelines of the Association for the Assessment and Accreditation for Laboratory Animal Care, International. Animals were provided sterile rodent chow and water ad libitum. All in vivo studies were carried out under approved institutional experimental animal care and use protocols.

HBP-ALL Cells were harvested from fresh culture before implanting in host mice, and washed once and re-suspended in sterile, serum-free medium. The cell suspension was adjusted appropriate density and supplemented with 50% Matrigel (BD

Biosciences, San Jose, CA) to facilitate tumor take. A total of 5-10 x 10⁶ cells in 200 μί were implanted subcutaneously into the hind-flank region of the mouse and allowed to grow to the designated size prior to the administration of antibody for each experiment.

For anti-tumor efficacy study, animals bearing HPB-AII tumors of 150-300 mm3 in size were randomized and divided into four groups receiving N248A at 1 mg, 3 mg and 10 mg per kilogram (kg) respectively, or receiving control antibody D16A at 5 mg per kg. The mAbs were injected subcutaneously once a week for 2 weeks. Animal body weight and tumor measurements were obtained every 2-3 days. Tumor volume (mm3) was measured with Vernier calipers and calculated using the formula: length (mm) x width (mm) x width (mm) x 0.4. The tumor volumes of drug-treated and vehicle-treated mice on the final day of study were used to calculate percent (5) inhibition values as 100- {1 - [(TreatedFinal day - TreatedDay 1 )/(ControlFinal day - ControlDay 1 )]}. For all tumor growth inhibition (TGI) experiments, 8 to 10 mice per dose group were used. A Student's t test was used to determine the P.

As shown in Figure 8, Notch 1 mAb, N248A, demonstrated robust antitumor activity in this model after 1 1 days' treatment, i.e. two weekly doses. The average tumor growth inhibition (TGI) in the 10 mg/kg group versus control mAb group is more than 77 %, which is highly significant in statistical term (P<0.01 ). TGI was roughly dose- dependent with an exception that the two lower dose groups, 1 mg/kg and 3 mg/kg, are too close to differentiate. The exact causes for this observation are unclear though it is likely due to high variability in tumor size of this model. N248A, as a human Notchl - specific inhibitor, was well-tolerated in mice, without causing significant weight loss, morbidity or mortality in any treatment groups.

Pharmacokinetics and pharmocodynamics (PK/PD) of Notch 1 mAb in mice

For PK/PD study, mice bearing tumors with size ranging 300-800 mm³ were administered a single dose of N248A at 5 mg/kg by subcutaneous injection. After administration of N248A mice were euthanized at time points of 6, 16 hours, and 1 , 2, 3, 5 days. Blood samples were drawn from the left cardiac ventricle using a syringe and transferred to tubes primed with heparin sulfate. In the meantime, the tumors were taken out by resection, snap-frozen and homogenized in cold 1X Cell Lysis Buffer (Cell Signaling Technologies, Boston MT). Proteins were extracted from the tumor lysate and the level of NICD in each tumor sample was determined using western blot analysis described above. The blood samples were subject to centrifugation to separate serum from blood cells. The serum level of N248A was assessed by ELISA method as described in Example 2. The ELISA plate was first coated with human Fc-specific mAb, which captures Notch 1-NRR-TM(-)/Fc antigen. The Notchl antigen in turn binds to Notchl mAb, N248A, in sera.

The PK curve indicated that N248A mAb reached maximum concentration (-235 nM) in mouse sera 24 hours after injection. The estimated half life is about 4.5 days (Figure 9). Evaluation of the direct marker for Notchl activation (i.e. NICD) showed that the tumor samples harvested from mice treated with N248A had a robust NICD reduction, which persisted until five days post dosing (Figure 10). In contrast, the control mAb, D16A, did not reduce NICD level (data not shown). The maximal inhibition of NICD by N248A at 5 mg/kg was approximately 80%.

Example 8: Cloning and sequences of Notchl mAb, N248A

The sequences of the variable regions of mAb N248A were determined. The antibody IgG subtype was first assessed using an Isostrip Mouse Monoclonal Antibody kit (Roche Diagnostics, Indianapolis, I N). The results indicated that N248A has an lgG1 heavy chain and a lambda light chain. For cloning and sequencing of mAb N248A, 1 x 10⁶ hybridoma cells were harvested and lysed to isolate total cellular RNA using RNeasy Mini Reagent kit and manufacturer's protocol (Qiagen, Valencia, CA). The first strand cDNA was synthesized on the RNA templates using Superscript III reverse transcriptase (InVitrogen). The cDNAs of the variable regions of light chain and heavy chain were amplified by PCR from the first strand cDNA using degenerate forward primers complimentary to the 5'-end of mouse lambda chain coding sequence and a reverse primer matching the constant region adjacent to the 3'-end of the variable region, or using degenerate forward primers complementary to the 5'-end of mouse lgG1 heavy chain coding sequence and a respective lgG1 constant region reverse primer. PCR cycling conditions were as follows: 1 cycle at 96°C for 1 minute, followed by 40 cycles at 95°C for 20 sec, 50°C for 20 sec, and 72°C for 30 seconds. The resulting PCR products were cloned into pCR-4-TOPO vector (Invitrogen), sequenced by conventional methods, and analyzed using Vector NTI Advance software,

(InVitrogen). The cloned antibody sequences were confirmed by direct comparison with the N-terminal sequences obtained from purified hybridoma-derived antibody, as determined by Mass spectrometry (Univ. of CA, Davis, Molecular Structure Facility). The compiled sequence results demonstrated that the variable region of mAb N248A heavy chain contains 121 amino acid residues, and the light chain contains 109 amino acid residues. Further analysis of the N248A mAb V_H sequence and V_L sequence using the Kabat system of CDR region determination led to the delineation of the heavy chain CDR1 , CDR2 and CDR3 and light change CDR1 , CDR2 and CDR3. The nucleotide and amino acid sequences of the heavy chain variable region CDR1 , CDR2 and CDR3 of mAb N248A are shown in Table 1. The nucleotide and amino acid sequences of the light chain variable region CDR1 , CDR2 and CDR3 of mAb N248A are also shown in Table 1.

Example 9: Humanized anti-Notchl Antibodies

The murine monoclonal antibody N248A was humanized and affinity matured to provide the antibody A12.2. As noted previously, the heavy chain and light chain sequence information of A12.2 is shown in Table 2. As noted below in Table 9, A12.2 has a K_on of 7.87 x10⁴ (1/Ms), a K_off of 2.48 x 10^"5 (1/s), and a K_D of 0.315 (nM), as determined at 25°C by Biacore analysis. In addition, several variants of A12.2 were prepared as shown below in Table 9. These A12.2 variants are indicated in Table 9, where the mutations in L-CDR1 , L-CDR3, H-CDR2, H-FW3 and/or H-CDR3 are shown with amino acid numbering in reference to the V_H and V_L A12.2 sequences shown in Table 2.

Table 9: Amino acid sequences and kinectic data for mAb A12.2 and its variants as determined at 25°C by Biacore analysis

Clone L-CDR1 L-CDR3 H-CDR2 H-FW3 H-CDR3 Kon (1/Ms) Koff (1/s) KD (nM)

A12.2 7.87E+04 2.48E-05 0.315

D1 L24S V94Y P56N I70F A105Y N.D. 8.30E-05 1.05 T31 S R107T

S32I

B1 L24S V94Y P56N I70F A105Y N.D. 1.30E-04 1.65 T31 S R107T

S32R

A1 1.2 I70F N.D. 1.38E-04 1.75

B2.2 L24S N.D. 1.40E-04 1.78 T31 S

S32I

H 1 R23S V94Y I70F A105Y N.D. 1.40E-04 1.78 L24F R107T

A10.2 P56H N.D. 1.49E-04 1.89

A9.2 P56H I70F N.D. 1.49E-04 1.89

B3.2 L24S I70F A105Y N.D. 1.50E-04 1.90 T31 S R107T

S32I

B1 .2 L24S I70F N.D. 1.57E-04 1.99 Clone L-CDR1 L-CDR3 H-CDR2 H-FW3 H-CDR3 Kon (1/Ms) Koff (1/s) KD (nM)

T31S

S32I

B4.2 L24S A105Y N.D. 1.60E-04 2.03 T31S R107T

S32I

B7.2 L24S V94Y I70F A105Y N.D. 1.65E-04 2.10 S25G R107T

S26L

B8.2 R23W V94Y I70F A105Y N.D. 1.79E-04 2.27

R107T

E1 L24S V94Y P56N I70F A105Y N.D. 2.20E-04 2.80 G27D R107T

A28L

C1 L24S V94Y P56N I70F A105Y N.D. 2.20E-04 2.80 S25H R107T

T26F

F4 L24S V94S P56N 170F A105Y N.D. 2.30E-04 2.92 S25T R107T

T26V

A7.2 170F A105Y N.D. 2.36E-04 3.00

R107T

F1 L24S V94Y P56N I70F A105Y N.D. 2.70E-04 3.43 S25A R107T

T26L

A1 L24S V94Y P56N I70F A105Y N.D. 2.80E-04 3.56 S25T R107T

T26V

A8.2 A105Y N.D. 3.81 E-04 4.84

R107T

A5.2 P56G I70F N.D. 4.08E-04 5.18

F57R

C4 L24S P56G I70F N.D. 4.40E-04 5.59

F57R

D4 L24S V94N P56G I70F N.D. 4.90E-04 6.23

F57R

F3 L24S V94Q P56G I70F N.D. 5.40E-04 6.86

F57R

B4 L24S V94S P56G I70F N.D. 5.60E-04 7.12

F57R

A3.2 P56G I70F R107A N.D. 6.27E-04 7.97

F57R

A1.2 P56G I70F S102K N.D. 6.86E-04 8.72

F57R A103P

A105Y

R107T

B5.2 L24S P56G I70F R107A N.D. 7.16E-04 9.10

F57R

E2 L24S V94N P56G I70F S102K N.D. 7.30E-04 9.28

F57R A103P

A105Y Clone L-CDR1 L-CDR3 H-CDR2 H-FW3 H-CDR3 Kon (1/Ms) Koff (1/s) KD (nM)

R107T

D2 L24S P56G I70F S102K N.D. 7.40E-04 9.40

F57R A103P

A105Y

R107T

C5 L24S V94S P56G I70F R107A N.D. 7.60E-04 9.66 S25T F57R

T26V

D3 L24S P56G I70F Y104G N.D. 7.80E-04 9.91

F57R A105Q

R107T

A4 L24S V94Q P56G I70F R107A N.D. 8.20E-04 10.42

F57R

B2 L24S V94Q P56G I70F S102K N.D. 8.40E-04 10.63

F57R A103P

A105Y

R107T

G3 L24S V94S P56G I70F R107A N.D. 8.50E-04 10.80

F57R

C2 L24S V94S P56G I70F S102K N.D. 9.20E-04 11.69

F57R A103P

A105Y

R107T

C3 L24S V94S P56G I70F Y104G N.D. 9.30E-04 11.82

F57R A105Q

R107T

G4 L24S V94S P56G I70F S102K N.D. 9.60-04 12.20 S25T F57R A103P

T26V A105Y

R107T

B3 L24S V94Q P56G I70F Y104G N.D. 1.10E-03 13.98

F57R A105Q

R107T

E3 L24S V94N P56G I70F Y104G N.D. 1.10E-03 13.98

F57R A105Q

R107T

A3 L24S V94N P56G I70F Y104W N.D. 1.20E-03 15.25

F57R A105S

R107T

H2 L24S P56G I70F Y104W N.D. 1.20E-03 15.25

F57R A105S

R107T

F2 L24S V94Q P56G I70F Y104W N.D. 1.20E-03 15.25

F57R A105S

R107T

G2 L24S V94S P56G I70F Y104W N.D. 1.30E-03 16.52

F57R A105S

R107T Clone L-CDR1 L-CDR3 H-CDR2 H-FW3 H-CDR3 Kon (1/Ms) Koff (1/s) KD (nM)

A5 L24S V94S P56G I70F Y104W N.D. 1.30E-03 16.52 S25T F57R A105S

T26V R107T

H4 L24S V94S P56N I70F S102K N.D. 1.30E-03 16.52 S25T A103P

T26V A105Y

R107T

E4 L24S V94S P56N I70F R107A N.D. 1.40E-03 17.79 S25T

T26V

B5 L24S V94S P56N I70F Y104W N.D. 1.90E-03 24.14 S25T A105S

T26V R107T

A6.2 P56G S102K N.D. 3.54E-03 44.98

F57R A103P

A105Y

R107T

A4.2 P56G R107A N.D. 5.74E-03 72.94

F57R

A2.2 P56G N.D. 7.93E-03 100.76

F57R

All CDRs are basec on Kaba t CDR definitions. Amino acid residues are numbered sequentially from the first residue of the mature heavy or light antibody variable region of A12.2 as shown in Table 2. All clones have sequences identical to A12.2 in heavy and light chain variable framework regions and in L-CDR2 and H-CDR1 , except as noted for framework H3 (H-FW3). K_D = k₀ff/k_on. All k₀ff values were determined in a screening mode except those that are underlined, which were obtained by global analysis of a hN 1 -NRR concentration series flowed across IgGs captured on the Biacore chip. Underlined K_D values were therefore determined experimentally by measuring k_on. Other K_D values are theoretical, based on an estimated k_on value of 7.9x10⁴ (1/Ms). N.D. = determined.

Example 10: Characterization of neutralization activity of A12.2 by luciferase reporter assays

To assess whether A12.2 can inhibit Notch ligand-induced signaling, N 1 dP-c16 cells (293/Notch1 -dPEST-CSLuc, as described in Example 1 ) were cultured on 96-well plates pre-coated with 50 μΙ_ of DLL4 ligand (diluted in PBS at 1 μg/mL) for 24 hours. A luciferase reporter assay was then performed as described previously in Example 1 . The results are shown in Figure 1 1 , which illustrates that A12.2 inhibit Jagged l -induced Notchl signaling with similar potency compared to N248A1 , while maximal inhibition by A12.2 is slightly greater (97% vs. 82%).

Example 11 : Analysis of A12.2 binding epitopes by domain swap with human Notch2

Binding epitopes of A12.2 were analyzed by domain swap and an ELISA binding assay, using the methods described in Examples 1 and 5. The results are shown in Figure 12, which illustrates that both N248A1 and A12.2 bind to similar epitopes in the Notch-1 Lin-A domain and the Notch-1 HD-C domain.

Example 12: A12.2 inhibits growth of HPB-ALL cells

A12.2 was analyzed for its ability to inhibit growth of HPB-ALL cells, using the methods described in Example 6. The results are shown in Figure 13, which illustrates the effect of A12.2 on the growth of HPB-ALL cells. In particular, seven day treatment of HPB-ALL cells with either N248A1 or A12.2 resulted in significant inhibition of cell growth with maximal inhibition of -57% (N248A1 ) and 65% (A12.2), respectively. The IC50 value for cell growth inhibition by N248A1 is approximately 1.15 μg/mL (-7.7 nM), and for A12.2 is 0.67 μg/mL (-4.5 nM).

Claims

Claims

1 . An antibody that specifically binds to human Notch-1 , comprising:

(i) an L-CDR1 amino acid sequence as set forth in SEQ ID NO:19, or a variant thereof in which 1 to 5 residues of SEQ ID NO: 19 are modified;

(ii) an L-CDR2 amino acid sequence as set forth in SEQ ID NO:20;

(iii) an L-CDR3 amino acid sequence as set forth in SEQ ID NO:21 , or a variant thereof in which 1 residue of SEQ ID NO:21 is modified;

(iv) an H-CDR1 amino acid sequence as set forth in SEQ ID NO:22, SEQ ID NO:23, or SEQ ID NO:24;

(ii) an H-CDR2 amino acid sequence as set forth in SEQ ID NO:25, or SEQ ID

NO:26, or a variant thereof in which 1 residue of SEQ ID NO:25 or SEQ ID NO: 26 is modified; and

(iii) an H-CDR3 amino acid sequence as set forth in SEQ ID NO:27, or a variant thereof in which 1 to 5 residues of SEQ ID NO:27 are modified.

2. The antibody according to claim 1 , comprising a V_H domain amino acid sequence as set forth in SEQ ID NO:18.

3. The antibody according to claim 1 , comprising a V_L domain amino acid sequence as set forth in SEQ ID NO: 17.

4. The antibody according to claim 1 , comprising a V_H domain amino acid sequence as set forth in SEQ ID NO:18 and a V_L domain amino acid sequence as set forth in SEQ ID NO:17.

5. The antibody according to claim 1 , comprising a heavy chain amino acid sequence as set forth in SEQ ID NO:37.

6. The antibody according to claim 1 , comprising a light chain amino acid sequence as set forth in SEQ ID NO:36.

7. The antibody according to claim 1 wherein said antibody is of isotype IgA, IgD, IgE, IgG, or IgM.

8. An antibody that specifically binds to human Notch-1 , wherein the heavy chain amino acid sequence of said antibody comprises the sequence set forth in SEQ ID

NO:37, the C-terminal lysine of SEQ ID NO:37 is optionally not present, and the light chain amino acid sequence comprises the sequence set forth in SEQ ID NO:36.

9. A nucleic acid that encodes the antibody according to claim 1 .

10. A nucleic acid comprising the sequence as set forth in SEQ ID NO:32 or SEQ ID NO:28.

1 1. A host cell comprising the nucleic acid according to claim 1.

12. A pharmaceutical composition comprising the antibody according to claim 1 and a pharmaceutically acceptable carrier.

13. A method of treating abnormal cell growth in a mammal in need thereof, comprising administering to said mammal the antibody according to claim 1.

14. An antibody according to claim 1 , for use in treating abnormal cell growth in a mammal in need thereof.