Anti-Notch2 antibody and method of use

Abstract

This invention provides an anti-Notch2 antibody and a method for using the same. [Solution] An isolated antibody that binds to human Notch2 is provided, which inhibits Jagged1-mediated signaling but does not inhibit DLL1-mediated signaling.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 053,034, filed on 17 July 2020, which is incorporated herein by reference in its entirety for any purpose.

[0002] Field of Invention The present invention relates to an anti-Notch2 antibody and a method for using the same. [Background technology]

[0003] background The Notch receptor family is an evolutionarily conserved class of transmembrane receptors that transmit signals influencing development in diverse organisms, including sea urchins and humans. Notch receptors and their ligands, Delta and Serrate (known as Jagged in mammals), are transmembrane proteins with a large extracellular domain containing epidermal growth factor (EGF)-like repeats. The number of Notch paralogs varies by species. For example, mammals have four Notch receptors (Notch1-Notch4), nematodes (Caenorhabditis elegans) have two (LIN-12 and GLP-1), and fruit flies (Drosophila melanogaster) have one (Notch). Notch receptors are proteolytically processed at the N-terminal site S1 of the transmembrane domain by a furin-like protease during transport to the cell surface, generating the extracellular Notch (ECN) subunit and the Notch transmembrane subunit (NTM). These two subunits remain non-covalently associated and constitute the surface receptor of mature heterodimeric cells. The Notch receptor and Notch signaling pathway are outlined, for example, in Aster et al., Annu. Rev. Pathol. Mech. Dis. 3:587-613, 2008, and Bolos et al., Endocrine Reviews 28:339-363, 2007.

[0004] The Notch2 ECN subunit contains 36 N-terminal EGF-like repeats followed by three tandem repeat Lin 12 / Notch repeat (LNR) modules preceding the S1 site. Each LNR module contains three disulfide bonds and a conserved group of acidic and polar residues expected to coordinate calcium ions. Within the EGF repeat region, there is a binding site for the activating ligand.

[0005] Binding of the Notch ligand to the ECN subunit initiates two consecutive proteolytic cleavages, mediated by regulated intramembrane proteolysis. The first cleavage at site S2 by a metalloproteinase (ADAM10 or ADAM17) makes the Notch transmembrane subunit more susceptible to a second cleavage at site S3, near the inner flap of the plasma membrane. The S3 cleavage, catalyzed by a multiprotein complex containing presenilin and nicatrin that promotes γ-secretase activity, releases the intracellular portion of the Notch transmembrane subunit, translocates it to the nucleus, and activates the transcription of the target gene. (For a review of Notch proteolytic cleavage, see, for example, Sisodia et al., Nat. Rev. Neurosci. 3:281-290, 2002.)

[0006] Five Notch ligands of the Jagged and Delta-like classes have been identified in humans (Jagged1 (also known as Serrate1), Jagged2 (also known as Serrate2), Delta-like1 (also known as DLL1), Delta-like3 (also known as DLL3), and Delta-like4 (also known as DLL4)). Each ligand is a single-pass transmembrane protein possessing a conserved N-terminal Delta, Serrate, and LAG-2 (DSL) motif essential for Notch binding. A series of EGF-like modules at the C-terminus, relative to the DSL motif, lies prior to the transmembrane segment. Unlike the Notch receptor, the ligands have a short cytoplasmic tail of 70–215 amino acids at the C-terminus. In addition, other types of ligands have been reported (e.g., DNER, NB3, and F3 / Contactin). (For a review of Notch ligands and ligand-mediated Notch activation, see, for example, D'Souza et al., Oncogene 27:5148–5167, 2008.)

[0007] The Notch pathway functions during a variety of developmental and physiological processes, including those affecting neurogenesis in flies and vertebrates. Generally, Notch signaling is involved in lateral inhibition, phylogenetic determination, and the establishment of boundaries between cell groups (see, e.g., Bray, Molecular Cell Biology 7:678-679, 2006). Inhibition of Jagged-Notch signaling has been shown to induce rapid loss of secretory bacilli and increase of ciliated cells in the mammalian airway. Blocking Jagged has also been shown to reverse goblet cell metaplasia in a preclinical asthma model (see Lafkas et al., Nature 528:127-131 (2015)).

[0008] Mucosal obstructive lung diseases are characterized by cough, sputum production, diffuse mucus obstruction, chronic inflammation, airway wall dilation, and frequent bacterial infections. In healthy individuals, the mucus layer of the lungs is rapidly transported from the distal airways towards the trachea. In individuals with mucosal obstructive diseases, epithelial defects in either ion liquid transport or mucin secretion, or both, result in high concentrations of mucus and failure of mucus transport, as well as mucus adhesion to the airway surface. This leads to mucus accumulation in the narrow airways that cannot be removed by coughing, resulting in airway obstruction, infection, and inflammation.

[0009] There is still a need for the treatment of mucosal obstructive lung diseases. The present invention described herein meets this need and provides other benefits.

Summary of the Invention

[0010] Summary The present invention provides anti-Notch2 antibodies and methods of using them.

[0011] In some embodiments, isolated antibodies that bind to human Notch2 and inhibit Jagged1-mediated signaling but do not inhibit DLL1-mediated signaling are provided. In some embodiments, isolated antibodies that bind to human Notch2 and inhibit Jagged1-mediated signaling to a greater extent than DLL1-mediated signaling are provided. In some embodiments, the antibody can achieve a maximum inhibition of 100% of Jagged1-mediated signaling and a maximum inhibition of less than 80%, or less than 70%, or less than 60% of DLL1-mediated signaling. In some embodiments, the antibody does not inhibit the binding of Jagged1 to Notch2. In some embodiments, the antibody does not inhibit the binding of DLL1 to Notch2. In some embodiments, when formatted as a bivalent IgG antibody comprising two heavy chains and two light chains, an isolated antibody that inhibits Jagged1-mediated signaling but does not inhibit DLL1-mediated signaling is provided.

[0012] In some embodiments, the antibody binds to an epitope within the EGF7 repeat of Notch2. In some embodiments, the antibody binds to an epitope between amino acids 260 and 296 of Notch2. In some embodiments, the antibody binds to a discontinuous epitope between amino acids 260 and 296 of Notch2.

[0013] In some embodiments, an isolated antibody that binds to Notch2 is provided, where the antibody binds to an epitope within the EGF7 repeat of Notch2. In some embodiments, the antibody binds to an epitope within amino acids 260-296 of Notch2. In some embodiments, the antibody binds to a discontinuous epitope within amino acids 260-296 of Notch2.

[0014] In some embodiments, the antibody that binds to Notch2 contacts arginine 268 (R268) of human Notch2. In some embodiments, the antibody does not bind to Notch2 containing lysine 268 (K268). In some embodiments, the antibody binds to a polypeptide containing the amino acid sequence of SEQ ID NO: 74, but not to a polypeptide containing the amino acid sequence of SEQ ID NO: 77. In some embodiments, the antibody binds to human Notch2 and cynomolgus monkey Notch2. In some embodiments, the antibody does not bind to mouse Notch2. In some embodiments, the antibody binds to guinea pig Notch2. In some embodiments, the antibody does not bind to human Notch1 or human Notch3.

[0015] In some embodiments, the antibody affinity (K) is less than 20 nM, less than 15 nM, less than 10 nM, or less than 5 nM, when determined by surface plasmon resonance. D It binds to human Notch2.

[0016] In some embodiments, the antibody inhibits Jagged1-mediated signaling with an IC50 of less than 20 nM, less than 15 nM, less than 10 nM, or less than 5 nM. In some embodiments, the inhibition of Jagged1-mediated signaling is determined using a high-content screening (HCS) assay.

[0017] In some embodiments, the antibody that binds to Notch2 includes: a) A heavy chain variable domain (VH) comprising (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 4, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 6 or 7, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 8, 9, 10, 11, or 12; and a light chain variable domain (VL) comprising (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 1, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 2, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 3; b) A heavy chain variable domain (VH) comprising (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 36, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 37, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 38; and a light chain variable domain (VL) comprising (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 33, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 34, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 35; c) A heavy chain variable domain (VH) comprising (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 44, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 45, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 46; and a light chain variable domain (VL) comprising (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 41, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 42, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 43; d) A heavy chain variable domain (VH) comprising (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 53, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 54, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 55, and a light chain variable domain (VL) comprising (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 49, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 50, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 51 or 52; or e) A heavy chain variable domain (VH) comprising (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 62, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 63, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 64, and a light chain variable domain (VL) comprising (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 59, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 60, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 61.

[0018] In some embodiments, the antibody includes: a) A VH sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 14; b) A VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 13; c) The VH sequence described in (a) and the VL sequence described in (b); d) A VH sequence having at least 95% sequence identity with an amino acid sequence selected from SEQ ID NOs: 17-24, 26, 28, 30, and 32; e) A VL sequence having at least 95% sequence identity with an amino acid sequence selected from SEQ ID NOs: 15, 16, 25, 27, 29, and 31; f)(d) VH sequence and (e) VL sequence; g) A VH sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 40; h) A VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 39; i) The VH sequence described in (g) and the VL sequence described in (h); j) A VH sequence having at least 95% sequence identity with an amino acid sequence selected from SEQ ID NOs: 102-106; k) A VL sequence having at least 95% sequence identity with an amino acid sequence selected from sequence numbers 98-100; l)(j) VH sequence and (k) VL sequence; m) A VH sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 48; n) A VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 47; o) The VH sequence described in (m) and the VL sequence described in (n); p) A VH sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 58; q) A VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 56 or 57; The VH sequence described in (r)(p) and the VL sequence described in (q); s) A VH sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 66; t) A VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 65; or The VH sequence described in (u)(s) and the VL sequence described in (t).

[0019] In some embodiments, the antibody includes: a) VH sequence containing the amino acid sequence of SEQ ID NO: 14; b) VL sequence containing the amino acid sequence of SEQ ID NO: 13; c) The VH sequence described in (a) and the VL sequence described in (b); d) A VH sequence containing an amino acid sequence selected from SEQ ID NOs: 17-24, 26, 28, 30, and 32; e) A VL sequence containing an amino acid sequence selected from SEQ ID NOs: 15, 16, 25, 27, 29, and 31; f)(d) VH sequence and (e) VL sequence; g) VH sequence containing the amino acid sequence of SEQ ID NO: 40; h) VL sequence containing the amino acid sequence of SEQ ID NO: 39; i) The VH sequence described in (g) and the VL sequence described in (h); j) A VH sequence containing an amino acid sequence selected from sequence numbers 101 to 106; k) A VL sequence containing an amino acid sequence selected from SEQ ID NOs. 98-100; l)(j) VH sequence and (k) VL sequence; m) VH sequence containing the amino acid sequence of SEQ ID NO: 48; n) VL sequence containing the amino acid sequence of SEQ ID NO: 47; o) The VH sequence described in (m) and the VL sequence described in (n); p) VH sequence containing the amino acid sequence of SEQ ID NO: 58; q) A VL sequence containing the amino acid sequence of SEQ ID NO: 56 or 57; The VH sequence described in (r)(p) and the VL sequence described in (q); s) VH sequence containing the amino acid sequence of SEQ ID NO: 66; t) A VL sequence containing the amino acid sequence of SEQ ID NO: 65; or The VH sequence described in (u)(s) and the VL sequence described in (t).

[0020] In some embodiments, the antibody includes: a) VH sequence containing the amino acid sequence of SEQ ID NO: 14; b) VL sequence containing the amino acid sequence of SEQ ID NO: 13; c) The VH sequence described in (a) and the VL sequence described in (b); d) A VH sequence containing an amino acid sequence selected from SEQ ID NOs: 17-24, 26, 28, 30, and 32; e) A VL sequence containing an amino acid sequence selected from SEQ ID NOs: 15, 16, 25, 27, 29, and 31; f)(d) VH sequence and (e) VL sequence; g) VH sequence containing the amino acid sequence of SEQ ID NO: 40; h) VL sequence containing the amino acid sequence of SEQ ID NO: 39; i) The VH sequence described in (g) and the VL sequence described in (h); j) A VH sequence containing an amino acid sequence selected from sequence numbers 101 to 106; k) A VL sequence containing an amino acid sequence selected from SEQ ID NOs. 98-100; l)(j) VH sequence and (k) VL sequence; m) VH sequence containing the amino acid sequence of SEQ ID NO: 48; n) VL sequence containing the amino acid sequence of SEQ ID NO: 47; o) The VH sequence described in (m) and the VL sequence described in (n); p) VH sequence containing the amino acid sequence of SEQ ID NO: 58; q) A VL sequence containing the amino acid sequence of SEQ ID NO: 56 or 57; The VH sequence described in (r)(p) and the VL sequence described in (q); s) VH sequence containing the amino acid sequence of SEQ ID NO: 66; t) A VL sequence containing the amino acid sequence of SEQ ID NO: 65; or The VH sequence described in (u)(s) and the VL sequence described in (t).

[0021] In some embodiments, the antibody includes: a) A VH sequence having at least 95% sequence identity with an amino acid sequence selected from SEQ ID NOs: 17-24, 26, 28, 30, and 32; b) A VL sequence having at least 95% sequence identity to an amino acid sequence selected from SEQ ID NOs: 15, 16, 25, 27, 29, and 31; or c) The VH sequence described in (a) and the VL sequence described in (b).

[0022] In some embodiments, the antibody includes: a) A VH sequence containing an amino acid sequence selected from SEQ ID NOs: 17-24, 26, 28, 30, and 32; b) A VL sequence containing an amino acid sequence selected from SEQ ID NOs. 15, 16, 25, 27, 29, and 31; or c) The VH sequence described in (a) and the VL sequence described in (b).

[0023] In some embodiments, the antibody is: a) Includes the VH sequence of sequence number 26 and the VL sequence of sequence number 25; b) Includes the VH sequence of sequence number 28 and the VL sequence of sequence number 27; c) Includes the VH sequence of SEQ ID NO: 30 and the VL sequence of SEQ ID NO: 29; or d) Includes the VH sequence of sequence number 32 and the VL sequence of sequence number 31.

[0024] In some embodiments, the antibody that binds to Notch2 is a monoclonal antibody. In some embodiments, the antibody is a humanized antibody or a chimeric antibody. In some embodiments, the antibody that binds to Notch2 is an antibody fragment that binds to Notch2. In some embodiments, the antibody fragment is selected from Fv, Fab, Fab', Fab'-SH, and F(ab')2. In some embodiments, the antibody fragment is Fab, Fab', or Fab'-SH. In some embodiments, the antibody is a full-length antibody.

[0025] In some embodiments, antibodies are provided that compete with the antibodies provided herein for binding to human Notch2.

[0026] In some embodiments, isolated nucleic acids encoding antibodies that bind to Notch2, as provided herein, are provided. In some embodiments, host cells containing nucleic acids are provided. In some embodiments, host cells expressing antibodies provided herein are provided. In some embodiments, a method for producing antibodies that bind to human Notch2 is provided, comprising culturing host cells under conditions suitable for antibody expression. In some embodiments, the method further comprises recovering the antibodies from the host cells. In some embodiments, antibodies produced by host cells are provided.

[0027] In some embodiments, a pharmaceutical composition is provided comprising an antibody that binds to Notch2 and a pharmaceutically acceptable carrier, as provided herein. In some embodiments, the pharmaceutical composition further comprises an additional therapeutic agent. In some embodiments, the additional therapeutic agent is selected from hypertonic saline, mannitol, pulmozyme, N-acetylcysteine, cysteamine, and bronchodilators.

[0028] In some embodiments, antibodies or pharmaceutical compositions that bind to Notch2 are provided herein for use as pharmaceuticals. In some embodiments, antibodies or pharmaceutical compositions that bind to Notch2 are provided herein for use in the treatment of mucosal-obstructive pulmonary diseases. In some embodiments, mucosal-obstructive pulmonary diseases are selected from chronic obstructive pulmonary disease (COPD), cystic fibrosis, primary ciliary dyskinesia, non-cystic fibrosis, bronchiectasis, and bronchiolitis.

[0029] In some embodiments, the use of antibodies or pharmaceutical compositions conjugated to Notch2 in the manufacture of a pharmacopoeia for the treatment of mucosal-obstructive pulmonary diseases is provided. In some embodiments, mucosal-obstructive pulmonary diseases are selected from chronic obstructive pulmonary disease (COPD), cystic fibrosis, primary ciliary dyskinesia, non-cystic fibrosis, bronchiectasis, and bronchiolitis. In some embodiments, the use of antibodies or pharmaceutical compositions conjugated to Notch2 in the manufacture of a pharmacopoeia for reducing the number of secretory cells in a subject is provided. In some embodiments, the pharmacopoeia converts secretory cells into ciliated cells. In some embodiments, the secretory cells are located in the lungs of the subject. In some embodiments, the secretory cells are goblet cells.

[0030] In some embodiments, a method is provided for treating a subject having a mucosal-obstructive pulmonary disease, comprising administering an effective amount of an antibody conjugated to Notch2, as provided herein, or a pharmaceutical composition provided herein, to the subject. In some embodiments, the mucosal-obstructive pulmonary disease is selected from chronic obstructive pulmonary disease (COPD), cystic fibrosis, primary ciliary dyskinesia, non-cystic fibrosis, bronchiectasis, and bronchiolitis. In some embodiments, a method is provided for reducing the number of secretory cells in a subject, comprising administering an effective amount of an antibody conjugated to Notch2, as provided herein, or a pharmaceutical composition provided herein, to deplete the secretory cells in the subject. In some embodiments, the method comprises converting the secretory cells to ciliated cells. In some embodiments, the secretory cells are located in the lungs of the subject. In some embodiments, the secretory cells are goblet cells. In some embodiments, the method further comprises administering an additional therapeutic agent to the subject. In some embodiments, the additional therapeutic agent is selected from hypertonic saline, mannitol, pulmozyme, N-acetylcysteine, cysteamine, and bronchodilators. [Brief explanation of the drawing]

[0031] [Figure 1] Figures 1A and 1B show the alignment of the light chain variable region (1A) and heavy chain variable region (1B) of rat anti-Notch2 antibody 1B2 and a specific humanized version thereof.

[0032] [Figure 2] Figures 2A and 2B show the light chain variable region (2A) and heavy chain variable region (2B) of rat anti-Notch2 antibody 3107.

[0033] [Figure 3] Figures 3A and 3B show the alignment of the light chain variable region (3A) and heavy chain variable region (3B) of rabbit anti-Notch2 antibodies 2338, 2430, 2430 (with a C95dS substitution in the light chain), and 2621.

[0034] [Figure 4] Figure 4 shows epitope binning of rat.1B2, rat.3107, rb.2338, rb.2430, rb.2621, and the anti-Notch 2 / 3 antibody OMP-59R5 (tarextumab, see U.S. Patent No. 8,226,943).

[0035] [Figure 5] Figures 5A-5F show the blockade of Jagged1-mediated Notch2 activity (5A, 5C, 5E) and the preservation of DLL1-mediated Notch2 activity (5B, 5D, 5F) in a co-culture assay including cells expressing the Notch2 receptor and cells expressing Jagged1 ligand (5A, 5C, 5E) or DLL1 ligand (5B, 5D, 5F). Figures 5A and 5B show the changes in the activity percentages of Jagged1-mediated and DLL1-mediated signaling, respectively, with increasing antibody concentrations for the anti-Notch2 antibody chimera 1B2 and its humanized versions hu1B2.v1.DFS, hu1B2.v101, hu1B2.v102, hu1B2.v103, and hu1B2.v104. Figures 5C and 5D show the changes in the activity percentages of Jagged1-mediated and DLL1-mediated signaling, respectively, with increasing antibody concentrations for the rat anti-Notch2 antibody 3107. Figures 5E and 5F show the changes in the activity percentages of Jagged1-mediated and DLL1-mediated signaling, respectively, with increasing antibody concentrations for rabbit anti-Notch2 antibodies 2338, 2621, and 2430.

[0036] [Figure 6]Figures 6A-6D show the mRNA expression of (6A) Muc5b, (6B) Muc5ac, and (6C) Scgb1a1 in gas-liquid interface (ALI) cultures of primary human bronchial epithelial cells contacted with anti-gD control antibody or rat / human chimeric anti-Notch2 antibody 1B2; and (6D) immunofluorescence analysis of ALI cultures treated with anti-gD control antibody (left) and ALI cultures treated with anti-Notch2 antibody 1B2 (right). Sections were stained with anti-Muc5b (green) for goblet cells, anti-acetylated α-tubulin (red) for ciliated cells, and DAPI (blue) for nuclear staining. A substantial decrease in goblet cells was observed in ALI cultures treated with anti-Notch2 antibody 1B2.

[0037] [Figure 7] Figures 7A–7B show the alignment of the light chain variable region (7A) and heavy chain variable region (7B) of rat anti-Notch2 antibody 3107 and a specific humanized version thereof. [Modes for carrying out the invention]

[0038] Detailed description of the invention I. Definition For the purposes of this specification, “receptor human framework” means a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. A human immunoglobulin framework or human consensus framework “derived” acceptor human framework may contain the same amino acid sequence or may contain modifications of the amino acid sequence. In some embodiments, the number of amino acid modifications is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is sequence-identical to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0039] "Affinity" refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can generally be represented by the dissociation constant (K D ). Affinity can be measured by methods common in the art, including those described herein. Specific exemplary and illustrative methods for measuring binding affinity are described below.

[0040] An "affinity matured" antibody refers to an antibody that has one or more changes in one or more complementarity determining regions (CDRs) compared to the parental antibody without such changes, and such modifications improve the affinity of the antibody for an antigen.

[0041] The terms "anti-Notch2 antibody" and "antibody that binds Notch2" refer to an antibody that can bind Notch2 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting Notch2. In some embodiments, the degree of binding of an anti-Notch2 antibody to an irrelevant non-Notch2 protein is less than about 10% of the binding of the antibody to Notch2, as measured, for example, by surface plasmon resonance (SPR). In certain embodiments, an antibody that binds Notch2 has a dissociation constant (K -8 ) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM or ≦0.001 nM (e.g., 10 -8 M or less, e.g., 10 -13 M to 10 -9 M, e.g., 10 -13 M). When an antibody has a K D of 1 μM or less, the antibody is said to "specifically bind" to Notch2. In some embodiments, an anti-Notch2 antibody binds to an epitope of Notch2 that is conserved among Notch2s from different species. ​​

[0042] The term "antibody" is used herein in its broadest sense and is not limited thereto, but encompasses a variety of antibody structures, including monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0043] An "antibody fragment" refers to a molecule other than an intact antibody, including a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv and scFab); single-domain antibodies (dAb); and multispecific antibodies formed from antibody fragments. For a review of specific antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0044] The term "epitope" refers to a site on an antigen, either proteinaceous or nonproteinaceous, to which an anti-Notch2 antibody binds. Epitopes can be formed from a continuous amino acid stretch site (linear epitopes) or from discontinuous amino acids (i.e., discontinuous epitopes or structural epitopes), and are formed in spatial proximity, for example, due to the folding of the antigen (i.e., by tertiary folding of a proteinaceous antigen). Linear epitopes generally remain bound to anti-Notch2 antibodies even after the proteinaceous antigen has been exposed to a denaturing agent, while structural epitopes are generally destroyed upon treatment with a denaturing agent. Epitopes contain at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial structure.

[0045] Screening for antibodies that bind to a specific epitope (i.e., antibodies that bind to the same epitope) may be performed using methods commonly used in the art, such as, but not limited to, alanine scanning, peptide blotting (Meth.Mol.Biol.248(2004)443-463), peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of antigens (see Prot.Sci.9(2000)487-496), and cross-blocking (see "Antibodies", Harlow and Lane, Cold Spring Harbor Press, Cold Spring Harb., NY).

[0046] Antigen structure-based antibody profiling (ASAP), also known as modification-assisted profiling (MAP), allows for the classification of numerous monoclonal antibodies that specifically bind to Notch2 based on their respective binding profiles to chemically or enzymatically modified antigen surfaces (see, for example, US 2004 / 0101920). Each classified antibody binds to the same epitope, which may be distinctly different from or partially overlapping epitopes represented by other classifications.

[0047] Furthermore, competitive binding can be used to easily determine whether an antibody binds to the same Notch2 epitope as the reference antibody, or whether it competes for binding with the reference anti-Notch2 antibody. For example, an antibody that "binds to the same epitope" as the reference anti-Notch2 antibody refers to an antibody that inhibits the binding of the reference anti-Notch2 antibody to the antigen by 50% or more in a competitive assay, and conversely, the reference antibody inhibits the binding of the antibody to the antigen by 50% or more in a competitive assay. Alternatively, for example, to determine whether an antibody binds to the same epitope as the reference anti-Notch2 antibody, the reference antibody can be bound to Notch2 in a saturated state. After removing the excess reference anti-Notch2 antibody, the ability of the anti-Notch2 antibody to bind to Notch2 is evaluated. If the anti-Notch2 antibody can bind to Notch2 after saturated binding of the reference anti-Notch2 antibody, it can be concluded that this anti-Notch2 antibody binds to a different epitope than the reference anti-Notch2 antibody. However, if an anti-Notch2 antibody cannot bind to Notch2 after saturated binding of a reference anti-Notch2 antibody, it is possible that this anti-Notch2 antibody is binding to the same epitope to which the reference anti-Notch2 antibody binds. Conventional experiments can be used to determine whether the antibody in question is binding to the same epitope or whether binding is simply being hindered for steric reasons (e.g., peptide mutation analysis or binding analysis using ELISA, RIA, surface plasmon resonance, flow cytometry, or other quantitative or qualitative antibody binding assays available in the art). This assay should be performed in two setups, i.e., with both antibodies saturated. If, in both setups, only the first (saturated) antibody is able to bind to Notch2, then it can be concluded that this anti-Notch2 antibody and the reference anti-Notch2 antibody are competing for binding to Notch2.

[0048] In some embodiments, if a competitive binding assay measures that a 1, 5, 10, 20, or 100-fold excess of one antibody inhibits the binding of the other by at least 50%, at least 75%, at least 90%, or even more than 99%, then the two antibodies are considered to bind to the same or overlapping epitopes. (See, for example, Junghans et al., Cancer Res. 50(1990) 1495-1502).

[0049] In some embodiments, two antibodies are considered to bind to the same epitope if substantially all amino acid mutations in an antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other antibody. Two antibodies are considered to have a “duplicate epitope” if only a subset of amino acid mutations that reduce or eliminate the binding of one antibody also reduces or eliminates the binding of the other antibody.

[0050] The term "chimeric" antibody refers to an antibody in which part of the heavy chain and / or light chain originates from a specific source or species, and the remaining part of the heavy chain and / or light chain originates from a different source or species.

[0051] The "class" of an antibody refers to the type of constant domain or constant region held by its heavy chain. Antibodies have five major classes: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In certain embodiments, the antibody is the IgG1 isotype. In certain embodiments, the antibody is the IgG1 isotype with P329G, L234A, and L235A mutations to reduce the effector function of the Fc region. In other embodiments, the antibody is the IgG2 isotype. In certain embodiments, the antibody is the IgG4 isotype with the S228P mutation in the hinge region to improve the stability of the IgG4 antibody. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The light chain of an antibody can be assigned to one of two types, called kappa (κ) or lambda (λ), based on the amino acid sequence of its constant domain.

[0052] "Effector function" refers to the biological activity resulting from the Fc region of an antibody, and it varies depending on the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cell-mediated cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0053] The “effective amount” of a drug, such as a pharmaceutical composition, refers to the amount that is effective in the dosage and duration required to achieve the desired therapeutic or preventive outcome.

[0054] The term “Fc region” is used herein to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. This term includes both the native sequence Fc region and the variant Fc region. In some embodiments, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Thus, by expression of a particular nucleic acid molecule encoding a full-length heavy chain, antibodies produced by host cells may contain the full-length heavy chain or a cleaved variant of the full-length heavy chain. This may be the case when the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbered according to the EU index). Therefore, the Fc region may or may not have a C-terminal lysine (Lys447), or a C-terminal glycine (Gly446) and lysine (Lys447). In some embodiments, the heavy chain containing the Fc region specified herein, as included in the antibody of the present invention, includes a further C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to the EU index). In some embodiments, the heavy chain containing the Fc region specified herein, as included in the antibody according to the present invention, includes a further C-terminal glycine residue (G446, numbered according to the EU index). Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system (also known as the EU index), as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0055] "Framework" or "FR" refers to variable domain residues other than the complementarity-determining region (CDR). The variable domain FR generally consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the CDR and FR sequences generally appear in VH (or VL) in the following sequence: FR1-CDR-H1(CDR-L1)-FR2-CDR-H2(CDR-L2)-FR3-CDR-H3(CDR-L3)-FR4.

[0056] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably in this specification and refer to antibodies that have a structure substantially similar to that of a natural antibody or that have a heavy chain containing an Fc region as defined herein.

[0057] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, and also include the offspring of such cells. Host cells include “transformers” and “transformed cells,” which include primary transformed cells and their offspring, regardless of passage number. Offspring may not have the exact same nucleic acid content as the parent cells, and may include mutations. In this specification, offspring of mutants having the same function or biological activity as those screened or selected in the initially transformed cells are included.

[0058] A "human antibody" is defined as an antibody produced by a human or human cell, or an antibody that has an amino acid sequence corresponding to a non-human antibody that utilizes a sequence encoding a human antibody, such as the human antibody repertoire. This definition of a human antibody explicitly excludes humanized antibodies that contain non-human antigen-binding residues.

[0059] The "Human Consensus Framework" is a framework representing the most commonly occurring amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from subgroups of variable domain sequences. Generally, the sequence subgroups are those described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In some embodiments, for VL, the subgroup is subgroup Kappa I or II, as described in Kabat et al. above. In some embodiments, for VH, the subgroup is subgroup I or III, as described in Kabat et al. above.

[0060] A "humanized" antibody refers to a chimeric antibody containing amino acid residues derived from non-human CDRs and amino acid residues derived from human FRs. In certain embodiments, a humanized antibody substantially contains all of at least one, typically two, variable domains, within which all or substantially all of the CDRs correspond to the CDRs of a non-human antibody, and all or substantially all of the FRs correspond to the FRs of a human antibody. A humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody. The "humanized form" of an antibody, for example, a non-human antibody, refers to an antibody that has undergone humanization.

[0061] As used herein, the terms “hypervariable region” or “HVR” mean each of the regions of an antibody variable domain that are hypervariable within a sequence and determine antigen-binding specificity, such as “complementarity-determining regions” (CDRs).

[0062] Generally, an antibody contains six CDRs, three located in the VH (CDR-H1, CDR-H2, CDR-H3) and three located in the VL (CDR-L1, CDR-L2, CDR-L3). Examples of CDRs used herein include: (a) Hypervariable loops formed at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)), (b) CDRs present at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) Antigen contact occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262:732-745 (1996)); and, (d) Positions 24–34 (L1), 50–56 (L2), and 89–97 (L3) of the CDR:VL domain as defined by the combination of Chothia and Kabat, and positions 26–35 (H1), 50–65 (H2), and 95–102 (H3) of the VH domain.

[0063] Unless otherwise specified, CDRs are determined in accordance with Kabat et al. above. Those skilled in the art will understand that the notation of CDRs may be determined in accordance with Chothia, McCallum, or any other scientifically recognized nomenclature system. In some embodiments, CDR residues include those identified in Figures 1-3 and / or the table of specific sequences herein.

[0064] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including but not limited to cytotoxic agents.

[0065] The “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0066] An "isolated" antibody is one that has been separated from its natural environment. In some embodiments, antibodies are purified to a purity higher than 95% or 99%, as determined by methods such as electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for evaluating antibody purity, see, for example, Flatman et al., J.Chromatogr.B 848:79-87 (2007).

[0067] The terms “nucleic acid molecule” or “polynucleotide” include any compound and / or substance containing a polymer of nucleotides. Each nucleotide is composed of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Often, nucleic acid molecules are described by their base sequence, where the bases represent the primary structure (linear structure) of the nucleic acid molecule. The base sequence is typically represented 5' to 3'. In this specification, the term nucleic acid molecule includes deoxyribonucleic acid (DNA), e.g., complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers containing two or more of these molecules. Nucleic acid molecules may be linear or cyclic. In addition, the term nucleic acid molecule includes both sense strands and antisense strands, as well as both single-stranded and double-stranded forms. Furthermore, nucleic acid molecules described herein may include naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides, including derivatized sugar or phosphate backbone links or chemically modified residues, include modified nucleotide bases. Nucleic acid molecules also include DNA and RNA molecules suitable as vectors for the direct expression of the antibodies of the present invention in vitro and / or in vivo in a host or patient, for example. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors may or may not be modified. For example, mRNA may be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule so that the mRNA can be injected into a target in vivo to produce an antibody (see, for example, Stadler et al., Nature Medicine 2017, published online on June 12, 2017, doi:10.1038 / nm.4356 or European Patent No. 2101823B1).

[0068] "Isolated" nucleic acids are nucleic acid molecules that have been separated from their natural environment. Isolated nucleic acids include nucleic acid molecules that are normally found inside cells, but these nucleic acid molecules are located outside of chromosomes or in chromosomal locations different from their natural chromosomal locations.

[0069] "Isolated nucleic acid encoding an anti-Notch2 antibody" means one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of an anti-Notch2 antibody, including such nucleic acid molecules(or molecules) in a single vector or separate vectors, and such nucleic acid molecules(or molecules) are located at one or more locations within a host cell.

[0070] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting the population are identical and / or bound to the same epitope, except for variant antibodies, such as those containing naturally occurring mutations or those that may arise during the production of a monoclonal antibody preparation, the exception of which such variants are generally present in trace amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed toward different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed toward a single determinant on one antigen. Thus, the modified term “monoclonal” indicates the characteristic of an antibody obtained from a substantially homogeneous collection of antibodies and should not be interpreted as requiring the production of the antibody by any particular method. For example, monoclonal antibodies according to the present invention can be produced by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, including but not limited to such methods and other exemplary methods for producing monoclonal antibodies described herein.

[0071] The term "mucosal-obstructive pulmonary disease" refers to a group of diseases characterized by diffuse mucous obstruction, chronic inflammation, airway wall dilatation, and frequent bacterial infections. In mucosal-obstructive pulmonary disease, high concentrations of mucus cannot be effectively transported from the distal airways to the trachea, causing mucus to adhere to the airway surface, leading to airflow obstruction, infection, and inflammation. Non-exclusive exemplary mucosal-obstructive pulmonary diseases include chronic obstructive pulmonary disease (COPD), cystic fibrosis, primary ciliary dyskinesia, non-cystic fibrosis, bronchiectasis, and bronchiolitis.

[0072] A "naked antibody" refers to an antibody that is not bound to a heterologous site (e.g., a cytotoxic site) or a radioactive label. Naked antibodies may be present in a pharmaceutical composition.

[0073] "Natural antibodies" refer to naturally occurring immunoglobulin molecules with various structures. For example, a natural IgG antibody is a heterotetrameric glycoprotein with approximately 150,000 daltons, containing two identical light chains and two identical heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable domain (VH), also called a variable heavy domain or heavy chain variable region, followed by three constant heavy domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable domain (VL), also called a variable light domain or light chain variable region, followed by a constant light (CL) domain.

[0074] As used herein, the term “Notch2” refers to any natural Notch2 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term also includes “full-length” untreated Notch2 and any form of Notch2 resulting from intracellular processing. The term also includes naturally occurring variants of Notch2, such as splice variants or allele variants. An exemplary amino acid sequence of human Notch2 is shown in UniProtKB / Swiss-Prot:Q04721.3 and Sequence ID No. 70 herein. An exemplary amino acid sequence of cynomolgus monkey Notch2 is shown in UniProt:A0A2K5U7N0_MACFA. Another exemplary cynomolgus monkey Notch2 is shown in Sequence ID No. 71 herein. An exemplary amino acid sequence of guinea pig Notch2 is shown in UniProt:H0VU21 and Sequence ID No. 72 herein. The amino acid sequence of an exemplary guinea pig Notch2 is shown in UniProt:O35516 and Sequence ID No. 73 herein. The amino acid sequence of an exemplary rat Notch2 is shown in UniProt:Q9QW30 and Sequence ID No. 81 herein.

[0075] The term “package insert” is used to refer to the instructions typically included in the commercial packaging of a therapeutic product, which contain information about the indications, usage, dosage, administration, combination therapy, contraindications, and / or warnings regarding the use of such therapeutic product.

[0076] The "amino acid sequence identity percentage (%)" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences for alignment purposes and introducing gaps if necessary to achieve the maximum sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for determining the amino acid sequence identity percentage can be achieved in various ways within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software, or FASTA program packages. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithm necessary to achieve the maximum alignment over the entire length of the sequences being compared. Alternatively, the identity percentage value can be generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and its source code is filed in the user documentation of the US Copyright Office (Washington DC, 20559), registered under US Copyright Registration No. TXU510087, and published in International Publication No. 2001 / 007611.

[0077] Unless otherwise noted, for the purposes of this specification, amino acid sequence identity values ​​are generated using the ggsearch program in FASTA package version 36.3.8c, or subsequently using the BLOSUM50 comparison matrix. The FASTA program package is described by WRPearson and DJLipman (1988), "Improved Tools for Biological Sequence Analysis," PNAS 85:2444-2448; WRPearson (1996), "Effective protein sequence comparison," Meth.Enzymol.266:227-258; and Pearson et al. (1997), Genomics 46:24-36, and is publicly available at www.fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml or www.ebi.ac.uk / Tools / sss / fasta. Alternatively, you can use the ggsearch(global protein:protein) program with default options (BLOSUM50;open:-10;ext:-2;Ktup=2) to compare sequences using a public server accessible at fasta.bioch.virginia.edu / fasta_www2 / index.cgi, ensuring that a global rather than local alignment is performed. The amino acid identity rate is given in the output alignment header.

[0078] The terms "pharmaceutical composition" or "pharmaceutical preparation" refer to a preparation in which the active ingredient contained therein is in a form that is effective in terms of its biological activity, and which does not contain any additional ingredients that are unacceptably toxic to the subject to which the pharmaceutical composition is to be administered.

[0079] "Pharmacologically acceptable carriers" refer to components in a pharmaceutical composition or preparation other than the active ingredient that are non-toxic to the target. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0080] As used herein, “treatment” (and its grammatical variations, e.g., “to treat” or “treating”) refers to a clinical intervention in an attempt to alter the original course of a disease in the individual being treated, and may be carried out for preventive purposes or during the course of a clinicopathological disease. Desired effects of treatment include preventing the onset or recurrence of the disease, reducing symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, achieving remission or mitigation of the condition, and achieving a recovered or improved prognosis. In some embodiments, the antibodies of the present invention are used to delay the onset of the disease or to slow the progression of the disease.

[0081] The term "variable region" or "variable domain" refers to a domain of the antibody heavy chain or antibody light chain involved in the binding of an antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, and each domain contains four conserved framework regions (FRs) and three complementarity-determining regions (CDRs) (e.g., Kindt et al., Kuby Immunology, 6). th See ed., WH Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen may be isolated by screening a library of complementary VL or VH domains using the VH or VL domain of the antibody that binds to the antigen, respectively. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0082] The term “vector” as used herein refers to a nucleic acid molecule capable of replicating another nucleic acid it is linked to. This term includes not only vectors as self-replicating nucleic acid structures, but also vectors that have been incorporated into the genome of a host cell into which they have been introduced. Certain vectors can direct the expression of a functionally linked nucleic acid. Such vectors are referred to herein as “expression vectors.”

[0083] II. Compositions and Methods In some embodiments, the present invention is partially based on an antibody that binds to Notch2 and inhibits Jagged1-mediated signaling but does not inhibit DLL1-mediated signaling. The antibody of the present invention is useful, for example, in the diagnosis or treatment of mucosal-occlusive pulmonary diseases.

[0084] A. Exemplary anti-Notch2 antibody In some embodiments, the present invention provides an antibody that binds to Notch2. In some embodiments, an isolated antibody that binds to Notch2 is provided. In some embodiments, the present invention provides an antibody that specifically binds to Notch2. In certain embodiments, the anti-Notch2 antibody is: ● Inhibits Jagged1-mediated signaling; ● Does not inhibit DLL1-mediated signaling; ● Does not inhibit the binding of Jagged1 to Notch2; ● Does not interfere with the binding of DLL1 to Notch2; ● Binds to an epitope within the EGF7 repeat of Notch2; ● Binds to epitopes within amino acids 260-296 of Notch2; ● Binds to discontinuous epitopes within amino acids 260-296 of Notch2; ● Human Notch2 contact arginine 268 (R268); ● Does not bind to Notch2 containing lysine 268 (268K); ● Binds to polypeptides containing the amino acid sequence of SEQ ID NO: 74, but does not bind to polypeptides containing the amino acid sequence of SEQ ID NO: 77; and / or ● For example, when determined by surface plasmon resonance, affinity (K) is less than 20 nM, less than 15 nM, less than 10 nM, or less than 5 nM. D It binds to human Notch2.

[0085] Antibodies containing one or more CDRs of antibody 1B2 or its humanized version. In some embodiments, the present invention provides an anti-Notch2 antibody comprising at least one, at least two, at least three, at least four, at least five, or all six CDRs selected from (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 6 or 7; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 8, 9, 10, 11, or 12; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 1; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 2; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 3.

[0086] In some embodiments, the present invention provides an antibody comprising at least one, at least two, or all three VH CDR sequences selected from (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 6 or 7, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 8, 9, 10, 11, or 12. In some embodiments, the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NO: 8, 9, 10, 11, or 12. In some embodiments, the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NO: 8, 9, 10, 11, or 12 and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 3. In further embodiments, the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NO: 8, 9, 10, 11, or 12, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 3, and CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2. In a further embodiment, the antibody comprises (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 4, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 8, 9, 10, 11, or 12.

[0087] In some embodiments, the present invention provides an antibody comprising at least one, at least two, or all three VL CDR sequences selected from (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 1, (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 2, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, the antibody comprises one, two, or three CDRs selected from (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 1, (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 2, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 3.

[0088] In some embodiments, the antibody of the present invention comprises (a) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 6 or 7, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 8, 9, 10, 11, or 12, and (b) a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 2, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 3.

[0089] In some embodiments, the present invention provides an antibody comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 6 or 7, (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 8, 9, 10, 11, or 12, (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 1, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 2, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 3.

[0090] In certain embodiments, any one or more amino acids of the anti-Notch2 antibody provided herein may be at the following CDR positions: CDR-H2 (Sequence ID 6): Position 2 CDR-H3 (Sequence ID 8): Positions 2, 4, 5, and / or 6 In certain embodiments, substitutions are conservative substitutions as provided herein. In certain embodiments, any one or more of the following substitutions may be any combination: CDR-H2 (Sequence ID 6): S2Q (S51Q according to Kabat numbering) CDR-H3 (Sequence ID 8): S2G (S96G according to Kabat numbering); R4K (R98K according to Kabat numbering); W5L (W99L according to Kabat numbering); and / or G6A (G100A according to Kabat numbering).

[0091] In any embodiment provided herein, the anti-Notch2 antibody is humanized. In some embodiments, the anti-Notch2 antibody further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework. In some embodiments, the anti-Notch2 antibody comprises a VH containing the FR1 sequence of SEQ ID NO: 92; the FR2 sequence of SEQ ID NO: 93 or 94; the FR3 sequence of SEQ ID NO: 95, 96 or 107; and / or the FR4 sequence of SEQ ID NO: 97. In some embodiments, the anti-Notch2 antibody comprises a VL containing the FR1 sequence of SEQ ID NO: 87; the FR2 sequence of SEQ ID NO: 88; the FR3 sequence of SEQ ID NO: 89 or 90; and / or the FR4 sequence of SEQ ID NO: 91.

[0092] In some embodiments, the anti-Notch2 antibody includes a VH domain comprising one or more heavy chain framework sequences selected from (a) heavy chain framework region 1 (HC-FR1) of SEQ ID NO: 92, (b) heavy chain framework region 2 (HC-FR2) of SEQ ID NO: 93 or 94, (c) heavy chain framework region 3 (HC-FR3) of SEQ ID NO: 95, 96, or 107, and (d) heavy chain framework region 4 (HC-FR4) of SEQ ID NO: 97.

[0093] In some embodiments, the anti-Notch2 antibody includes a VH domain containing HC-FR1 of SEQ ID NO: 92. In some embodiments, the anti-Notch2 antibody includes a VH domain containing HC-FR2 of SEQ ID NO: 93 or 94. In some embodiments, the anti-Notch2 antibody includes a VH domain containing HC-FR3 of SEQ ID NO: 95, 96, or 107. In some embodiments, the anti-Notch2 antibody includes a VH domain containing HC-FR4 of SEQ ID NO: 97.

[0094] In some embodiments, the anti-Notch2 antibody includes a VH domain containing HC-FR1 with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 92. In some embodiments, the VH domain contains HC-FR1 with at least 95% sequence identity with SEQ ID NO: 92. In some embodiments, the VH domain contains HC-FR1 with at least 98% sequence identity with SEQ ID NO: 92.

[0095] In some embodiments, the anti-Notch2 antibody includes a VH domain containing HC-FR2 with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 93 or 94. In some embodiments, the VH domain contains HC-FR2 with at least 95% sequence identity with SEQ ID NO: 93 or 94. In some embodiments, the VH domain contains HC-FR2 with at least 98% sequence identity with SEQ ID NO: 93 or 94.

[0096] In some embodiments, the anti-Notch2 antibody includes a VH domain containing HC-FR3 with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NOs. 95, 96, or 107. In some embodiments, the VH domain contains HC-FR3 with at least 95% sequence identity with SEQ ID NOs. 95, 96, or 107. In some embodiments, the VH domain contains HC-FR3 with at least 98% sequence identity with SEQ ID NOs. 95, 96, or 107.

[0097] In some embodiments, the anti-Notch2 antibody includes a VH domain containing HC-FR4 with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 97. In some embodiments, the VH domain contains HC-FR4 with at least 95% sequence identity with SEQ ID NO: 97. In some embodiments, the VH domain contains HC-FR4 with at least 98% sequence identity with SEQ ID NO: 97.

[0098] In some embodiments, the anti-Notch2 antibody includes a VL domain comprising one or more light chain framework sequences selected from (a) light chain framework region 1 (LC-FR1) of SEQ ID NO: 87, (b) light chain framework region 2 (LC-FR2) of SEQ ID NO: 88, (c) light chain framework region 3 (LC-FR3) of SEQ ID NO: 89 or 90, and (d) light chain framework region 4 (LC-FR4) of SEQ ID NO: 91.

[0099] In some embodiments, the anti-Notch2 antibody includes a VL domain containing LC-FR1 of SEQ ID NO: 87. In some embodiments, the anti-Notch2 antibody includes a VL domain containing LC-FR2 of SEQ ID NO: 88. In some embodiments, the anti-Notch2 antibody includes a VL domain containing LC-FR3 of SEQ ID NO: 89 or 90. In some embodiments, the anti-Notch2 antibody includes a VL domain containing LC-FR4 of SEQ ID NO: 91.

[0100] In some embodiments, the anti-Notch2 antibody includes a VL domain containing LC-FR1 with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 87. In some embodiments, the VL domain contains LC-FR1 with at least 95% sequence identity with SEQ ID NO: 87. In some embodiments, the VL domain contains LC-FR1 with at least 98% sequence identity with SEQ ID NO: 87.

[0101] In some embodiments, the anti-Notch2 antibody includes a VL domain containing LC-FR2 with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 88. In some embodiments, the VL domain contains LC-FR2 with at least 95% sequence identity with SEQ ID NO: 88. In some embodiments, the VL domain contains LC-FR2 with at least 98% sequence identity with SEQ ID NO: 88.

[0102] In some embodiments, the anti-Notch2 antibody includes a VL domain containing LC-FR3 with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 89 or 90. In some embodiments, the VL domain contains LC-FR3 with at least 95% sequence identity with SEQ ID NO: 89 or 90. In some embodiments, the VL domain contains LC-FR3 with at least 98% sequence identity with SEQ ID NO: 89 or 90.

[0103] In some embodiments, the anti-Notch2 antibody includes a VL domain containing LC-FR4 with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 91. In some embodiments, the VL domain includes LC-FR1 with at least 95% sequence identity with SEQ ID NO: 91. In some embodiments, the VL domain includes LC-FR1 with at least 98% sequence identity with SEQ ID NO: 91.

[0104] In some embodiments, the anti-Notch2 antibody comprises one or more VH CDR sequences of SEQ ID NOs: 14, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, or 32. In another embodiment, the anti-Notch2 antibody comprises one or more VL CDR sequences of SEQ ID NOs: 13, 15, 16, 25, 27, 29, or 31. In yet another embodiment, the anti-Notch2 antibody comprises the VH CDR sequence of SEQ ID NOs: 14, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, or 32 and the VL CDR sequence of SEQ ID NOs: 13, 15, 16, 25, 27, 29, or 31.

[0105] In a further embodiment, the anti-Notch2 antibody comprises the CDR-H1, CDR-H2, and CDR-H3 amino acid sequences of the VH domain of SEQ ID NOs. 14, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, or 32, and the CDR-L1, CDR-L2, and CDR-L3 amino acid sequences of the VL domain of SEQ ID NOs. 13, 15, 16, 25, 27, 29, or 31.

[0106] In some embodiments, the anti-Notch2 antibody comprises one or more heavy chain CDR amino acid sequences of the VH domain of SEQ ID NOs. 14, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, or 32, and a framework of sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% to the framework amino acid sequences of the VH domain of SEQ ID NOs. 14, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, or 32. In some embodiments, the anti-Notch2 antibody comprises three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NOs. 14, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, or 32, and a framework of sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% to the framework amino acid sequence of the VH domain of SEQ ID NOs. 14, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, or 32. In some embodiments, the anti-Notch2 antibody comprises three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NOs. 14, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, or 32, and a framework with at least 95% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NOs. 14, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, or 32. In some embodiments, the anti-Notch2 antibody comprises three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NOs. 14, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, or 32, and a framework with at least 98% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NOs. 14, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, or 32.

[0107] In some embodiments, the anti-Notch2 antibody comprises one or more light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 13, 15, 16, 25, 27, 29, or 31, and a framework of sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% to the framework amino acid sequence of the VL domain of SEQ ID NO: 13, 15, 16, 25, 27, 29, or 31. In some embodiments, the anti-Notch2 antibody comprises three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 13, 15, 16, 25, 27, 29, or 31, and a framework with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 13, 15, 16, 25, 27, 29, or 31. In some embodiments, the anti-Notch2 antibody comprises three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 13, 15, 16, 25, 27, 29, or 31, and a framework with at least 95% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 13, 15, 16, 25, 27, 29, or 31. In some embodiments, the anti-Notch2 antibody comprises three light chain CDR amino acid sequences of the VL domain of SEQ ID NOs. 13, 15, 16, 25, 27, 29, or 31, and a framework with at least 98% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NOs. 13, 15, 16, 25, 27, 29, or 31.

[0108] In some embodiments, the anti-Notch2 antibody includes (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 4; (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 6 or 7; (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 8, 9, 10, 11, or 12; (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 1; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 2; and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 3, as well as SEQ ID NOs: 14, 17, 18, 19, 20, 2 The material comprises a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of 1, 22, 23, 24, 26, 28, 30, or 32, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NOs. 13, 15, 16, 25, 27, 29, or 31. In some embodiments, the VH domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NOs. 14, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, or 32. In some embodiments, the VL domain has at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NOs: 13, 15, 16, 25, 27, 29, or 31.

[0109] In some embodiments, the anti-Notch2 antibody includes (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 4; (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 6 or 7; (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 8, 9, 10, 11, or 12; (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 1; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 2; and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 3, as well as SEQ ID NOs: 14, 17, 18, 19, 20, 21, 22, 23, 24 The antibody comprises a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NOs. 13, 15, 16, 25, 27, 29, or 31; and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NOs. 13, 15, 16, 25, 27, 29, or 31; and the antibody binds specifically to Notch2. In some embodiments, the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NOs. 14, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, or 32. In some embodiments, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NOs. 13, 15, 16, 25, 27, 29, or 31. In some embodiments, the antibody has a dissociation constant (K) of the antibody containing the VH sequence of SEQ ID NOs. 14, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, or 32 and the VL sequence of SEQ ID NOs. 13, 15, 16, 25, 27, 29, or 31. D Compared to the dissociation constant (K), the dissociation constant (K) is reduced by up to 10 times or increased by up to 10 times. D It connects to Notch2 which has ).

[0110] In some embodiments, the anti-Notch2 antibody contains a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NOs. 14, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, or 32. In some embodiments, the anti-Notch2 antibody contains a heavy chain variable domain (VH) sequence having at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NOs. 14, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, or 32. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to a reference sequence, but an anti-Notch2 antibody containing that sequence retains the ability to bind to Notch2. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NOs: 14, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, or 32. In certain embodiments, the substitutions, insertions, or deletions occur in the region outside the CDR (i.e., in the FR). Optionally, the anti-Notch2 antibody includes the VH sequence of SEQ ID NOs. 14, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, or 32, including post-translational modifications of that sequence. In certain embodiments, the VH includes one, two, or three CDRs selected from: (a) CDR-H1 containing the amino acid sequence of SEQ ID NOs. 4, (b) CDR-H2 containing the amino acid sequence of SEQ ID NOs. 6 or 7, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NOs. 8, 9, 10, 11, or 12. In some embodiments, an anti-Notch2 antibody is provided, which comprises a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NOs. 13, 15, 16, 25, 27, 29, or 31.In some embodiments, the anti-Notch2 antibody contains a light chain variable domain (VL) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NOs. 13, 15, 16, 25, 27, 29, or 31. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the anti-Notch2 antibody containing that sequence retains the ability to bind to Notch2. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NOs. 13, 15, 16, 25, 27, 29, or 31. In certain embodiments, the substitutions, insertions, or deletions occur in the region outside the CDR (i.e., in the FR). Optionally, the anti-Notch2 antibody includes the VL sequence of SEQ ID NOs. 13, 15, 16, 25, 27, 29, or 31, including post-translational modifications of that sequence. In certain embodiments, the VL includes one, two, or three CDRs selected from: (a) CDR-L1 containing the amino acid sequence of SEQ ID NOs. 1, (b) CDR-L2 containing the amino acid sequence of SEQ ID NOs. 2, and (c) CDR-L3 containing the amino acid sequence of SEQ ID NOs. 3.

[0111] In some embodiments, an anti-Notch2 antibody is provided, which comprises a VH sequence as in any of the embodiments provided above, and a VL sequence as in any of the embodiments provided above. In some embodiments, the antibody comprises the VH sequence and the VL sequence (including post-translational modifications of those sequences) in SEQ ID NOs: 14, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, or 32 and SEQ ID NOs: 13, 15, 16, 25, 27, 29, or 31, respectively.

[0112] Antibody containing one or more CDRs of antibody 3107 In some embodiments, the present invention provides an anti-Notch2 antibody comprising at least one, at least two, at least three, at least four, at least five, or all six CDRs selected from (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 36; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 37; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 38; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 33; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 34; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 35.

[0113] In some embodiments, the present invention provides an antibody comprising at least one, at least two, or all three VH CDR sequences selected from (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 36, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 37, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 38. In some embodiments, the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NO: 38. In some embodiments, the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NO: 38 and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 35. In further embodiments, the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NO: 38, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 35, and CDR-H2 comprising the amino acid sequence of SEQ ID NO: 37. In further embodiments, the antibody comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 36, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 37, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 38.

[0114] In some embodiments, the present invention provides an antibody comprising at least one, at least two, or all three VL CDR sequences selected from (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 33, (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 34, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the antibody comprises one, two, or three CDRs selected from (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 33, (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 34, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 35.

[0115] In some embodiments, the antibody of the present invention comprises (a) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 36, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 37, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 38, and (b) a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 33, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 34, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 35.

[0116] In some embodiments, the present invention provides antibodies comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 36, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 37, (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 38, (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 33, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 34, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 35.

[0117] In some embodiments, the anti-Notch2 antibody comprises one or more of the VH CDR sequences of SEQ ID NO: 40. In another embodiment, the anti-Notch2 antibody comprises one or more of the VL CDR sequences of SEQ ID NO: 39. In yet another embodiment, the anti-Notch2 antibody comprises the VH CDR sequence of SEQ ID NO: 40 and the VL CDR sequence of SEQ ID NO: 39.

[0118] In a further embodiment, the anti-Notch2 antibody comprises the CDR-H1, CDR-H2, and CDR-H3 amino acid sequences of the VH domain of SEQ ID NO: 40 and the CDR-L1, CDR-L2, and CDR-L3 amino acid sequences of the VL domain of SEQ ID NO: 39.

[0119] In some embodiments, the anti-Notch2 antibody comprises one or more heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 40 and a framework of sequence identity with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% relative to framework amino acid sequences of the VH domain selected from SEQ ID NO: 40 and 101-106. In some embodiments, the anti-Notch2 antibody comprises three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 40 and a framework of sequence identity with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% relative to framework amino acid sequences of the VH domain selected from SEQ ID NO: 40 and 101-106. In some embodiments, the anti-Notch2 antibody comprises a framework with at least 95% sequence identity to the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 40 and a framework amino acid sequence of the VH domain selected from SEQ ID NOs: 40 and 101-106. In some embodiments, the anti-Notch2 antibody comprises a framework with at least 98% sequence identity to the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 40 and a framework amino acid sequence of the VH domain selected from SEQ ID NOs: 40 and 101-106.

[0120] In some embodiments, the anti-Notch2 antibody comprises one or more light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 39 and a framework of sequence identity with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% relative to a framework amino acid sequence of the VL domain selected from SEQ ID NO: 39 and 98-100. In some embodiments, the anti-Notch2 antibody comprises three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 39 and a framework of sequence identity with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% relative to a framework amino acid sequence of the VL domain selected from SEQ ID NO: 39 and 98-100. In some embodiments, the anti-Notch2 antibody comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 39 and a framework with at least 95% sequence identity to a framework amino acid sequence of the VL domain selected from SEQ ID NO: 39 and 98-100. In some embodiments, the anti-Notch2 antibody comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 39 and a framework with at least 98% sequence identity to a framework amino acid sequence of the VL domain selected from SEQ ID NO: 39 and 98-100.

[0121] In some embodiments, the anti-Notch2 antibody includes (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 36; (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 37; (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 38; (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 33; (e) CDR-L2 containing the amino acid sequence of sequence number 34; and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 35, as well as SEQ ID NOs. 40 and 101 The device comprises a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an amino acid sequence selected from ~106, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an amino acid sequence selected from SEQ ID NOs. 39 and 98~100. In some embodiments, the VH domain has at least 95% sequence identity with an amino acid sequence selected from SEQ ID NOs. 40 and 101~106. In some embodiments, the VL domain has at least 95% sequence identity with an amino acid sequence selected from SEQ ID NOs. 39 and 98~100.

[0122] In some embodiments, the anti-Notch2 antibody is selected from (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 36; (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 37; (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 38; (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 33; (e) CDR-L2 containing the amino acid sequence of sequence number 34; and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 35, as well as SEQ ID NOs. 40 and 101-106. The antibody comprises a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs. 39 and 98-100; the antibody binds specifically to Notch2. In some embodiments, the VH domain has at least 95% sequence identity to an amino acid sequence selected from SEQ ID NOs. 40 and 101-106. In some embodiments, the VL domain has at least 95% sequence identity to an amino acid sequence selected from SEQ ID NOs. 39 and 98-100. In some embodiments, the antibody has a dissociation constant (K) of the antibody containing the VH sequence of SEQ ID NOs. 40 and the VL sequence of SEQ ID NOs. 39. D Compared to the dissociation constant (K), the dissociation constant (K) is reduced by up to 10 times or increased by up to 10 times. D It connects to Notch2 which has ).

[0123] In some embodiments, the anti-Notch2 antibody contains a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs. In some embodiments, the anti-Notch2 antibody contains a heavy chain variable domain (VH) sequence having at least 95% sequence identity to an amino acid sequence selected from SEQ ID NOs. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the anti-Notch2 antibody containing that sequence retains the ability to bind to Notch2. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in any one of SEQ ID NOs. 40 and 101-106. In certain embodiments, the substitution, insertion, or deletion occurs in the outer region of the CDR (i.e., at the FR). Optionally, the anti-Notch2 antibody contains a VH sequence selected from SEQ ID NOs. 40 and 101-106, including post-translational modifications of that sequence. In certain embodiments, the VH contains one, two, or three CDRs selected from: (a) CDR-H1 containing the amino acid sequence of SEQ ID NOs. 36, (b) CDR-H2 containing the amino acid sequence of SEQ ID NOs. 37, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NOs. 38.

[0124] In some embodiments, an anti-Notch2 antibody is provided, comprising a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an amino acid sequence selected from SEQ ID NOs. In some embodiments, the anti-Notch2 antibody comprises a light chain variable domain (VL) sequence having at least 95% sequence identity with an amino acid sequence selected from SEQ ID NOs. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to a reference sequence, but the anti-Notch2 antibody containing that sequence retains the ability to bind to Notch2. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in any one of SEQ ID NOs: 39 and 98-100. In certain embodiments, the substitution, insertion, or deletion occurs in the outer region of the CDR (i.e., in the FR). Optionally, the anti-Notch2 antibody contains a VL sequence selected from SEQ ID NOs: 39 and 98-100, which includes post-translational modifications of that sequence. In certain embodiments, the VL contains one, two, or three CDRs selected from: (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 33, (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 34, and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 35.

[0125] In some embodiments, an anti-Notch2 antibody is provided, which comprises a VH sequence as in any of the embodiments provided above, and a VL sequence as in any of the embodiments provided above. In some embodiments, the antibody comprises the VH and VL sequences of SEQ ID NO: 40 and SEQ ID NO: 39, respectively, which include post-translational modifications of those sequences. In some embodiments, the antibody comprises a VH sequence selected from SEQ ID NOs: 101-106 and a VL sequence selected from 98-100, which include post-translational modifications of those sequences.

[0126] Antibodies containing one or more CDRs of antibody 2338 In some embodiments, the present invention provides an anti-Notch2 antibody comprising at least one, at least two, at least three, at least four, at least five, or all six CDRs selected from (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 44; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 45; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 46; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 41; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 42; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 43.

[0127] In some embodiments, the present invention provides an antibody comprising at least one, at least two, or all three VH CDR sequences selected from (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 44, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 45, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 46. In some embodiments, the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NO: 46. In some embodiments, the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NO: 46 and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 43. In further embodiments, the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NO: 46, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 43, and CDR-H2 comprising the amino acid sequence of SEQ ID NO: 45. In further embodiments, the antibody comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 44, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 45, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 46.

[0128] In some embodiments, the present invention provides an antibody comprising at least one, at least two, or all three VL CDR sequences selected from (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 41, (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 42, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, the antibody comprises one, two, or three CDRs selected from (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 41, (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 42, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 43.

[0129] In some embodiments, the antibody of the present invention comprises (a) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 44, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 45, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 46, and (b) a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 41, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 42, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 43.

[0130] In some embodiments, the present invention provides antibodies comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 44, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 45, (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 46, (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 41, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 42, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 43.

[0131] In some embodiments, the anti-Notch2 antibody comprises one or more of the VH CDR sequences of SEQ ID NO: 48. In another embodiment, the anti-Notch2 antibody comprises one or more of the VL CDR sequences of SEQ ID NO: 47. In yet another embodiment, the anti-Notch2 antibody comprises the VH CDR sequence of SEQ ID NO: 48 and the VL CDR sequence of SEQ ID NO: 47.

[0132] In a further embodiment, the anti-Notch2 antibody comprises the CDR-H1, CDR-H2, and CDR-H3 amino acid sequences of the VH domain of SEQ ID NO: 48 and the CDR-L1, CDR-L2, and CDR-L3 amino acid sequences of the VL domain of SEQ ID NO: 47.

[0133] In some embodiments, the anti-Notch2 antibody comprises one or more heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 48 and a framework of sequence identity with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% relative to the framework amino acid sequence of the VH domain of SEQ ID NO: 48. In some embodiments, the anti-Notch2 antibody comprises three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 48 and a framework of sequence identity with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% relative to the framework amino acid sequence of the VH domain of SEQ ID NO: 48. In some embodiments, the anti-Notch2 antibody comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 48 and a framework with at least 95% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 48. In some embodiments, the anti-Notch2 antibody comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 48 and a framework with at least 98% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 48.

[0134] In some embodiments, the anti-Notch2 antibody comprises one or more light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 47 and a framework of sequence identity with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% relative to the framework amino acid sequence of the VL domain of SEQ ID NO: 47. In some embodiments, the anti-Notch2 antibody comprises three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 47 and a framework of sequence identity with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% relative to the framework amino acid sequence of the VL domain of SEQ ID NO: 47. In some embodiments, the anti-Notch2 antibody comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 47 and a framework with at least 95% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 47. In some embodiments, the anti-Notch2 antibody comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 47 and a framework with at least 98% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 47.

[0135] In some embodiments, the anti-Notch2 antibody comprises (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 44; (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 45; (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 46; (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 41; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 42; and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 43, as well as a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 48, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 47. In some embodiments, the VH domain has at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 48. In some embodiments, the VL domain has at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 47.

[0136] In some embodiments, the anti-Notch2 antibody comprises (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 44; (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 45; (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 46; (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 41; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 42; and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 43, as well as a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 48, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 47; and the antibody specifically binds to Notch2. In some embodiments, the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 48. In some embodiments, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 47. In some embodiments, the antibody has a dissociation constant (K) of the antibody, which includes the VH sequence of SEQ ID NO: 48 and the VL sequence of SEQ ID NO: 47. D Compared to the dissociation constant (K), the dissociation constant (K) is reduced by up to 10 times or increased by up to 10 times. D It connects to Notch2 which has ).

[0137] In some embodiments, the anti-Notch2 antibody contains a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 48. In some embodiments, the anti-Notch2 antibody contains a heavy chain variable domain (VH) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 48. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the anti-Notch2 antibody containing that sequence retains the ability to bind to Notch2. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 48. In certain embodiments, substitutions, insertions, or deletions occur in the region outside the CDR (i.e., at the FR). Optionally, the anti-Notch2 antibody comprises the VH sequence of SEQ ID NO: 48, which includes post-translational modifications of that sequence. In certain embodiments, the VH comprises one, two, or three CDRs selected from: (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 44, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 45, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 46. In some embodiments, an anti-Notch2 antibody is provided, which comprises a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 47. In some embodiments, the anti-Notch2 antibody comprises a light chain variable domain (VL) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 47. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the anti-Notch2 antibody containing that sequence retains the ability to bind to Notch2.In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 47. In certain embodiments, the substitutions, insertions, or deletions occur in the region outside the CDR (i.e., in the FR). Optionally, the anti-Notch2 antibody contains the VL sequence of SEQ ID NO: 47, which includes post-translational modifications of that sequence. In certain embodiments, the VL contains one, two, or three CDRs selected from: (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 41, (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 42, and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 43.

[0138] In some embodiments, an anti-Notch2 antibody is provided, which comprises a VH sequence as in any of the embodiments provided above, and a VL sequence as in any of the embodiments provided above. In some embodiments, the antibody comprises the VH and VL sequences of SEQ ID NO: 48 and SEQ ID NO: 47, respectively, which include post-translational modifications of those sequences.

[0139] Antibodies containing one or more CDRs of antibody 2430 In some embodiments, the present invention provides an anti-Notch2 antibody comprising at least one, at least two, at least three, at least four, at least five, or all six CDRs selected from (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 53; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 54; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 55; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 49; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 50; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 51 or 52.

[0140] In some embodiments, the present invention provides an antibody comprising at least one, at least two, or all three VH CDR sequences selected from (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 53, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 54, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 55. In some embodiments, the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NO: 55. In some embodiments, the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NO: 55 and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 51 or 52. In further embodiments, the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NO: 55, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 51 or 52, and CDR-H2 comprising the amino acid sequence of SEQ ID NO: 54. In a further embodiment, the antibody comprises (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 53, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 54, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 55.

[0141] In some embodiments, the present invention provides an antibody comprising at least one, at least two, or all three VL CDR sequences selected from (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 49, (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 50, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 51 or 52. In some embodiments, the antibody comprises one, two, or three CDRs selected from (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 49, (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 50, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 51 or 52.

[0142] In some embodiments, the antibody of the present invention comprises (a) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 53, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 54, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 55, and (b) a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 49, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 50, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 51 or 52.

[0143] In some embodiments, the present invention provides an antibody comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 53, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 54, (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 55, (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 49, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 50, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 51 or 52.

[0144] In some embodiments, the anti-Notch2 antibody comprises one or more of the VH CDR sequences of SEQ ID NO: 58. In another embodiment, the anti-Notch2 antibody comprises one or more of the VL CDR sequences of SEQ ID NO: 56 or 57. In yet another embodiment, the anti-Notch2 antibody comprises the VH CDR sequence of SEQ ID NO: 58 and the VL CDR sequence of SEQ ID NO: 56 or 57.

[0145] In a further embodiment, the anti-Notch2 antibody comprises the CDR-H1, CDR-H2, and CDR-H3 amino acid sequences of the VH domain of SEQ ID NO: 58 and the CDR-L1, CDR-L2, and CDR-L3 amino acid sequences of the VL domain of SEQ ID NO: 56 or 57.

[0146] In some embodiments, the anti-Notch2 antibody comprises one or more heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 58 and a framework of sequence identity with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% relative to the framework amino acid sequence of the VH domain of SEQ ID NO: 58. In some embodiments, the anti-Notch2 antibody comprises three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 58 and a framework of sequence identity with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% relative to the framework amino acid sequence of the VH domain of SEQ ID NO: 58. In some embodiments, the anti-Notch2 antibody comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 58 and a framework with at least 95% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 58. In some embodiments, the anti-Notch2 antibody comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 58 and a framework with at least 98% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 58.

[0147] In some embodiments, the anti-Notch2 antibody comprises one or more light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 56 or 57 and a framework of sequence identity with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% relative to the framework amino acid sequence of the VL domain of SEQ ID NO: 56 or 57. In some embodiments, the anti-Notch2 antibody comprises three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 56 or 57 and a framework of sequence identity with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% relative to the framework amino acid sequence of the VL domain of SEQ ID NO: 56 or 57. In some embodiments, the anti-Notch2 antibody comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 56 or 57 and a framework with at least 95% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 56 or 57. In some embodiments, the anti-Notch2 antibody comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 56 or 57 and a framework with at least 98% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 56 or 57.

[0148] In some embodiments, the anti-Notch2 antibody comprises (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 53; (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 54; (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 55; (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 49; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 50; and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 51 or 52, as well as a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 58, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 56 or 57. In some embodiments, the VH domain has at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the VL domain has at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 56 or 57.

[0149] In some embodiments, the anti-Notch2 antibody includes (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 53; (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 54; (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 55; (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 49; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 50; and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 51 or 52, as well as SEQ ID NO: 58 The antibody comprises a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 58, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 56 or 57; the antibody binds specifically to Notch2. In some embodiments, the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 56 or 57. In some embodiments, the antibody comprises an antibody with a dissociation constant (K) of the VH sequence of SEQ ID NO: 58 and the VL sequence of SEQ ID NO: 56 or 57. D Compared to the dissociation constant (K), the dissociation constant (K) is reduced by up to 10 times or increased by up to 10 times. D It connects to Notch2 which has ).

[0150] In some embodiments, the anti-Notch2 antibody contains a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the anti-Notch2 antibody contains a heavy chain variable domain (VH) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 58. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the anti-Notch2 antibody containing that sequence retains the ability to bind to Notch2. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 58. In certain embodiments, substitutions, insertions, or deletions occur in the region outside the CDR (i.e., at the FR). Optionally, the anti-Notch2 antibody includes the VH sequence of SEQ ID NO: 58, which includes post-translational modifications of that sequence. In certain embodiments, the VH includes one, two, or three CDRs selected from: (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 53, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 54, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 55. In some embodiments, an anti-Notch2 antibody is provided, which includes a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 56 or 57. In some embodiments, the anti-Notch2 antibody comprises a light chain variable domain (VL) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 56 or 57. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the anti-Notch2 antibody containing that sequence retains the ability to bind to Notch2.In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 56 or 57. In certain embodiments, the substitutions, insertions, or deletions occur in the outer region of the CDR (i.e., in the FR). Optionally, the anti-Notch2 antibody contains the VL sequence of SEQ ID NO: 56 or 57, which includes post-translational modifications of that sequence. In certain embodiments, the VL contains one, two, or three CDRs selected from: (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 49, (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 50, and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 51 or 52.

[0151] In some embodiments, an anti-Notch2 antibody is provided, which comprises a VH sequence as in any of the embodiments provided above, and a VL sequence as in any of the embodiments provided above. In some embodiments, the antibody comprises the VH and VL sequences of SEQ ID NO: 58 and SEQ ID NO: 56 or 57, respectively, which include post-translational modifications of those sequences.

[0152] Antibodies containing one or more CDRs of antibody 2621 In some embodiments, the present invention provides an anti-Notch2 antibody comprising at least one, at least two, at least three, at least four, at least five, or all six CDRs selected from (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 62; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 63; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 64; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 59; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 60; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 61.

[0153] In some embodiments, the present invention provides an antibody comprising at least one, at least two, or all three VH CDR sequences selected from (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 62, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 63, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 64. In some embodiments, the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NO: 59. In some embodiments, the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NO: 60 and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 61. In further embodiments, the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NO: 64, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 61, and CDR-H2 comprising the amino acid sequence of SEQ ID NO: 63. In further embodiments, the antibody comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 62, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 63, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 64.

[0154] In some embodiments, the present invention provides an antibody comprising at least one, at least two, or all three VL CDR sequences selected from (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 59, (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 60, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 61. In some embodiments, the antibody comprises one, two, or three CDRs selected from (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 59, (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 60, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 61.

[0155] In some embodiments, the antibody of the present invention comprises (a) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 62, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 63, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 64, and (b) a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 59, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 60, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 61.

[0156] In some embodiments, the present invention provides antibodies comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 62, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 63, (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 64, (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 59, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 60, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 61.

[0157] In some embodiments, the anti-Notch2 antibody comprises one or more of the VH CDR sequences of SEQ ID NO: 66. In another embodiment, the anti-Notch2 antibody comprises one or more of the VL CDR sequences of SEQ ID NO: 65. In yet another embodiment, the anti-Notch2 antibody comprises the VH CDR sequence of SEQ ID NO: 66 and the VL CDR sequence of SEQ ID NO: 65.

[0158] In a further embodiment, the anti-Notch2 antibody comprises the CDR-H1, CDR-H2, and CDR-H3 amino acid sequences of the VH domain of SEQ ID NO: 66 and the CDR-L1, CDR-L2, and CDR-L3 amino acid sequences of the VL domain of SEQ ID NO: 65.

[0159] In some embodiments, the anti-Notch2 antibody comprises one or more heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 66 and a framework of sequence identity with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% relative to the framework amino acid sequence of the VH domain of SEQ ID NO: 66. In some embodiments, the anti-Notch2 antibody comprises three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 66 and a framework of sequence identity with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% relative to the framework amino acid sequence of the VH domain of SEQ ID NO: 66. In some embodiments, the anti-Notch2 antibody comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 66 and a framework with at least 95% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 66. In some embodiments, the anti-Notch2 antibody comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 66 and a framework with at least 98% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 66.

[0160] In some embodiments, the anti-Notch2 antibody comprises one or more light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 65 and a framework of sequence identity with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% relative to the framework amino acid sequence of the VL domain of SEQ ID NO: 65. In some embodiments, the anti-Notch2 antibody comprises three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 65 and a framework of sequence identity with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% relative to the framework amino acid sequence of the VL domain of SEQ ID NO: 65. In some embodiments, the anti-Notch2 antibody comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 65 and a framework with at least 95% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 65. In some embodiments, the anti-Notch2 antibody comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 65 and a framework with at least 98% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 65.

[0161] In some embodiments, the anti-Notch2 antibody comprises (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 62; (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 63; (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 64; (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 59; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 60; and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 61, as well as a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 66, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 65. In some embodiments, the VH domain has at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 66. In some embodiments, the VL domain has at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 65.

[0162] In some embodiments, the anti-Notch2 antibody comprises (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 62; (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 63; (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 64; (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 59; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 60; and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 61, as well as a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 66, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 65; and the antibody specifically binds to Notch2. In some embodiments, the VH domain has at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 66. In some embodiments, the VL domain has at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 65. In some embodiments, the antibody has a dissociation constant (K) of the antibody, which includes the VH sequence of SEQ ID NO: 66 and the VL sequence of SEQ ID NO: 65. D Compared to the dissociation constant (K), the dissociation constant (K) is reduced by up to 10 times or increased by up to 10 times. D It connects to Notch2 which has ).

[0163] In some embodiments, the anti-Notch2 antibody contains a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 66. In some embodiments, the anti-Notch2 antibody contains a heavy chain variable domain (VH) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 66. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the anti-Notch2 antibody containing that sequence retains the ability to bind to Notch2. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 66. In certain embodiments, substitutions, insertions, or deletions occur in the region outside the CDR (i.e., at the FR). Optionally, the anti-Notch2 antibody includes the VH sequence of SEQ ID NO: 66, which includes post-translational modifications of that sequence. In certain embodiments, the VH includes one, two, or three CDRs selected from: (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 62, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 63, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 64. In some embodiments, an anti-Notch2 antibody is provided, which includes a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 65. In some embodiments, the anti-Notch2 antibody comprises a light chain variable domain (VL) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 65. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the anti-Notch2 antibody containing that sequence retains the ability to bind to Notch2.In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 65. In certain embodiments, the substitutions, insertions, or deletions occur in the region outside the CDR (i.e., in the FR). Optionally, the anti-Notch2 antibody contains the VL sequence of SEQ ID NO: 65, which includes post-translational modifications of that sequence. In certain embodiments, the VL contains one, two, or three CDRs selected from: (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 59, (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 60, and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 61.

[0164] In some embodiments, an anti-Notch2 antibody is provided, which comprises a VH sequence as in any of the embodiments provided above, and a VL sequence as in any of the embodiments provided above. In some embodiments, the antibody comprises the VH and VL sequences of SEQ ID NO: 66 and SEQ ID NO: 65, respectively, which include post-translational modifications of those sequences.

[0165] In further embodiments, the present invention provides antibodies that bind to the same epitopes as the anti-Notch2 antibodies provided herein. For example, in certain embodiments, antibodies are provided that bind to the same epitopes as the anti-Notch2 antibody comprising the VH sequence of SEQ ID NO: 32 and the VL sequence of SEQ ID NO: 31. In certain embodiments, anti-Notch2 antibodies are provided that bind to epitopes within the EGF7 repeat of Notch2. In some embodiments, anti-Notch2 antibodies are provided that bind to epitopes within amino acids 260-296 (SEQ ID NO: 70) of Notch2. In some embodiments, anti-Notch2 antibodies are provided that bind to epitopes within amino acids 260-296 (SEQ ID NO: 70) of Notch2.

[0166] In a further embodiment, the present invention provides antibodies that compete with the anti-Notch2 antibodies provided herein for binding to Notch2. For example, in a particular embodiment, an antibody is provided that competes with the anti-Notch2 antibody comprising the VH sequence of SEQ ID NO: 32 and the VL sequence of SEQ ID NO: 31 for binding to Notch2.

[0167] In further embodiments of the present invention, the anti-Notch2 antibody according to any of the above embodiments is a monoclonal antibody comprising a chimeric antibody, a humanized antibody, or a human antibody. In some embodiments, the anti-Notch2 antibody is an antibody fragment, e.g., Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In some embodiments, the antibody is a full-length antibody, e.g., an intact IgG1 antibody, IgG2 antibody, IgG3 antibody, or IgG4 antibody, or other antibody classes or isotypes as defined herein.

[0168] In a further embodiment, an anti-Notch2 antibody according to any of the above embodiments may incorporate any of the features described in Sections 1 to 8 below, either individually or in combination:

[0169] 1. Antibody affinity In certain embodiments, the antibodies provided herein are ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (for example, 10 -8 M or less, for example, 10 -8 M~10 -13 M, for example 10 -9 M~10 -13 The dissociation constant (K) of M D ) has.

[0170] In some embodiments, K DThis is measured using the BIACORE® surface plasmon resonance assay. For example, assays using BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) are performed at 25°C for approximately 10 response units (RUs) using an immobilized antigen CM5 chip. In some embodiments, the carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate at pH 4.8, then injected at a flow rate of 5 μl / min to achieve approximately 10 response units (RUs) of the coupled protein. After antigen injection, 1M ethanolamine is injected to block unreacted groups. For kinetic analysis, Fab's 2-fold serial dilutions (0.78 nM to 500 nM) are injected at a flow rate of approximately 25 μL / min into PBS containing 0.05% polysorbate 20 (TWEEN-20®) surfactant (PBST) at 25°C. The association rate (k on ) and dissociation rate (k off The equilibrium dissociation constant (K) is calculated using a simple one-to-one (1:1) Langmuir coupled model (BIACORE® Evaluation Software version 3.2) by simultaneously fitting the association sensorgram and dissociation sensorgram. D ) is k off / k on It is calculated as a ratio. For example, see Chen et al. J.Mol.Biol.293:865-881(1999).

[0171] In another exemplary assay using the BIAcore® T200 machine, for example, an antibody with a human IgG1 constant region is captured on a Protein A chip to achieve approximately 300 RU. In some such embodiments, serial dilutions of the purified antigen are injected at a flow rate of 100 μL / min at 37°C into HBS-P buffer containing an additional 3 mM CaCl2. The binding rate (ka) and dissociation rate (kd) are calculated using a 1:1 Langmuir binding model (e.g., BIAcore® T200 evaluation software version 2.0). Equilibrium dissociation constant (K) D ) can be calculated as the ratio kd / ka.

[0172] The on-velocity was determined by the above surface plasmon resonance assay to be 10 6 M -1 s -1 If it exceeds this value, this ON rate can be determined by using a fluorescence quenching technique, which measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm, emission = 340 nm, 16 nm band-passing) of a 20 nM anti-antigen antibody (Fab type) in PBS (pH 7.2) at 25°C in the presence of gradually increasing concentrations of antigen, measured with a spectrometer such as an Aviv Instruments stop-flow spectrophotometer equipped with a stirred cuvette or an 8000 series SLM-AMINCO™ spectrophotometer (ThermoSpectronic).

[0173] Alternatively, K D This is measured by radiolabeled antigen-binding assay (RIA). In some embodiments, the RIA is performed using Fab versions of the antibody of interest and its antigen. For example, the solution binding affinity of Fab to the antigen is measured in the presence of a titration series of unlabeled antigens at the lowest concentration. 125I) Fab is equilibrated with labeled antigen, and then measured by capturing the bound antigen with a plate coated with anti-Fab antibody (see, for example, Chen et al., "J.Mol.Biol." Vol. 293, pp. 865-881 (1999)). To establish assay conditions, MICROTITER® multiwell plates (Thermo Scientific) are coated overnight with 5 μg / mL of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and then blocked with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23°C). In a non-adsorbent plate (Nunc#269620), 100 pM or 26 pM [ 125 Mix the [I]-antigen with serial dilutions of the Fab of interest (e.g., consistent with the evaluation of the anti-VEGF antibody Fab-12 in Presta et al., "Cancer Res.", Vol. 57, pp. 4593-4599 (1997)). Then incubate the Fab of interest overnight, but incubation can be extended for a longer period (e.g., about 65 hours) to ensure equilibrium is reached. Then transfer the mixture to a capture plate for incubation at room temperature (e.g., 1 hour). Next, remove the solution and wash the plate eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate is dry, add 150 μL / well of scintillant (MICROSCINT-20®, Packard) and count the plate on a TOPCOUNT® gamma counter (Packard) for 10 minutes. Select a concentration of each Fab that yields less than 20% of the maximum binding for use in competitive binding assays.

[0174] 2. Antibody fragment In certain embodiments, the antibodies provided herein are antibody fragments.

[0175] In some embodiments, the antibody fragment is a Fab, Fab', Fab'-SH, or F(ab')2 fragment, particularly a Fab fragment. When an intact antibody is digested with papain, two identical antigen-binding fragments (so-called "Fab" fragments) are obtained, each containing the variable domains of the heavy and light chains (VH and VL, respectively), as well as the constant domain (CL) and the first constant domain (CH1) of the heavy chain. Thus, a "Fab fragment" is an antibody fragment having a light chain containing the VL and CL domains, and a heavy chain fragment containing the VH and CH1 domains. A "Fab' fragment" is different from a Fab fragment by adding residues at the carboxyl terminus of the CH1 domain, which contains one or more cysteines, from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine ​​residues (multiple valencies) of the constant domain retain a free thiol group. Pepsin treatment yields an F(ab')2 fragment having two antigen-binding sites (two Fab fragments) and a portion of the Fc region. For a description of the Fab and F(ab')2 fragments, which contain salvage receptor-binding epitope residues and have a longer in vivo half-life, please refer to U.S. Patent No. 5,869,046.

[0176] In some embodiments, the antibody fragment is a diabody, triabody, or tetrabody. A diabody is an antibody fragment having two antigen-binding sites that may be bivalent or bispecific. See, for example, European Patent No. 404,097, WO1993 / 01161, Hudson et al., Nat. Med. 9:129-134 (2003), and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0177] In a further embodiment, the antibody fragment is a single-stranded Fab fragment. The "single-stranded Fab fragment" or "scFab" is a polypeptide comprising an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL), and a linker, wherein the antibody domain and the linker have one of the following orders from the N-terminus to the C-terminus: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL. In particular, the linker is a polypeptide of at least 30 amino acids, preferably 32 to 50 amino acids. The single-stranded Fab fragment is stabilized by a native disulfide bond between the CL domain and the CH1 domain. In addition, these single-stranded Fab fragments can be further stabilized by the formation of interchain disulfide bonds via the insertion of cysteine ​​residues (for example, at position 44 of the variable heavy chain and position 100 of the variable light chain, according to Kabat numbering).

[0178] In some embodiments, the antibody fragment is a single-stranded variable fragment (scFv). A "single-stranded variable fragment" or "scFv" is a fusion protein of the variable domains of the heavy chain (VH) and light chain (VL) of an antibody, linked by a linker. In particular, the linker is a short polypeptide of 10 to 25 amino acids, usually rich in glycine for flexibility and serine or threonine for solubility, and can be linked at either the N-terminus of VH or the C-terminus of VL. This protein can retain the specificity of the original antibody despite the removal of the constant region and the introduction of a linker. For a review of scFv fragments, see, for example, Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994). See also International Publication No. 93 / 16185 and U.S. Publications No. 5,571,894 and No. 5,587,458.

[0179] In some embodiments, the antibody fragment is a single-domain antibody. A "single-domain antibody" is an antibody fragment that contains all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, the single-domain antibody is a human single-domain antibody (see Domantis, Inc., Waltham, MA; for example, U.S. Patent No. 6,248,516B1).

[0180] Antibody fragments can be produced by various techniques, including, but not limited to, proteolytic digestion of intact antibodies and recombinant production by recombinant host cells (e.g., Escherichia coli), as described herein.

[0181] 3. Chimeric antibodies and humanized antibodies In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567 and in Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody includes a non-human variable region (e.g., a variable region derived from mouse, rat, hamster, rabbit, or non-human primate, e.g., monkey) and a human constant region. In further examples, a chimeric antibody is a “class-switched” antibody in which the class or subclass is changed from those of the parent antibody. A chimeric antibody includes its antigen-binding fragment.

[0182] In certain embodiments, chimeric antibodies are humanized antibodies. Typically, non-human antibodies are humanized to reduce their immunogenicity against humans while retaining the specificity and affinity of the parent non-human antibody. Humanized antibodies typically contain one or more variable domains (CDRs or parts thereof) derived from the non-human antibody, and FRs (or parts thereof) derived from the human antibody sequence. Humanized antibodies also optionally contain at least a portion of the human constant region. In some embodiments, several FR residues in the humanized antibody are replaced with corresponding residues from the non-human antibody (e.g., the antibody from which the CDR residues are derived) to restore or improve antibody specificity or affinity, for example.

[0183] Humanized antibodies and their production methods are reviewed in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described below: Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patents No. 5,821,337, No. 7,527,791, No. 6,982,321, and No. 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (Describes specificity-determining region (SDR) grafts); Padlan, Mol.Immunol.28:489-498 (1991) (Describes resurfacing); Dall'Acqua et al., Methods 36:43-60 (2005) (Describes "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br.J.Cancer,83:252-260 (2000) (Describes the "guided selection approach" to FR shuffling).

[0184] The following are, but are not limited to, human framework regions that can be used for humanization: framework regions selected using the "best fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from human antibody consensus sequences of specific subtypes of heavy chain or light chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); human maturation (somatic mutation) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening of the FR library (see, e.g., Baca et al. See al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996).

[0185] 4. Human antibodies In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be prepared using a variety of techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0186] Human antibodies can be prepared by administering immunogens to transgenic animals modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals typically contain, or have extrachromosomal, or randomly integrated human immunoglobulin loci, replacing endogenous immunoglobulin loci. In such transgenic mice, endogenous immunoglobulin loci are generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 describing XENOMOUSE® technology; U.S. Patent No. 5,770,429 describing HuMab® technology; U.S. Patent No. 7,041,870 describing KM MOUSE® technology; and U.S. Patent Application Publication 2007 / 0061900 describing VelociMouse® technology. The human variable region from intact antibodies produced by such animals may be further modified, for example, by combining it with a different human constant region.

[0187] Human antibodies can also be produced by hybridoma-based methods. Human myeloma cell lines and mouse-human xenomyeloma cell lines for producing human monoclonal antibodies have been described. (See, for example, Kozbor J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991)). Human antibodies produced via human B-cell hybridoma technology have also been described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Further methods include, for example, U.S. Patent No. 7,189,826 (describes the production of monoclonal human IgM antibodies derived from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describes human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0188] Human antibodies can also be generated by isolating variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences can then be combined with desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0189] 5. Antibodies derived from the library In certain embodiments, the antibodies provided herein are obtained from a library. The antibodies of the present invention can be isolated by screening a combinatorial library for one or more antibodies having desired activity. Methods for screening combinatorial libraries are reviewed, for example, by Lerner et al. in Nature Reviews 16:498-508 (2016). For example, various methods for producing phage display libraries and screening such libraries for antibodies having desired binding properties are known in the art. Such methods can be found, for example, in Frenzel et al.'s mAbs 8:1177-1194 (2016); Bazan et al.'s Human Vaccines and Immunotherapeutics 8:1817-1828 (2012); Zhao et al.'s Critical Reviews in Biotechnology 36:276-289 (2016); Hoogenboom et al.'s Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001); and Marks and Bradbury's Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003).

[0190] In certain phage display methods, the repertoire of VH and VL genes are individually cloned by polymerase chain reaction (PCR), randomly recombined in a phage library, and then screened for antigen-binding phages, as described by Winter et al. in Annual Review of Immunology 12:433-455 (1994). The phages typically present antibody fragments either as single-stranded Fv (scFv) fragments or as Fab fragments. Libraries from immunization sources provide high-affinity antibodies against immunogens without the need to construct hybridomas. Alternatively, a naive repertoire can be cloned (e.g., from humans) to obtain a single antibody source against a wide range of non-self to self antigens without immunization, as described by Griffiths et al. in EMBO Journal 12:725-734 (1993). Ultimately, naive libraries can also be constructed synthetically by cloning an unrealigned V gene segment from stem cells, as described by Hoogenboom and Winter in Journal of Molecular Biology 227:381-388 (1992), and by encoding a highly variable CDR3 region using PCR primers containing random sequences to achieve realignment in vitro. Patent publications describing human antibody phage libraries include, for example, U.S. Patent Nos. 5,750,373, 7,985,840, 7,785,903 and 8,679,490, and U.S. Patent Application Publications 2005 / 0079574, 2007 / 0117126, 2007 / 0237764 and 2007 / 0292936.

[0191] Further examples of methods known in the art for screening combinatorial libraries for antibodies having desired activity or multiple activity include ribosome and mRNA display, as well as methods for antibody display and selection in bacterial, mammalian, insect, or yeast cells. Methods for yeast surface display are outlined, for example, in Scholler et al. in Methods in Molecular Biology 503:135-56 (2012), Cherf et al. in Methods in Molecular Biology 1319:155-175 (2015), and Zhao et al. in Methods in Molecular Biology 889:73-84 (2012). Methods for ribosome display are described, for example, in He et al. in Nucleic Acids Research 25:5132-5134 (1997) and Hanes et al. in PNAS 94:4937-4942 (1997).

[0192] Antibodies or antibody fragments isolated from a human antibody library are considered human antibodies or human antibody fragments in this specification.

[0193] 6. Multispecific antibodies In certain embodiments, the antibodies provided herein are multispecific antibodies, e.g., bispecific antibodies. A multispecific antibody is a monoclonal antibody that has binding specificity to at least two different sites, i.e., different epitopes on different antigens or different epitopes on the same antigen. In certain embodiments, a multispecific antibody has three or more binding specificities. In certain embodiments, one binding specificity is for Notch2 and the other is for any other antigen. In certain embodiments, a bispecific antibody may bind to two (or more) different epitopes of Notch2. Multispecific (e.g., bispecific) antibodies can also be used as cytotoxic agents or to localize cells to cells expressing Notch2. Multispecific antibodies may be prepared as full-length antibodies or antibody fragments.

[0194] Techniques for producing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)) and "knob-in-hole" operations (see, for example, U.S. Patent No. 5,731,168 and Atwell et al., J.Mol.Biol.270:26 (1997)). Multispecific antibodies are also produced by manipulating the electrostatic steering effect to create antibody Fc heterodimer molecules (see, e.g., International Publication No. 2009 / 089004); crosslinking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980 and Brennan et al., Science, 229:81 (1985)); generating bispecific antibodies using leucine zippers (see, e.g., Kostelny et al., J.Immunol., 148(5):1547-1553 (1992) and International Publication No. 2011 / 034605); using common light chain techniques to avoid light chain mispairing problems (see, e.g., International Publication No. 98 / 50431); and using "diabody" techniques to produce bispecific antibody fragments (see, e.g., Hollinger et al.) It can be prepared by using single-stranded Fv(sFv) dimers (see, for example, Gruber et al., J.Immunol., 152:5368 (1994)); and by preparing a trispecific antibody as described, for example, Tutt et al. J.Immunol. 147:60 (1991).

[0195] For example, this also includes manipulated antibodies having three or more antigen-binding sites, such as "octopus antibodies," or DVD-Ig (see, for example, International Publication Nos. 2001 / 77342 and International Publication Nos. 2008 / 024715). Other examples of multispecific antibodies having three or more antigen-binding sites can be found in International Publication Nos. 2010 / 115589, 2010 / 112193, 2010 / 136172, 2010 / 145792, and 2013 / 026831. Bispecific antibodies or their antigen-binding fragments also include "dual-acting FAbs" or "DAFs" that contain antigen-binding sites that bind to Notch2 and another different antigen, or to two different epitopes of Notch2 (see, for example, U.S. Patent Application Publication 2008 / 0069820 and International Publication 2015 / 095539).

[0196] Multispecific antibodies may also be provided in an asymmetric form with domain crossovers on one or more binding arms of the same antigen specificity, i.e., by exchanging VH / VL domains (see, e.g., International Publications 2009 / 080252 and 2015 / 150447), CH1 / CL domains (see International Publication 2009 / 080253), or complete Fab arms (see International Publications 2009 / 080251, 2016 / 016299, PNAS, Schaefer et al., 108(2011)1187-1191, and Klein et al., MAbs 8(2016)1010-20). In some embodiments, multispecific antibodies include cross-Fab fragments. The terms “cross-Fab fragment,” “xFab fragment,” or “crossover Fab fragment” refer to Fab fragments in which either the variable or constant regions of the heavy and light chains are exchanged. The cross-Fab fragment includes a polypeptide chain composed of a light chain variable region (VL) and a heavy chain constant region 1 (CH1), and a polypeptide chain composed of a heavy chain variable region (VH) and a light chain constant region (CL). The asymmetric Fab arm can also be manipulated by introducing charged or uncharged amino acid mutations into the domain interface to direct the correct Fab pairing. See, for example, International Publication 2016 / 172485.

[0197] Various further molecular formats of multispecific antibodies are known in the art and are included herein (see, for example, Spiess et al., Mol Immunol 67(2015) 95-106).

[0198] Certain types of multispecific antibodies similarly included herein are bispecific antibodies designed to simultaneously bind to target cells, such as surface antigens on tumor cells, and to activated invariant components of the T cell receptor (TCR) complex, such as CD3, for retargeting T cells and killing target cells. Thus, in certain embodiments, the antibodies provided herein are multispecific antibodies, particularly bispecific antibodies, in which one of their binding specificities is for Notch2 and the other is for CD3.

[0199] Examples of bispecific antibody formats that may be useful for this purpose include so-called "BiTE" (bispecific T cell engager) molecules in which two scFv molecules are fused by a flexible linker (see, e.g., International Publication Nos. 2004 / 106381, 2005 / 061547, 2007 / 042261, and 2008 / 119567, Nagorsen and Baeuerle, Exp Cell Res 317, 1255-1260 (2011)), diabodies (Holliger et al., Prot Eng 9, 299-305 (1996)) and their derivatives, such as tandem diabodies ("TandAb", Kipriyanov et al., J Mol Biol This specification includes, but is not limited to, T cell bispecific antibody formats, such as 293,41-56(1999), “DART” (Dual Affinity Retargeting) molecules based on the diabody format but characterized by a C-terminal disulfide crosslink for stabilization (Johnson et al., J Mol Biol 399,436-449(2010)), and so-called triomabs, which are all-hybrid mouse / rat IgG molecules (Seimetz et al., Cancer Treat Rev 36,458-467(2010)). Specific T cell bispecific antibody formats included herein are described in International Publication Nos. 2013 / 026833, 2013 / 026839, and 2016 / 020309; Bacac et al., Oncoimmunology 5(8)(2016)e1203498.

[0200] 7. Antibody variants In certain embodiments, amino acid sequence variants of antibodies provided herein are intended. For example, it may be desirable to alter the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of antibodies may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from residues in the amino acid sequence of the antibody, and / or insertions into residues in the amino acid sequence of the antibody, and / or substitutions of residues in the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be carried out so as to reach the final construct, insofar as the final construct has the desired characteristics (e.g., antigen binding).

[0201] a) Substitution, insertion, and deletion variants In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include CDRs and FRs. Conservative substitutions are shown in Table 1 under the heading "Preferred Substitutions." More substantial substitutions are provided in Table 1 under the heading "Exemplary Substitutions" and are further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest, and the product can be screened for desired activity, e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC. [Table 1]

[0202] Amino acids can be classified according to their general side-chain properties. (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basicity: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0203] Non-conservative substitution involves exchanging a member of one class with a member of another class.

[0204] Certain substitution variants involve substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized antibody or a human antibody). Generally, the resulting variant(s) selected for further testing have modifications (e.g., improvements) to certain biological properties (e.g., increased affinity, decreased immunogenicity) compared to the parent antibody, and / or substantially retain certain biological properties of the parent antibody. Exemplary substitution variants are affinity-matured antibodies, which can be readily produced, for example, using affinity maturation techniques based on phage display, as described herein. In short, one or more CDR residues are mutated, the variant antibody is displayed to a phage, and it is screened for specific biological activity (e.g., binding affinity).

[0205] To improve antibody affinity, modifications (e.g., substitutions) may be made in the CDR, for example. Such modifications may be made in CDR "hot spots," i.e., residues encoded by codons that frequently undergo mutations during the somatic cell maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and / or residues that come into contact with the antigen, and the resulting variant VH or VL is tested for binding affinity. Affinity maturation by secondary library construction and reselection from there is described, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some aspects of affinity maturation, diversity is introduced into the variable genes selected for maturation by one of various methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then constructed. Next, this library is screened to identify antibody variants with the desired affinity. Another method for introducing diversity involves CDR-directed methods in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3, in particular, are often targeted.

[0206] In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, provided that such modifications do not substantially reduce the antibody's ability to bind to the antigen. For example, conservative modifications that do not substantially reduce binding affinity (e.g., conservative substitutions as provided herein) may be made within a CDR. Such modifications may, for example, be outside the antigen-contact residue in the CDR. In the specific variant VH and VL sequences described above, each CDR is either unmodified or has one, two, or three or fewer amino acid substitutions.

[0207] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, 244:1081-1085. This method identifies target residues or groups (e.g., charged residues such as arg, asp, his, lys, and glu) and substituted them with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the antibody-antigen interaction is affected. Further substitutions may be introduced at amino acid positions that exhibit functional sensitivity to the initial substitution. Alternatively, or additionally, the crystalline structure of the antigen-antibody complex can be used to identify contact points between the antibody and antigen. Such contact residues and adjacent residues may be targeted as candidate substitutions or removed. Variants may be screened to determine whether they possess the desired properties.

[0208] Amino acid sequence insertions include amino-terminus and / or carboxyl-terminus fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of one or more amino acid residues. An example of terminal insertion is an antibody with an N-terminal methionyl residue. Other insertion variants of antibody molecules include the fusion of the N-terminus or C-terminus of an antibody to an enzyme (e.g., ADEPT (for antibody-directed enzyme prodrug therapy)) or polypeptide, which increases the serum half-life of the antibody.

[0209] b) Glycosylated variants In certain embodiments, the antibody provided herein is modified to increase or decrease the degree of glycosylation of the antibody. The addition or deletion of glycosylation sites to an antibody can be conveniently achieved by modifying the amino acid sequence so that one or more glycosylation sites are created or removed.

[0210] If the antibody contains an Fc region, the oligosaccharide attached to the antibody may be modified. Natural antibodies produced by mammalian cells typically contain branched or bibranched oligosaccharides that are commonly bound to Asn297 of the CH2 domain of the Fc region by an N-bond. See, for example, Wright et al. TIBTECH 15:26-32 (1997). Oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose bound to the GlcNAc of the "stem" of the bibranched oligosaccharide structure. In some embodiments, modification of the oligosaccharide in the antibody of the present invention may be carried out to generate antibody variants having specific improved properties.

[0211] In some embodiments, antibody variants are provided that have an oligosaccharide structure lacking non-fucosylated oligosaccharides, i.e., fucose binding (direct or indirect) to the Fc region. Such non-fucosylated oligosaccharides (also called "afucosylated" oligosaccharides) are N-linked oligosaccharides that lack a fucose residue to which a first GlcNAc is bound in the stem of a branched oligosaccharide structure. In some embodiments, antibody variants are provided in which the proportion of non-fucosylated oligosaccharides in the Fc region is increased compared to the natural or parent antibody. For example, the proportion of non-fucosylated oligosaccharides may be at least about 20%, at least about 40%, at least about 60%, at least about 80%, or possibly about 100% (i.e., no fucosylated oligosaccharides are present). The proportion of non-fucosylated oligosaccharides is the (average) amount of fucose-less oligosaccharides relative to the total of all oligosaccharides bound to Asn297 (e.g., complex, hybrid, and high-mannose structures), as measured by MALDI-TOF mass spectrometry, for example, as described in International Publication No. 2006 / 082515. Asn297 refers to the asparagine residue located at approximately position 297 of the Fc region (EU numbering of Fc region residues); however, due to minor sequence changes in the antibody, Asn297 may be located upstream or downstream of position 297, i.e., approximately ±3 amino acids between positions 294 and 300. Such antibodies with an increased proportion of non-fucosylated oligosaccharides in the Fc region may have improved FcγRIIIa receptor binding and / or improved effector function, particularly improved ADCC function. See, for example, U.S. Patent Application Publication No. 2003 / 0157108; and No. 2004 / 0093621.

[0212] Examples of cell lines capable of producing antibodies with reduced fucosylation include Lec13CHO cells with insufficient protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Patent Application Publication No. 2003 / 0157108; and International Publication No. 2004 / 056312, particularly Example 11), and knockout cell lines, such as FUT8 knockout CHO cells of the alpha-1,6-fucosyltransferase gene (e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614-622 (2004); Kanda, Y. et al.) Examples include cells in which GDP-fucose synthesis or transporter protein activity is reduced or eliminated (see, for example, U.S. Patent Publication Nos. 2004259150, 2005031613, 2004132140, and 2004110282).

[0213] In a further embodiment, the antibody variant is provided with a bifurcated oligosaccharide, for example, in which a bifurcated oligosaccharide bound to the Fc region of the antibody is bifurcated by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function, as described above. Examples of such antibody variants are described, for example, in Umana et al., Nat Biotechnol 17, 176-180 (1999); Ferrara et al., Biotechn Bioeng 93, 851-861 (2006); International Publication No. 99 / 54342, International Publication No. 2004 / 065540, and International Publication No. 2003 / 011878.

[0214] Antibody variants are also provided that have at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function. Examples of such antibody variants are described, for example, in International Publications 1997 / 30087; 1998 / 58964; and 1999 / 22764.

[0215] c) Fc region variant In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibodies presented herein to create an Fc region variant. The Fc region variant may include a human Fc region sequence (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc region) containing amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0216] In certain embodiments, the present invention conceives antibody variants that, by possessing some, but not all, effector functions, are desirable candidates for applications where the in vivo half-life of the antibody is important, but certain effector functions (e.g., complement-dependent cell-mediated cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC)) are unnecessary or harmful. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / loss of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to confirm that the antibody lacks FcγR binding (and therefore is likely to lack ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcR in hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for evaluating the ADCC activity of the target molecule are described in U.S. Patent No. 5,500,362 (see, for example, Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be employed (see, for example, ACTI® non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA) and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells.Alternatively, or in addition, the target ADCC activity can be evaluated in vitro in an animal model, for example, as disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). Furthermore, a C1q binding assay may be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in International Publication Nos. 2006 / 029879 and 2005 / 100402. To evaluate complement activation, a CDC assay can be performed (see, for example, Gazzano-Santoro et al., J.Immunol.Methods 202:163(1996); Cragg, MS et al., Blood 101:1045-1052(2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743(2004)). FcRn binding and in vitro clearance / half-life determination can also be performed using methods known in the art (see, for example, Petkova, S B et al., Int'l.Immunol.18(12):1759-1769(2006); WO2013 / 120929Al).

[0217] Antibodies with reduced effector function include those having one or more substitutions at residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region (U.S. Patent No. 6,737,056). Such Fc mutants include those having two or more substitutions at amino acid positions 265, 269, 270, 297, and 327, and include the so-called "DANA" Fc mutant in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581).

[0218] Specific antibody variants exhibiting improved or reduced binding to FcR are described. (See, for example, U.S. Patent No. 6,737,056; International Publication No. 2004 / 056312; and Shields et al., J. Biol. Chem. 9(2):6591-6604(2001).)

[0219] In certain embodiments, the antibody variant includes an Fc region having one or more amino acid substitutions that improve ADCC, for example, substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region.

[0220] In certain embodiments, the antibody variant includes an Fc region having one or more amino acid substitutions that reduce FcγR binding, e.g., Fc region positions 234 and 235 (residues in EU numbering). In some embodiments, the substitutions are L234A and L235A (LALA). In certain embodiments, the antibody variant further includes D265A and / or P329G in the Fc region derived from the human IgG1 Fc region. In some embodiments, the substitutions are L234A, L235A, and P329G (LALA-PG) in the Fc region derived from the human IgG1 Fc region. (See, for example, International Publication No. 2012 / 130831.) In some embodiments, the substitutions are L234A, L235A, and D265A (LALA-DA) in the Fc region derived from the human IgG1 Fc region.

[0221] In some embodiments, changes occur within the Fc region that result in alterations (i.e., either enhancement or reduction) of C1q binding and / or complement-dependent cell-mediated cytotoxicity (CDC), as described, for example, in U.S. Patent No. 6,194,551, International Publication No. 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0222] Antibodies that have increased half-lives and improved binding to the embryonic Fc receptor (FcRn) and play a role in transferring maternal IgG to the fetus (Guyer et al., J.Immunol. 117:587 (1976) and Kim et al., J.Immunol. 24:249 (1994)) are described in U.S. Patent Application Publication No. 2005 / 0014934 (Hinton et al.). These antibodies contain an Fc region having one or more substitutions therein that improve the binding of the Fc region to FcRn. Such Fc variants include substitutions of one or more of the Fc region residues: 238, 252, 254, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, for example, substitution of Fc region residue 434 (see, for example, U.S. Patent No. 7,371,826; Dall'Acqua, WF, et al. J. Biol. Chem. 281 (2006) 23514-23524).

[0223] The Fc region residues crucial to the mouse Fc-mouse FcRn interaction have been identified by site-directed mutagenesis (see, for example, Dall'Acqua, WF, et al. J.Immunol 169(2002) 5171-5180). Residues I253, H310, H433, N434, and H435 (EU index numbering) are involved in the interaction (Medesan, C., et al., Eur.J.Immunol.26(1996) 2533; Firan, M., et al., Int.Immunol.13(2001) 993; Kim, JK, et al., Eur.J.Immunol.24(1994) 542). Residues I253, H310, and H435 were found to be decisive in the interaction between human Fc and mouse FcRn (Kim, JK, et al., Eur. J. Immunol. 29 (1999) 2819). Studies of the human Fc-human FcRn complex have shown that residues I253, S254, H435, and Y436 are decisive in the interaction (Firan, M., et al., Int. Immunol. 13 (2001) 993; Shields, RL, et al., J. Biol. Chem. 276 (2001) 6591-6604). Yeung, YA, et al. (J.Immunol.182(2009)7667-7671) reported and investigated various mutations in residues 248-259, 301-317, 376-382, and 424-437.

[0224] In certain embodiments, the antibody variant includes an Fc region having one or more amino acid substitutions that reduce FcRn binding, e.g., mutations at Fc region positions 253 and / or 310 and / or 435 (residues in EU numbering). In certain embodiments, the antibody variant includes an Fc region having amino acid substitutions at positions 253, 310, and 435. In some embodiments, the substitutions are I253A, H310A, and H435A in the Fc region derived from the human IgG1 Fc region. See, for example, Grevys, A., et al., J.Immunol. 194 (2015) 5497-5508.

[0225] In certain embodiments, the antibody variant includes an Fc region having one or more amino acid substitutions that reduce FcRn binding, e.g., mutations at positions 310 and / or 433 and / or 436 (residues in EU numbering) of the Fc region. In certain embodiments, the antibody variant includes an Fc region having amino acid substitutions at positions 310, 433, and 436. In some embodiments, the substitutions are H310A, H433A, and Y436A in the Fc region derived from the human IgG1 Fc region. (See, for example, International Publication No. 2014 / 177460.)

[0226] In certain embodiments, the antibody variant includes an Fc region having one or more amino acid substitutions that increase FcRn binding, e.g., mutations at Fc region positions 252 and / or 254 and / or 256 (residues in EU numbering). In certain embodiments, the antibody variant includes an Fc region having amino acid substitutions at positions 252, 254, and 256. In some embodiments, the substitutions are M252Y, S254T, and T256E in the Fc region derived from the human IgG1 Fc region. For other examples of Fc region variants, see also Duncan & Winter, Nature 322:738-40 (1988), U.S. Patent No. 5,648,260, U.S. Patent No. 5,624,821, and International Publication No. 94 / 29351.

[0227] The C-terminus of the heavy chain of an antibody as reported herein may be a complete C-terminus ending with the amino acid residues PGK. The C-terminus of the heavy chain may be a shortened C-terminus with one or two of the C-terminal amino acid residues removed. In one preferred embodiment, the C-terminus of the heavy chain is the shortened C-terminal end PG. In some embodiments of all the embodiments reported herein, an antibody comprising a heavy chain containing the C-terminal CH3 domain specified herein contains a C-terminal glycine-lysine dipeptide (amino acid positions G446 and K447, EU index numbering). In some embodiments of all the embodiments reported herein, an antibody comprising a heavy chain containing the C-terminal CH3 domain specified herein contains a C-terminal glycine residue (amino acid position G446, EU index numbering).

[0228] d) Cysteine-engineered antibody variants In certain embodiments, it may be desirable to generate a cysteine-engineered antibody, for example, a THIOMAB™, in which one or more residues of the antibody are replaced with cysteine residues. In certain embodiments, the replaced residues occur at accessible sites of the antibody. By replacing these residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and can be used to conjugate the antibody to other sites such as a drug moiety or a linker-drug moiety to create an immunoconjugate, as further described herein. Cysteine-engineered antibodies can be generated, for example, as described in U.S. Patent Nos. 7,521,541, 8,309,30, 7,855,275, 9,000,130, or International Publication No. 2016 / 040856.

[0229] 8. Immunoconjugates The invention also provides an immunoconjugate comprising an anti-Notch2 antibody herein conjugated to one or more therapeutic agents such as a cytotoxic agent, a chemotherapeutic agent or chemotherapeutic drug, a growth inhibitor, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), or a radioisotope.

[0230] In some embodiments, the immunoconjugate is an antibody-drug conjugate (ADC) in which the antibody is conjugated to one or more of the aforementioned therapeutic agents. The antibody is typically connected to one or more of the therapeutic agents using a linker. An overview of ADC technology, including examples of therapeutic agents, therapeutic drugs, and linkers, is described in Pharmacol Review 68:3-19 (2016).

[0231] In some embodiments, the immunoconjugate comprises an antibody described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (derived from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogelin, restrictocin, phenomycin, enomycin, and trichothecene.

[0232] In some embodiments, the immunoconjugate comprises an antibody described herein conjugated to a radioactive atom to form a radioactive conjugate. A variety of radioisotopes are available for the production of radioactive conjugates. Examples include At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212Examples include radioactive isotopes of Lu. When used for detection, radioactive conjugates may include radioactive atoms for scintigraphy, such as tc99m or I123, or spin labels for nuclear magnetic resonance (NMR) imaging (also known as MRI) (here again, iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron, etc.).

[0233] Conjugates of antibodies and cytotoxic agents can be prepared using various bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imide esters (e.g., dimethyladipimidate HCl), active esters (e.g., disuccinimidyl sberate), aldehydes (e.g., glutaraldehyde), bisazide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and diactive fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, lysine immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for the conjugation of radioactive nucleotides to antibodies. See International Publication No. 94 / 11026. The linker may be a “cleavable linker” that facilitates the release of cytotoxic drugs within cells. For example, acid-unstable linkers, peptidase-sensitive linkers, photo-unstable linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al., “Cancer Res.” Vol. 52, pp. 127-131 (1992); U.S. Patent No. 5,208,020).

[0234] The immunoconjugates or ADCs used herein are, but are not limited to, conjugates prepared using commercially available crosslinking reagents, including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, as well as SVSB (succinimidyl-(4-vinylsulfone)benzoate).

[0235] B. Recombination methods and compositions Antibodies can be produced using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. For these methods, one or more isolated nucleic acids encoding antibodies are provided.

[0236] In the case of a natural antibody or natural antibody fragment requiring two nucleic acids, one is for the light chain or fragment thereof, and the other is for the heavy chain or fragment thereof. Such nucleic acids encode the amino acid sequence containing the VL and / or VH of the antibody (e.g., the light chain and / or heavy chain(s) of the antibody). These nucleic acids may be on the same expression vector or on different expression vectors.

[0237] In the case of a bispecific antibody having a heterodimer heavy chain requiring four nucleic acids, one is for the first light chain, one for the first heavy chain containing the first heteromonomer Fc region polypeptide, one for the second light chain, and one for the second heavy chain containing the second heteromonomer Fc region polypeptide. The four nucleic acids may be contained in one or more nucleic acid molecules or expression vectors. Such nucleic acids encode amino acid sequences containing the first VL and / or the first VH containing the first heteromonomer Fc region and / or the second VL and / or the second VH containing the second heteromonomer Fc region of the antibody (e.g., the first and / or second light chains and / or the first and / or second heavy chains of the antibody). These nucleic acids may be on the same expression vector or different expression vectors, and typically these nucleic acids are located on two or three expression vectors, i.e., one vector may contain two or more of these nucleic acids. An example of these bispecific antibodies is a cross-Mab (see, e.g., Schaefer, W. et al., PNAS, 108(2011)11187-1191). For example, one heteromonomer heavy chain contains a so-called "knob mutation" (T366W and optionally one of S354C or Y349C), and the other contains a so-called "hole mutation" (T366S, L368A and Y407V and optionally Y349C or S354C) (see, e.g., Carter, P. et al., Immunotechnol. 2(1996)73) (according to EU numbering).

[0238] In some embodiments, isolated nucleic acids encoding antibodies used in the methods reported herein are provided.

[0239] In some embodiments, a method is provided for producing an anti-Notch2 antibody, comprising culturing host cells containing nucleic acids encoding the antibody under conditions suitable for antibody expression, and optionally recovering the antibody from the host cells (or host cell culture).

[0240] For recombinant production of anti-Notch2 antibodies, for example, the nucleic acids encoding the aforementioned antibodies are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using common procedures (for example, by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antibody).

[0241] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, especially when glycosylation or Fc effector function is not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patents 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, KA, In: Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, which describes the expression of antibody fragments in Escherichia coli.) After expression, antibodies may be isolated from bacterial cell paste in appropriate fractions and further purified.

[0242] In addition to prokaryotes, eukaryotes such as filamentous fungi and yeasts are suitable as cloning or expression hosts for antibody-encoding vectors, including strains of fungi and yeast in which the glycosylation pathway has been "humanized," resulting in the production of antibodies with partially or completely human glycosylation patterns. See Gerngross, TU, Nat. Biotech. 22 (2004) 1409-1414; and Li, H. et al., Nat. Biotech. 24 (2006) 210-215.

[0243] Furthermore, suitable host cells for expressing (glycosylated) antibodies are derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains have been identified, and these may be used in combination with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0244] Plant cell cultures can also be used as hosts. See, for example, U.S. Patents 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLANTIBODIES® technology for antibody production in transgenic plants).

[0245] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspensions may be useful. Other examples of useful mammalian host cell lines include the CV1 monkey kidney cell line transformed by SV40 (COS-7), human embryonic kidney cells (e.g., 293 cells or 293T cells as described in Graham, F. Let al., J. Gen Virol. 36 (1977) 59-74, baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells as described in Mather, JP, Biol. Reprod. 23 (1980) 243-252), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT060562), and TRI cells (e.g., Mather, JP et al., Annals). These include MRC5 cells and FS4 cells (described in NYAcad.Sci.383(1982)44-68). Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells (Urlaub, G. et al., Proc. Natl. Acad.Sci. USA 77(1980)4216-4220), including DHFR-CHO cells, and myeloma cell lines, such as Y0, NS0, and Sp2 / 0. For a review of specific mammalian host cells suitable for antibody production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0246] In some embodiments, the host cell is a eukaryote, such as a Chinese hamster ovary (CHO) cell or a lymphocyte (e.g., Y0, NS0, Sp20 cell).

[0247] C. Assay The anti-Notch2 antibodies provided herein may be identified, screened, or characterized for their physical / chemical properties and / or biological activity by various assays known in the art.

[0248] 1. Binding assays and other assays In some embodiments, the antibody of the present invention is tested for its antigen-binding activity by known methods such as ELISA and Western blotting.

[0249] In some embodiments, a competitive assay may be used to identify antibodies that compete with one or more of the antibodies rat.1B2 or its humanized version, rat.3107, rb.2338, rb.2430, and / or rb.2621 provided herein for binding to Notch2. In certain embodiments, such competitive antibodies bind to the same epitope (e.g., a linear or structural epitope) to which rat.1B2 or its humanized version, rat.3107, rb.2338, rb.2430, and / or rb.2621 binds. Detailed illustrative methods for mapping the epitopes to which antibodies bind are provided in Morris (1996), "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).

[0250] In an exemplary competition assay, immobilized Notch2 is incubated in a solution containing a first labeled antibody that binds to Notch2 (e.g., rat.1B2 or its humanized version, rat.3107, rb.2338, rb.2430, or rb.2621) and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to Notch2. The second antibody may be present in a hybridoma supernatant. As a control, immobilized Notch2 is incubated in a solution containing the first labeled antibody but not in a solution containing the second unlabeled antibody. After incubation under conditions that permit binding of the first antibody to Notch2, excess unbound antibody is removed and the amount of label associated with the immobilized Notch2 is measured. If the amount of label associated with the immobilized Notch2 is substantially decreased in the test sample compared to the control sample, this indicates that the second antibody is competing with the first antibody for binding to Notch2. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0251] In an exemplary epitope binding assay, surface plasmon resonance is used to determine competition between antibodies. For example, a first antibody (e.g., rat.1B2 or its humanized version, rat.3107, rb.2338, rb.2430, or rb.2621) is immobilized onto an SPR sensor prism CMD 200M chip using amino coupling. An analyte is injected for 4 minutes, e.g., at 50 nM, and then the second antibody is injected for 4 minutes, e.g., at 10 μg / ml. The assay can be performed at 25° C in a running buffer of HBS-T buffer (0.01 M HEPES at pH 7.4, 0.15 M NaCl, 0.05% surfactant P20, 5 mM CaCl2). Binding data can be processed using the Wasatch binding software tool Epitope (Carterra USA).

[0252] 2. Activity assay In some embodiments, assays are provided for identifying anti-Notch2 antibodies having specific biological activity. For example, assays are provided for identifying anti-Notch2 antibodies that inhibit Jagged1-mediated signaling but leave DLL1-mediated signaling substantially intact. Assays are also provided for identifying anti-Notch2 antibodies that reduce the number of secretory cells in vitro and / or in vivo.

[0253] Example 5 describes a non-limiting, exemplary assay for identifying anti-Notch2 antibodies that inhibit Jagged1-mediated signaling but leave DLL1-mediated signaling substantially intact. Generally, in some embodiments, the test antibody is added to a culture of human cells expressing human Notch2 (e.g., cell line U87-MG). The culture is then brought into contact with cells expressing Jagged1 or DLL1. Ligand-dependent Notch2 activation results in a Notch2-ICD translocation in Notch2-expressing cells. After incubation, the co-cultured cells are fixed, permeabilized, and then brought into contact with the anti-Notch2 ICD antibody. After removing unbound anti-Notch2 ICD antibody, the bound antibody is detected, for example, using a labeled anti-Ig antibody. If the anti-Notch2 test antibody inhibits Jagged1-mediated signaling but not DLL1-mediated signaling, co-culture with DLL1-expressing cells produces a substantially larger signal than co-culture with Jagged1-expressing cells.

[0254] In some embodiments, anti-Notch2 antibodies are assayed to determine whether they reduce the number of secretory cells. A non-limiting, exemplary assay for selecting an antibody with this activity is described in Example 8. Generally, in some embodiments, a gas-liquid interface (ALI) culture of primary human bronchial epithelial cells (HBECs) is established and cultured for several weeks until they are fully differentiated, for example, as indicated by the visual pulsation of cilia. The test anti-Notch2 antibody is added to the culture medium in the lower chamber of the ALI culture. After about 7 days, the ALI culture is analyzed. RNA is extracted from the culture sample and assayed for the expression of genes indicating secretory cells, e.g., Muc5b, Muc5ac, and Scgb1a1. The culture can also be analyzed histologically by fixing and embedding it in paraffin. Sections are stained with antibodies against markers for secretory cells (e.g., Muc5b) and ciliated cells (e.g., tubulin). By comparing cultures incubated with and without the test anti-Notch2 antibody, we identify anti-Notch2 antibodies that reduce the number of secretory cells, such as goblet cells.

[0255] D. Methods and compositions for diagnosis and detection In certain embodiments, any of the anti-Notch2 antibodies provided herein are useful for detecting the presence of Notch2 in a biological sample. As used herein, the term “detection” encompasses quantitative or qualitative detection. In certain embodiments, the biological sample includes biological fluids, cells, or tissues such as sputum, secretory cells, airway epithelial cells, immune cells, lung cells or tissues, or bronchial cells or tissues.

[0256] In some embodiments, anti-Notch2 antibodies are provided for use in diagnostic or detection methods. In further embodiments, methods for detecting the presence of Notch2 in a biological sample are provided. In certain embodiments, the method includes contacting a biological sample with the anti-Notch2 antibody described herein under conditions that allow binding of the anti-Notch2 antibody to Notch2, and detecting whether a complex is formed between the anti-Notch2 antibody and Notch2. Such methods may be in vitro or in vivo. In some embodiments, for example, if Notch2 is a biomarker for patient selection, anti-Notch2 antibodies are used to select subjects eligible for therapy with the anti-Notch2 antibody.

[0257] In certain embodiments, labeled anti-Notch2 antibodies are provided. Labels include, but are not limited to, directly detectable labels or moieties (e.g., fluorescent, plastid, electron-dense, chemiluminescent, and radioactive labels), and moieties indirectly detectable by, for example, enzymatic reactions or molecular interactions (e.g., enzymes or ligands). Exemplary labels include, but are not limited to, radioactive isotopes. 32 P, 14 C, 125 I, 3 H and 131Examples include fluorophores, such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luceriferases, such as firefly luciferase and bacterial luciferase (U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatases, β-galactosidase, glucoamylase, lysozyme, sugar oxidases, such as glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase, heterocyclic oxidases, such as uricase, and xanthine oxidases that oxidize dye precursors by linking with enzymes using hydrogen peroxide, such as HRP, lactoperoxidase, or microperoxidase, biotin / avidin, spin-labeled, bacteriophage-labeled, and stable free radicals.

[0258] E. Pharmaceutical Compositions In further embodiments, pharmaceutical compositions are provided, for example, for use in any of the following therapeutic methods, comprising one of the antibodies provided herein. In some embodiments, the pharmaceutical composition comprises one of the antibodies provided herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises one of the antibodies provided herein and at least one additional therapeutic agent, for example, one of those described below.

[0259] The anti-Notch2 antibody pharmaceutical compositions described herein are prepared in the form of lyophilized compositions or aqueous solutions by mixing such antibody having a desired degree of purity with one or more pharmaceutically acceptable carriers ("Remington's Pharmaceutical Sciences," 16th edition, Osol, A. ed. (1980)). Pharmaceutically acceptable carriers are generally nontoxic to the recipient at the dosage and concentration used and include buffers such as histidine, phosphates, citrates, acetates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol, etc.); and low molecular weight (less than approximately 10 residues) poly Peptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers as herein further include intervening drug dispersants such as soluble neutrally active hyaluronidase glycoproteins (sHASEGP), e.g., human soluble PH-20 hyaluronidase glycoproteins such as rHuPH20 (HYLENEX®, Halozyme, Inc.). Specific exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Application Publications 2005 / 0260186 and 2006 / 0104968. In some embodiments, sHASEGP is combined with one or more further glycosaminoglycansases (e.g., chondroitinases).

[0260] An example of a lyophilized antibody composition is described in U.S. Patent No. 6,267,958. Examples of aqueous antibody compositions are described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter of which comprises a histidine-acetate buffer.

[0261] The pharmaceutical compositions herein may also include multiple active ingredients required for the specific symptom being treated, preferably those having complementary activities that do not adversely affect each other. For example, it may be desirable to further provide agents that can reduce the viscoelasticity of mucus. In some embodiments, additional therapeutic agents are selected from hypertonic saline, mannitol, pulmozyme, N-acetylcysteine, cysteamine, and bronchodilators. Such active ingredients are preferably present in combination in amounts effective for the intended purpose.

[0262] The active ingredient may be encapsulated in microcapsules prepared, for example, by coacervation technology or interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively), or encapsulated in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions. Such technologies are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980).

[0263] Sustained-release pharmaceutical compositions can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing antibodies, where these matrices are in the form of molded articles, such as films or microcapsules.

[0264] Pharmaceutical compositions used for in vivo administration are generally sterile. Sterilization can be easily achieved, for example, by filtration using a sterile filtration membrane.

[0265] F. Treatment methods and routes of administration Any of the anti-Notch2 antibodies provided herein can be used in therapeutic methods.

[0266] In some embodiments, anti-Notch2 antibodies are provided for pharmaceutical use. In further embodiments, anti-Notch2 antibodies are provided for use in the treatment of mucosal-occlusive pulmonary disease. In particular embodiments, anti-Notch2 antibodies are provided for use in therapeutic methods. In particular embodiments, the present invention provides anti-Notch2 antibodies for use in a method of treating an individual having mucosal-occlusive pulmonary disease, comprising administering an effective amount of anti-Notch2 antibodies to the individual. In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent (e.g., one, two, three, four, five, or six additional therapeutic agents) to the individual, as described below, for example. In further embodiments, the present invention provides anti-Notch2 antibodies for use in reducing the number of secretory cells, such as goblet cells, in an individual, for example, in the lungs of the individual. In certain embodiments, the present invention provides an anti-Notch2 antibody for use in a method for reducing the number of secretory cells, such as goblet cells, in an individual, for example, in the individual's lungs, comprising administering an effective amount of the anti-Notch2 antibody to the individual to reduce the number of secretory cells, such as goblet cells, in the individual, for example, in the individual's lungs. By reducing the number of secretory cells, such as goblet cells, in the lungs, mucus production in the lungs is reduced and / or clearance or mucus is increased, thereby alleviating one or more symptoms of, for example, mucosal-obstructive lung disease. In some embodiments, treatment with the anti-Notch2 antibody provided herein improves FEV1 (forced expiratory volume per second), reduces shortness of breath, and / or reduces cough in subjects having mucosal-obstructive lung disease.

[0267] In further embodiments, the present invention provides the use of an anti-Notch2 antibody in the manufacture or preparation of a pharmaceutical. In some embodiments, the pharmaceutical is for the treatment of mucosal-occlusive lung disease. In further embodiments, the pharmaceutical is for use in a method of treating mucosal-occlusive lung disease, comprising administering an effective amount of the pharmaceutical to an individual having mucosal-occlusive lung disease. In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent, e.g., one described below, to the individual. In further embodiments, the pharmaceutical is for reducing the number of secretory cells, e.g., goblet cells, in an individual, e.g., in the lungs of the individual. In further embodiments, the pharmaceutical is for use in a method of reducing the number of secretory cells, e.g., goblet cells, in an individual, e.g., in the lungs of the individual, e.g., comprising administering an effective amount of the pharmaceutical to the individual for reducing the number of secretory cells, e.g., goblet cells, in an individual, e.g., in the lungs of the individual.

[0268] In further embodiments, the present invention provides a method for treating mucosal-occlusive lung disease. In some embodiments, the method comprises administering an effective amount of anti-Notch2 antibody to an individual having such mucosal-occlusive lung disease. In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent, as described below, to the individual.

[0269] In further embodiments, the present invention provides a method for reducing the number of secretory cells, such as goblet cells, in an individual, for example, in the lungs of that individual. In some embodiments, the method comprises administering an effective amount of anti-Notch2 antibody to an individual in order to reduce the number of secretory cells, such as goblet cells, in an individual, for example, in the lungs of that individual. In some embodiments, “individual” is a human.

[0270] Non-limiting exemplary mucosal obstructive pulmonary diseases that can be treated with the anti-Notch2 antibodies provided herein include chronic obstructive pulmonary disease (COPD), cystic fibrosis, primary ciliary dyskinesia, non-cystic fibrosis, bronchiectasis, and bronchiolitis.

[0271] The "individual" or "subject" in any of the above embodiments may be a human being.

[0272] In further embodiments, the present invention provides a pharmaceutical composition comprising one of the anti-Notch2 antibodies provided herein for use, for example, in any of the therapeutic methods described above. In some embodiments, the pharmaceutical composition comprises one of the anti-Notch2 antibodies provided herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises one of the anti-Notch2 antibodies provided herein and at least one additional therapeutic agent, for example, one of those described below.

[0273] The antibodies of the present invention may be administered alone or used in combination therapy. For example, such combination therapy may include administering the antibodies of the present invention and administering at least one additional therapeutic agent (e.g., additional therapeutic agents 1, 2, 3, 4, 5, or 6). In certain embodiments, the combination therapy may include administering the antibodies of the present invention and administering at least one additional therapeutic agent, such as a mucosal viscoelasticity-reducing agent. In some embodiments, the additional therapeutic agent may be selected from hypertonic saline, mannitol, pulmozyme, N-acetylcysteine, cysteamine, and bronchodilators.

[0274] Such combination therapies described above encompass combined administration (where two or more therapeutic agents are contained in the same or separate pharmaceutical composition) and separate administration, in which case the administration of the antibody of the present invention may be performed prior to, simultaneously with, and / or subsequently to the administration of the additional therapeutic agent or drug. In some embodiments, the administration of the anti-Notch2 antibody and the administration of the additional therapeutic agent occur within about one month of each other, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days. In some embodiments, the antibody and the additional therapeutic agent are administered to the patient on day one of treatment.

[0275] The antibodies (and any additional therapeutic agents) of the present invention may be administered by any suitable means, including parenteral, intrapulmonary, intranasal, and, if desired for topical treatment, intralesional administration. Parenteral administration includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Dosage may be by any preferred route, e.g., injection such as intravenous or subcutaneous injection, or intrapulmonary (e.g., inhalation) or intranasal delivery, depending in part whether the administration is short-term or long-term. Various dosing schedules, including but not limited to single doses, multiple doses over various time points, bolus administration, and pulse infusion, are contemplated herein.

[0276] The antibodies of the present invention will be formulated, administered, and given in a manner consistent with good medical practice. Factors to be considered in this regard include the specific disorder being treated, the specific mammal being treated, the clinical symptoms of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the administration schedule, and other factors known to the healthcare professional. The antibodies will be formulated, optionally but not necessarily, with one or more drugs currently used to prevent or treat the disorder in question. The effective amount of such other drugs will depend on the amount of antibody present in the pharmaceutical composition, the type of disease or treatment, and the other factors mentioned above. These will generally be used by the same dosages and routes of administration as described herein, or at about 1-99% of the dosages described herein, or by any dosage and route that is empirically / clinically deemed appropriate.

[0277] For the prevention or treatment of disease, the appropriate dosage of the antibody of the present invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease being treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapies, the patient's medical history and response to the antibody, and the discretion of the attending physician. The antibody is appropriately administered to the patient in a single dose or over a series of treatments. Such doses may be administered intermittently, for example, weekly or every three weeks (for example, so that the patient receives about 2 to about 20 doses, or for example, about 6 doses of the antibody). A larger initial dose may be administered, followed by one or more smaller doses. However, other drug regimens may be useful. The progress of this treatment is readily monitored by conventional techniques and assays.

[0278] G. Manufactured articles In some aspects of the present invention, a manufactured article is provided containing a substance useful for the treatment, prevention and / or diagnosis of the above-mentioned disorders. The manufactured article comprises a container and a label or package insert inserted into or accompanying the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container may be formed from a variety of materials such as glass or plastic. The container holds the composition to be used alone or in combination with another composition effective for treating, preventing and / or diagnosing the symptoms and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a subcutaneous needle). At least one activator in the composition is the antibody of the present invention. The label or insert indicates that the composition is used to treat a selected symptom. Furthermore, the manufactured article comprises (a) a first container containing the composition, the composition containing the antibody of the present invention, and (b) a second container containing the composition, the composition further containing a cytopathogenic or other therapeutic agent. The manufactured article in this embodiment of the present invention may further include a package insert indicating that the composition may be used to treat a particular condition. Alternatively, or in addition thereto, the manufactured article may further comprise a second (or third) container containing pharmaceutically acceptable buffers, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further comprise other materials desirable from a commercial and user standpoint, such as other buffers, diluents, filters, needles, syringes, etc. [Examples]

[0279] Examples The following are examples of the methods and compositions of the present invention. Considering the general description provided above, it will be understood that various other embodiments may be implemented.

[0280] Example 1: Preparation of rabbit and rat anti-Notch2 antibodies New Zealand white rabbits were co-immunized with human and mouse extracellular domain (ECD) constructs containing Notch2 EGF repeats 6-10 (huNotch2-EGF6-10 and muNotch2-EGF6-10), and single B cells were isolated using a modified protocol based on Offner et al. PLoS ONE 9(2), 2014. The modified workflow included direct FACS sorting of IgG+huNotch2+ B cells into single wells. The B cell culture supernatant was assayed by ELISA for binding to human Notch2 and unrelated control proteins. Notch2-specific B cells were lysed, immediately frozen at -80°C, and stored until molecular cloning. The variable regions (VH and VL) of each monoclonal antibody derived from rabbit B cells were cloned from mRNA extracted as previously described (Offner et al. PLoS ONE 9(2), 2014) into expression vectors containing a human constant region with the N297G mutation. Individual recombinant chimeric rabbit / human antibodies were expressed in Expi293 cells and then purified with protein A. The purified anti-Notch2 antibodies were subjected to functional activity assays and kinetic screening as described herein.

[0281] Rats were immunized with the MBP-huNotch2 EGF6-10 + MBP-huNotch2 EGF7-9 combination, or primed with MBP-huNotch2 EGF6-10 and boosted with huNotch2-EGF6-10 to generate hybridomas using modified fusion partners (Price et al. J Immunol Methods 2009). Various conditions were optimized to allow sorting of individual IgG+huNotch2+ hybridomas into single wells, followed by further culture. The resulting hybridoma supernatants were assayed by ELISA, positive samples were purified with protein A, and then functionally and kinetically characterized. Specific rat monoclonal antibodies were sequenced and cloned into the constant region containing the N297G mutation. Individual recombinant chimeric rat / human antibodies were expressed in Expi293 cells and subsequently purified with protein A. The purified anti-Notch2 antibody was then subjected to functional activity assays and kinetic screenings as described herein.

[0282] Example 2: Kinetic analysis and epitope binning using array-based surface plasmon resonance Using an array-based SPR imaging system (Carterra USA), a panel of five monoclonal antibodies prepared in Example 1 (rat.1B2, rat.3107, rb.2338, rb.2430, and rb.2621) and the anti-Notch 2 / 3 antibody OMP-59R5 (tarextumab, see U.S. Patent No. 8,226,943) were epitope-binned. The purified antibodies were diluted to 10 μg / ml in 10 mM sodium acetate buffer (pH 4.5). Using amine coupling, the antibodies were directly immobilized on an SPR sensor prism CMD200M tip (XanTec Bioanalytics GmbH, Germany) using a Continuous Flow Microspotter (Carterra, USA) to create an array of six antibodies. For analysis, IBIS MX96 SPRi (Carterra, USA) was used to evaluate binding to the immobilized ligands. For kinetic analysis, human Notch2 was injected at a 3-fold dilution of 0–300 nM for 3 minutes, followed by a 10-minute dissociation period. For epitope binning, human Notch2 was first injected at 50 nM for 4 minutes, followed by a second injection of individual monoclonal antibodies at 10 μg / ml for 4 minutes. The surface was regenerated with 10 mM glycine pH 1.5 between cycles. The experiment was performed at 25°C in a running buffer of HBS-T buffer (HEPES at 0.01 M pH 7.4, 0.15 M NaCl, 0.05% surfactant P2O, 5 mM CaCl2). Binning data were processed using the Wasatch binning software tool Epitope (Carterra USA).

[0283] The results are shown in Figure 4. Antibodies rat.1B2, rat.3107, rb.2338, rb.2430, and rb.2621 were determined to be located in different epitope bins than the anti-Notch 2 / 3 antibody OMP-59R5.

[0284] Example 3: Humanization of rat anti-Notch2 antibody Rat monoclonal antibodies 1B2 and 3107 were humanized as described below. Residue numbers are based on Kabat et al., Sequences of proteins of immunological interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

[0285] Variants constructed during the humanization of 1B2 and 3107 were evaluated in the form of human IgG. The hypervariable regions from each antibody (positions 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the VL domain, and positions 26-35 (H1), 50-65 (H2), and 95-102 (H3) in the VH domain) were transplanted into various receptor frameworks. For rat 1B2, the VL CDR was transplanted into KV1-12*01 and the VH CDR was transplanted into HV3-73*01. Furthermore, the glycosylation site of CDR-H2 Asn54-Phe55-Ser56 was mutated to Asp54-Phe55-Ser56. For rat 3107, the VL CDR was transplanted into KV2-30*02 and the VH CDR into HV1-2*01. All VL and VH Vernier positions from the parental antibody were also transplanted into their respective human germline frameworks. Grafts in which all rat amino acids are in the Vernier position are called L1H1 (hu.1B2.L1H1 and hu.3107.L1H1).

[0286] The binding affinity of the hu.1B2.L1H1 antibody was compared to that of its chimeric parent clone. The rat Vernier positions of the version L1H1 antibody were returned to human residues, and the contribution of each rat Vernier position to the binding affinity to huNOTCH2 was evaluated. Four further light chain Vernier variants L2-L5 and eight further heavy chain Vernier variants H2-H9 were generated. Ser43 and Tyr71 on the light chain (L7) and Val24, Ala49, Ser76, and Leu78 on the heavy chain (H14) were determined to be key rat Vernier residues based on the binding affinity evaluation of the above variant antibodies (data not shown). The binding affinity was determined as discussed in Example 6 below. Chimera 1B2 is 5.21E-9 M K D The combination is hu1B2.L7H14 is 6.13E-9 M K D They were joined together.

[0287] The binding affinity of the hu.3107.L1H1 antibody was compared to that of its chimeric parent clone. The rat Vernier positions of the version L1H1 antibody were replaced with human residues, and the contribution of each rat Vernier position to the binding affinity to huNOTCH2 was evaluated. One further light chain variant (L2) and ten further heavy chain variants H2-H11 were generated.

[0288] To increase the affinity of the anti-Notch2 humanized antibody based on 3107, four heavy chain sequence variants were constructed based on binding affinity evaluation and HCS efficacy of the humanized antibody (data not shown): H12 in HV1-2*01 having P45, T48, A67, V71, S75, and T76; H13 in HV1-2*01 having P45, T48, A67, V71, and T76; H14 and H15 are found in HV5-51*01, which has the same Vernier residues as H12 and H13, respectively. For the light chain, germline KV4-1*1 was used as a CDR graft (L7). Furthermore, V2 and F36 on the light chain were determined to be rat Vernier residues that maintain efficacy in the HCS assay and were transplanted into Germline KV4-1*01 (L6). The HCS assay was performed substantially as described in Example 5.

[0289] Example 4: Affinity improvement of humanized 1B2 antibody To enhance the potency of 1B2-based anti-Notch2 humanized antibodies, 560 single-point mutation variants were generated using L7H10 as a template. The resulting antibodies were screened by surface plasmon resonance and ranked according to their dissociation rate. Five mutations in the heavy chain (A50G, S51Q, I57R, S96H, and R98F) and three mutations in the light chain (S31V, Q55H, and L96I) resulted in slower dissociation rates. To identify good combinations of mutations, 80 variants were generated using individual sets and combination sets of mutations and evaluated by surface plasmon resonance characterization. S51Q was identified as a mutation that improved dissociation rate.

[0290] To further improve the affinity of 1B2, L1H1 with S51Q and N54D mutations was used as a template for phage display affinity maturation. Briefly, a total of four phage libraries were constructed and presented as monovalent Fab on the surface of M13 bacteriophage. The first set of libraries consisted of two CDR NNK walks (one for CDR-H1, H2, and H3, and one for CDR-L1, L2, and L3), with one position in each of the three CDRs simultaneously randomized. The second set consisted of two hard-randomized libraries with either CDR-L3 or CDR-H3 entirely mutated.

[0291] For affinity-enhancing selection, phage libraries were subjected to four rounds of solution sorting with increased stringency and using cold human Notch2 EGF6-10 as a competitor. Enrichment was observed in the CDR-H3 hard randomized libraries. After comparing the parental sequences with the enriched clones, several CDR-H3 mutations were identified. A total of 54 combination variants were reformatted to human IgG1 for antibody production and further BIAcore binding kinetic analysis and HCS assay. The HCS assay was performed substantially as described in Example 5. hu1B2.v2, hu1B2.v4, hu1B2.v9, and hu1B2.v8 were identified as showing the greatest improvement in both affinity and potency in the HCS assay. The CDR-H3 of these four variants was transplanted into the Vernier polished humanized variant L7H14 to produce hu1B2.v101, hu1B2.v102, hu1B2.v103, and hu1B2.v104, respectively. The binding affinity was determined as discussed in Example 6 below. hu1B2.L7H14 had an affinity of 6.13E-9M for hu.Notch2, while hu1B2.v101, hu1B2.v102, hu1B2.v103, and hu1B2.v014 had affinities of 2.84E-09, 3.37E-09, 3.08E-09, and 3.09E-09, respectively. None of the variants showed binding to human Notch1, human Notch3, or human Notch4 by surface plasmon resonance. Nonspecific binding of each anti-Notch2 variant was measured by ELISA using baculovirus particles (Hotzel et al. MAbs 2012). Hu1B2.v102 and hu1B2.v104 were assayed for molecular liabilities using thermal stress and AAPH oxidative stress tests (see Dion et al. J.Pharm.Sci 2018, 107(2), 550). No liabilities were identified.

[0292] Example 5: High-content screening (HCS) assay to identify antibodies that block Jagged1 signaling but not DLL1 signaling. Human cell line U87-MG, which endogenously expresses high levels of huNotch2(N2), was harvested and seeded at 4,000 cells / well in Cell Carrier ultra 384-well plates (Perkin Elmer, Waltham, MA). The plates were incubated in a 37°C CO2 incubator for 2-5 hours. During this incubation period, antibody (Ab) test samples were manually prepared using initial dilutions, and then 10 points of 3-fold or 3.5-fold serial dilutions were performed using a Bravo automated liquid handler (Agilent, Santa Clara, CA). The diluted Ab samples were transferred to a double set of plates containing U-87-MG cells. After adding diluted Ab, 3T3-Jag1 or OP9-DLL1 cells were harvested, and each ligand cell line was seeded at 4,000 cells per well on top of Ab-treated U-87-MG cells. The cells were incubated to induce ligand-dependent Notch-2 activation and N2-ICD translocation in the U-87-MG cells.

[0293] After incubation for 16–22 hours, each co-culture of receptor and ligand-expressing cells was fixed with 4% paraformaldehyde for 10 minutes, the plates were washed with PBS, and then the cells were permeabilized with 0.05% saponin (Sigma-Aldrich, San Louis, MO) in PBS + 0.05% BSA buffer for 1 hour. After permeabilization, the plates were washed, rabbit anti-N2-ICD mAb D76A6 (Cell Signaling Technology, Danvers, MA) was diluted in 0.05% saponin-containing PBS / BSA buffer, added to the plates, and incubated overnight at 4°C.

[0294] The following day, the plates were washed and stained with a buffer containing detection AF-647 conjugate anti-rabbit detection Ab (Jackson-Immunoresearch, West Grove, PA) and Hoechst-33342 dye (Thermo Fisher Scientific, Waltham, MA), and then incubated at room temperature for 2 hours with gentle shaking. After staining the cells, the plates were washed with washing buffer, then PBS was added to each well, and then the plates were imaged.

[0295] Six images were acquired from each well using an OperaPhenix High Content imaging system (Perkin Elmer, Waltham, MA) with a 20x immersion objective lens. Analysis was performed using the Columbus software image analysis tool (Perkin Elmer, Waltham, MA) to identify the nuclear region and the surrounding ring region, and to calculate the signal intensity. A threshold was obtained to calculate the N2-ICD nuclear translocation positive population from the maximum inhibitory control sample. The results from the Columbus software analysis were uploaded to the Geneda Screener application (Lexington, MA), where a normalization process was set up using the translocation percentage obtained from the neutral control, subtracted by the maximum inhibitory control, and the IC50 value was calculated.

[0296] The results of data analysis from 3T3-Jag1 and OP9-DLL1 co-culture sets were compared and used to identify Notch2 antibodies that block Jagged1-mediated activation but not DLL1-mediated activation, and to optimize humanized versions of these antibodies. Exemplary results are shown in Figures 5A to 5F. All tested antibodies blocked Jagged1-mediated activation but not DLL1-mediated activation. Table 2 summarizes the IC50 values ​​of each antibody for blocking Jagged1-mediated signaling. [Table 2]

[0297] In another experiment, rat antibody 3107 and its humanized version 3107 were tested using the HCS assay, essentially as described above. All antibodies tested in this experiment contained human IgG1 with the N297G mutation. Both 3107 and the humanized variant blocked Jagged1-mediated activation but not DLL1-mediated activation (data not shown). Table 3 summarizes the IC50 values ​​for each antibody regarding the blockade of Jagged1-mediated signaling. [Table 3]

[0298] Example 6: Kinetic analysis using BIAcore (trademark) Antibody binding affinity was determined using a BIAcore® T200 machine. Rabbit antibodies were expressed as chimeric antibodies possessing a rabbit variable domain and a human constant domain. Rat antibodies were expressed as chimeric antibodies possessing a rat variable domain and a human constant domain. Humanized antibodies were expressed with a human IgG1 backbone. For kinetic measurements, antibodies were captured using research-grade Protein A chips (GE Healthcare) to achieve approximately 300 RU. Tenfold serial dilutions of huNotch2-EGF6-10 were injected at a flow rate of 100 μL / min at 37°C into HBS-P buffer containing an additional 3 mM CaCl2. Binding rate (ka) and dissociation rate (kd) were calculated using a 1:1 Langmuir binding model (BIAcore® T200 evaluation software version 2.0). The equilibrium dissociation constant (KD) was calculated as the ratio kd / ka. The results are shown in Table 4. [Table 4]

[0299] In another experiment, the binding affinity of the humanized version of the rat 3107 antibody was determined, essentially as described above. The results are shown in Table 5. [Table 5]

[0300] The binding of anti-Notch2 antibodies to constructs containing Notch2 from further species and different EGF repeat regions was evaluated using BIAcore®. For this experiment, antibodies with human constant regions were captured on a Protein A chip to achieve approximately 200 RU. Tenfold serial dilutions of various antigens were injected at a flow rate of 100 μL / min into HBS-P buffer containing an additional 3 mM CaCl2 at 37°C. The experimental results are summarized in Table 6. [Table 6]

[0301] Other humanized versions of 1B2 (hu1B2.L1H1.DFS, hu1B2.v4L7, hu1B2.v8L7, hu1B2.v9L7, hu.1B2.DFS.H14L7) showed similar binding profiles to hu.1B2.v102 and hu.1B2.v104 in Table 4 above. Based on the binding characteristics of the anti-Notch2 antibodies shown in Table 4, rat.3107, the humanized version of rat.1B2, rb.2338, rb.2430, and rb.2621 bind to the epitope within human Notch2 EGF7. Furthermore, while all the tested antibodies showed little to no binding to huNotch2-EGF6-12.R268K or muNotch2-EGF6-10, they did bind to huNotch2-EGF6-10 and muNotch2-EGF6-12.K268R, suggesting that the antibodies contact arginine at position 268 of human Notch2.

[0302] Example 7: Inhibition of Jagged1 and DLL1 signaling by anti-Notch2 Fab A specific anti-Notch2 antibody was reformatted as a monovalent Fab, and assays were performed for Jagged1 and DLL1 signaling inhibition using the HCS assay described in Example 5.

[0303] Using data analysis results from 3T3-Jag1 and OP9-DLL1 co-culture sets, Jagged1 IC50 was calculated to determine the maximum percentage of Jagged1 and DLL1 signaling inhibition by each Fab. Table 7 shows the maximum Jagged1 and DLL1 signaling inhibition observed for each Fab. [Table 7]

[0304] Surprisingly, both hu1B2.v8 and hu1B2.v104 were selective for inhibiting Jagged1 signaling in bivalent antibody format, but when reformatted as monovalent Fab, both hu1B2.v8 and hu1B2.v104 inhibited DLL1 signaling, but with reduced maximum inhibition compared to Jagged1 signaling inhibition. In contrast, monovalent Fab formats hu1B2.v1.DFS, hu1B2.v101, and hu1B2.v103 retained Jagged1-specific signaling inhibitory activity and did not inhibit DLL1 (data not shown). While not intended to be bound by any particular theory, the difference in selectivity between Fab formats hu1B2.v8 and hu1B2.v104 and Fab formats hu1B2.v1.DFS, hu1B2.v101, and hu1B2.v103 may be due to differences in the CDR-H3 sequence. Hu1B2.v8 and hu1B2.v104 use the CDR-H3 sequence DGGK. LA They share LDA (sequence number 11), while hu1B2.v1.DFS, hu1B2.v101, and hu1B2.v103 share the CDR-H3 sequence DSGR. WG LDA (Sequence ID 8), DGGR WG LDA (SEQ ID NO: 9) and DGGK WG Each has LDA (sequence number 12).

[0305] Example 8: Reduction of secretory cells by anti-Notch2 antibody Gas-liquid interface (ALI) culture: Primary human bronchial epithelial cells (HBECs) are seeded in a 0.4 μm pore PET transwell (Corning #7369) and cultured in Pneumacult Ex-Plus medium (StemCell Technologies #05040) under immersion conditions until confluence. Once confluence is reached, the medium is removed from the upper chamber to expose the HBECs to air, and the medium in the lower chamber is replaced with Pneumacult ALI basal medium (StemCell Technologies #05001). The cells are cultured for 3-4 weeks and fully differentiated when the cilia are clearly pulsating.

[0306] Antibody treatment and sample analysis: Antibodies were added to the basal medium at a concentration of 50 mg / ml. Antibodies were replenished by changing the medium in the lower chamber (three times a week). On day 7, ALI cultures were collected for RNA analysis and histology. RNA was extracted using the Qiagen RNA extraction kit (#74106) for RNA analysis. After cDNA synthesis using iScript cDNA synthesis (Biorad #1708891), gene expression analysis was performed for the genes Muc5b, Muc5ac, and Scgb1a1 (Taqman Assays). For histological analysis, Transwell cells were fixed with formalin and embedded in paraffin. Samples were sectioned and stained with anti-Muc5b (goblet cells), anti-acetylated α-tubulin (ciliary cells), and DAPI (nuclear staining).

[0307] As shown in Figures 6A–6D, treatment with anti-Notch2 antibody 1B2 reduced the mRNA expression of Muc5b, Muc5ac, and Scgb1a1 in HBEC ALI cultures. Treatment with anti-Notch2 antibody 1B2 also reduced the appearance of goblet cells, as detected by immunofluorescence using anti-Muc5b antibody. These results indicate that inhibition of Jagged-Notch2 signaling is sufficient to significantly reduce secretory goblet cells in cultures.

[0308] Although the above invention has been described in some detail by description and examples for the purpose of clarifying understanding, the description and examples should not be construed as limiting the scope of the invention. All patent and scientific literature disclosures cited herein are expressly incorporated in their entirety by reference. IV. Table of specific sequences JPEG2026102663000009.jpg236170JPEG2026102663000010.jpg245170JPEG2026102663000011.j pg248170JPEG2026102663000012.jpg246170JPEG2026102663000013.jpg247170JPEG20261026630 00014.jpg247170JPEG2026102663000015.jpg249170JPEG2026102663000016.jpg249170JPEG202 6102663000017.jpg245170JPEG2026102663000018.jpg247170JPEG2026102663000019.jpg147170

Claims

1. An isolated antibody that binds to human Notch2 and inhibits Jagged1-mediated signaling but does not inhibit DLL1-mediated signaling.

2. An isolated antibody that binds to human Notch2 and inhibits Jagged1-mediated signaling to a greater extent than DLL1-mediated signaling.

3. The isolated antibody according to claim 2, which can achieve 100% maximum inhibition of Jagged1-mediated signaling and less than 80%, less than 70%, or less than 60% maximum inhibition of DLL1-mediated signaling.

4. The isolated antibody according to claim 2 or 3, wherein the antibody is a Fab fragment.

5. The isolated antibody according to claim 4, which, when formatted as a bivalent IgG antibody containing two heavy chains and two light chains, inhibits Jagged1-mediated signaling but does not inhibit DLL1-mediated signaling.

6. An isolated antibody according to any one of claims 1 to 5, which does not inhibit the binding of Jaggered1 to Notch2.

7. An isolated antibody according to any one of claims 1 to 6, which does not inhibit the binding of DLL1 to Notch2.

8. An isolated antibody according to any one of claims 1 to 7, which binds to an epitope in the EGF7 repeat of Notch2.

9. An isolated antibody according to any one of claims 1 to 8, which binds to an epitope in amino acids 260 to 296 of Notch2.

10. An isolated antibody according to any one of claims 1 to 8, which binds to a discontinuous epitope in amino acids 260 to 296 of Notch2.

11. An isolated antibody that binds to Notch2, specifically an antibody that binds to an epitope within the EGF7 repeat of Notch2.

12. An isolated antibody that binds to Notch2, specifically an antibody that binds to an epitope within amino acids 260-296 of Notch2.

13. An isolated antibody that binds to Notch2, specifically to a discontinuous epitope within amino acids 260-296 of Notch2.

14. An isolated antibody according to any one of claims 1 to 13, which contacts human Notch2 arginine 268 (R268).

15. An isolated antibody according to claim 14, which does not bind to Notch2 and contains lysine 268 (K268).

16. An isolated antibody according to any one of claims 1 to 15, which binds to a polypeptide containing the amino acid sequence of SEQ ID NO: 74 and does not bind to a polypeptide containing the amino acid sequence of SEQ ID NO:

77.

17. An isolated antibody according to any one of claims 1 to 16, which binds to human Notch2 and cynomolgus monkey Notch2.

18. An isolated antibody according to any one of claims 1 to 17, which does not bind to mouse Notch2.

19. An isolated antibody according to any one of claims 1 to 18, which binds to guinea pig Notch2.

20. An isolated antibody according to any one of claims 1 to 19, which does not bind to human Notch1 or human Notch3.

21. When determined by surface plasmon resonance, affinity (K) is less than 20 nM, less than 15 nM, less than 10 nM, or less than 5 nM. Ｄ An isolated antibody according to any one of claims 1 to 20, which binds to human Notch2 via )

22. An isolated antibody according to any one of claims 1 to 21, which inhibits Jagged1-mediated signaling with an IC50 of less than 20 nM, less than 15 nM, less than 10 nM, or less than 5 nM.

23. The isolated antibody according to claim 22, wherein inhibition of Jagged1-mediated signaling is determined using a high-content screening (HCS) assay.

24. a) A heavy chain variable domain (VH) comprising (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 4, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 6 or 7, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 8, 9, 10, 11, or 12, and a light chain variable domain (VL) comprising (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 1, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 2, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 3; b) A heavy chain variable domain (VH) comprising (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 36, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 37, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 38, and a light chain variable domain (VL) comprising (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 33, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 34, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 35; c) A heavy chain variable domain (VH) comprising (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 44, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 45, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 46, and a light chain variable domain (VL) comprising (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 41, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 42, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 43; d) A heavy chain variable domain (VH) comprising (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 53, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 54, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 55, and (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 49, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 50, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 51 or 52; or e) A heavy chain variable domain (VH) comprising (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 62, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 63, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 64, and a light chain variable domain (VL) comprising (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 59, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 60, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO:

61. An isolated antibody according to any one of claims 1 to 23, comprising:

25. An isolated antibody that binds to human Notch2, a) A heavy chain variable domain (VH) comprising (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 4, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 6 or 7, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 8, 9, 10, 11, or 12, and a light chain variable domain (VL) comprising (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 1, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 2, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 3; b) A heavy chain variable domain (VH) comprising (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 36, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 37, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 38, and a light chain variable domain (VL) comprising (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 33, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 34, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 35; c) A heavy chain variable domain (VH) comprising (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 44, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 45, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 46, and a light chain variable domain (VL) comprising (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 41, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 42, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 43; d) A heavy chain variable domain (VH) comprising (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 53, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 54, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 55, and (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 49, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 50, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 51 or 52; or e) A heavy chain variable domain (VH) comprising (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 62, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 63, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 64, and a light chain variable domain (VL) comprising (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 59, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 60, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO:

61. Antibodies containing antibodies.

26. a) A VH sequence having at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 14; b) A VL sequence having at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 13; c) The VH sequence described in (a) and the VL sequence described in (b); d) A VH sequence having at least 95% sequence identity with an amino acid sequence selected from SEQ ID NOs: 17-24, 26, 28, 30, and 32; e) A VL sequence having at least 95% sequence identity with an amino acid sequence selected from SEQ ID NOs: 15, 16, 25, 27, 29, and 31; f) The VH sequence described in (d) and the VL sequence described in (e); g) A VH sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 40; h) A VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 39; i) The VH sequence described in (g) and the VL sequence described in (h); j) A VH sequence having at least 95% sequence identity with an amino acid sequence selected from SEQ ID NOs: 102-106; k) A VL sequence having at least 95% sequence identity with an amino acid sequence selected from SEQ ID NOs: 98-100; l) The VH sequence described in (j) and the VL sequence described in (k); m) A VH sequence having at least 95% sequence identity with the amino acid sequence of Sequence ID No. 48; n) A VL sequence having at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 47; o) The VH sequence described in (m) and the VL sequence described in (n); p) A VH sequence having at least 95% sequence identity with the amino acid sequence of Sequence ID No. 58; q) A VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 56 or 57; r) The VH sequence described in (p) and the VL sequence described in (q); s) A VH sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 66; t) A VL sequence having at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 65; or The VH sequence described in (u)(s) and the VL sequence described in (t) An isolated antibody according to any one of claims 1 to 25, comprising:

27. a) VH sequence containing the amino acid sequence of SEQ ID NO: 14; b) A VL sequence containing the amino acid sequence of SEQ ID NO: 13; c) The VH sequence described in (a) and the VL sequence described in (b); d) A VH sequence containing an amino acid sequence selected from SEQ ID NOs: 17-24, 26, 28, 30, and 32; e) A VL sequence containing an amino acid sequence selected from SEQ ID NOs: 15, 16, 25, 27, 29, and 31; f) The VH sequence described in (d) and the VL sequence described in (e); g) VH sequence containing the amino acid sequence of SEQ ID NO: 40; h) VL sequence containing the amino acid sequence of SEQ ID NO: 39; i) The VH sequence described in (g) and the VL sequence described in (h); j) A VH sequence containing an amino acid sequence selected from sequence numbers 101 to 106; k) A VL sequence containing an amino acid sequence selected from SEQ ID NOs: 98-100; l) The VH sequence described in (j) and the VL sequence described in (k); m) VH sequence containing the amino acid sequence of SEQ ID NO: 48; n) VL sequence containing the amino acid sequence of SEQ ID NO: 47; o) The VH sequence described in (m) and the VL sequence described in (n); p) VH sequence containing the amino acid sequence of Sequence ID No. 58; q) A VL sequence containing the amino acid sequence of SEQ ID NO: 56 or 57; r) The VH sequence described in (p) and the VL sequence described in (q); s) VH sequence containing the amino acid sequence of SEQ ID NO: 66; t) A VL sequence containing the amino acid sequence of SEQ ID NO: 65; or The VH sequence described in (u)(s) and the VL sequence described in (t) An isolated antibody according to any one of claims 1 to 26, comprising:

28. An isolated antibody that binds to human Notch2, a) VH sequence containing the amino acid sequence of SEQ ID NO: 14; b) A VL sequence containing the amino acid sequence of SEQ ID NO: 13; c) The VH sequence described in (a) and the VL sequence described in (b); d) A VH sequence containing an amino acid sequence selected from SEQ ID NOs: 17-24, 26, 28, 30, and 32; e) A VL sequence containing an amino acid sequence selected from SEQ ID NOs: 15, 16, 25, 27, 29, and 31; f) The VH sequence described in (d) and the VL sequence described in (e); g) VH sequence containing the amino acid sequence of SEQ ID NO: 40; h) VL sequence containing the amino acid sequence of SEQ ID NO: 39; i) The VH sequence described in (g) and the VL sequence described in (h); j) A VH sequence containing an amino acid sequence selected from sequence numbers 101 to 106; k) A VL sequence containing an amino acid sequence selected from SEQ ID NOs: 98-100; l) The VH sequence described in (j) and the VL sequence described in (k); m) VH sequence containing the amino acid sequence of SEQ ID NO: 48; n) VL sequence containing the amino acid sequence of SEQ ID NO: 47; o) The VH sequence described in (m) and the VL sequence described in (n); p) VH sequence containing the amino acid sequence of Sequence ID No. 58; q) A VL sequence containing the amino acid sequence of SEQ ID NO: 56 or 57; r) The VH sequence described in (p) and the VL sequence described in (q); s) VH sequence containing the amino acid sequence of SEQ ID NO: 66; t) A VL sequence containing the amino acid sequence of SEQ ID NO: 65; or The VH sequence described in (u)(s) and the VL sequence described in (t) Antibodies containing antibodies.

29. a) A VH sequence having at least 95% sequence identity with an amino acid sequence selected from SEQ ID NOs: 17-24, 26, 28, 30, and 32; b) A VL sequence having at least 95% sequence identity with an amino acid sequence selected from SEQ ID NOs: 15, 16, 25, 27, 29, and 31; c) The VH sequence described in (a) and the VL sequence described in (b); d) A VH sequence having at least 95% sequence identity with an amino acid sequence selected from SEQ ID NOs: 102-106; e) A VL sequence having at least 95% sequence identity with an amino acid sequence selected from SEQ ID NOs: 98-100; or f) The VH sequence described in (d) and the VL sequence described in (e) An isolated antibody according to any one of claims 1 to 25, comprising:

30. a) A VH sequence comprising an amino acid sequence selected from SEQ ID NOs: 17-24, 26, 28, 30, and 32; b) A VL sequence containing an amino acid sequence selected from SEQ ID NOs: 15, 16, 25, 27, 29, and 31; c) The VH sequence described in (a) and the VL sequence described in (b); d) A VH sequence containing an amino acid sequence selected from SEQ ID NOs: 101-106; e) A VL sequence containing an amino acid sequence selected from SEQ ID NOs: 98-100; or f) The VH sequence described in (d) and the VL sequence described in (e) An antibody according to any one of claims 1 to 25 and 29, comprising:

31. a) Includes the VH sequence of sequence number 26 and the VL sequence of sequence number 25; b) Including the VH sequence of sequence number 28 and the VL sequence of sequence number 27; c) comprising the VH sequence of sequence number 30 and the VL sequence of sequence number 29; or d) Including the VH sequence of sequence number 32 and the VL sequence of sequence number 31, An isolated antibody according to any one of claims 1 to 25.

32. An isolated antibody that binds to human Notch2, a) Includes the VH sequence of sequence number 26 and the VL sequence of sequence number 25; b) Including the VH sequence of sequence number 28 and the VL sequence of sequence number 27; c) comprising the VH sequence of sequence number 30 and the VL sequence of sequence number 29; or d) An antibody containing the VH sequence of SEQ ID NO: 32 and the VL sequence of SEQ ID NO:

31.

33. An isolated antibody according to any one of claims 1 to 32, which is a monoclonal antibody.

34. An isolated antibody according to any one of claims 1 to 33, which is a human antibody, a humanized antibody, or a chimeric antibody.

35. An isolated antibody according to any one of claims 1 to 34, which is an antibody fragment that binds to Notch2.

36. The antibody fragments are Fv, Fab, Fab', Fab'-SH and F(ab') ２ An isolated antibody according to claim 35, selected from the above.

37. The isolated antibody according to claim 36, wherein the antibody fragment is Fab, Fab', or Fab'-SH.

38. An isolated antibody according to any one of claims 1 to 3 and 6 to 37, which is a full-length antibody.

39. The isolated antibody according to claim 38, which is a full-length IgG antibody.

40. The isolated antibody according to claim 39, which is an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody.

41. An isolated antibody that competes with the antibody described in any one of claims 1 to 40 for binding to human Notch2.

42. An isolated nucleic acid encoding an antibody according to any one of claims 1 to 41.

43. A host cell comprising the nucleic acid described in claim 42.

44. A host cell expressing the antibody described in any one of claims 1 to 41.

45. A method for producing an antibody that binds to human Notch2, comprising culturing the host cell described in claim 43 or claim 44 under conditions suitable for the expression of the antibody.

46. The method according to claim 45, further comprising recovering the antibody from the host cell.

47. An antibody produced by the method described in claim 45 or claim 46.

48. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 41 and a pharmaceutically acceptable carrier.

49. The pharmaceutical composition according to claim 48, further comprising an additional therapeutic agent.

50. The pharmaceutical composition according to claim 49, wherein the additional therapeutic agent is selected from hypertonic saline, mannitol, pulmozyme, N-acetylcysteine, cysteamine, and a bronchodilator.

51. An antibody according to any one of claims 1 to 41 or a pharmaceutical composition according to any one of claims 48 to 50, for use as a pharmaceutical.

52. An antibody according to any one of claims 1 to 41 or a pharmaceutical composition according to any one of claims 48 to 50 for use in the treatment of mucosal-obstructive pulmonary disease.

53. The antibody for use according to claim 52, wherein the mucosal obstructive pulmonary disease is selected from chronic obstructive pulmonary disease (COPD), cystic fibrosis, primary ciliary dyskinesia, non-cystic fibrosis, bronchiectasis, and bronchiolitis.

54. Use of an antibody according to any one of claims 1 to 41 or a pharmaceutical composition according to any one of claims 48 to 50 in the manufacture of a pharmaceutical for treating mucosal-obstructive pulmonary disease.

55. The use according to claim 54, wherein the mucosal obstructive pulmonary disease is selected from chronic obstructive pulmonary disease (COPD), cystic fibrosis, primary ciliary dyskinesia, non-cystic fibrosis, bronchiectasis, and bronchiolitis.

56. Use of an antibody according to any one of claims 1 to 41 or a pharmaceutical composition according to any one of claims 48 to 50 in the manufacture of a pharmaceutical for reducing the number of secretory cells in a target.

57. The use according to claim 56, wherein the pharmaceutical agent converts secretory cells into ciliated cells.

58. The use according to claim 56 or claim 57, wherein the secretory cells are located in the lung of the subject.

59. The use according to any one of claims 56 to 58, wherein the secretory cells are goblet cells.

60. A method for treating a subject having a mucosal-obstructive pulmonary disease, comprising administering to the subject an effective amount of an antibody according to any one of claims 1 to 41 or a pharmaceutical composition according to any one of claims 48 to 50.

61. The method according to claim 60, wherein the mucosal obstructive pulmonary disease is selected from chronic obstructive pulmonary disease (COPD), cystic fibrosis, primary ciliary dyskinesia, non-cystic fibrosis, bronchiectasis, and bronchiolitis.

62. A method for reducing the number of secretory cells in a target, comprising administering an effective amount of an antibody according to any one of claims 1 to 41 or a pharmaceutical composition according to any one of claims 48 to 50 to an individual to deplete the secretory cells in the target.

63. The method according to claim 62, comprising converting secretory cells into ciliated cells.

64. The method according to claim 62 or claim 63, wherein the secretory cells are located in the lung of the subject.

65. The method according to any one of claims 62 to 64, wherein the secretory cell is a goblet cell.

66. The method according to any one of claims 60 to 65, further comprising administering an additional therapeutic agent to the subject.

67. The method according to claim 66, wherein the additional therapeutic agent is selected from hypertonic saline, mannitol, pulmozyme, N-acetylcysteine, cysteamine, and bronchodilators.

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