NOTCH4 antibodies, compositions and methods for treating airway inflammation

Abstract

Antibodies specific for Notch4 and related methods of treating conditions such as airway inflammation.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 378,479, filed on October 5, 2022, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0003] Sequence Listing

[0004] This specification incorporates by reference in its entirety a sequence listing that is electronically submitted in XML format. The XML file, created on October 4, 2023, is named ALC - 050WO_ST26.xml and is 133 kb in size. Background of the Invention

[0005] Chronic airway inflammation is a common feature of many conditions such as asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), and bronchopulmonary dysplasia (BPD). Current strategies for treating these chronic diseases largely focus on systemically inhibiting pro - inflammatory pathways. A challenge with such strategies is to effectively shut down part of the immune response without making the patient vulnerable to opportunistic pathogens or disabled by side effects. However, due to the different profiles of the inflammatory response, so far, no single strategy has proven to be widely effective, and the response rates of existing therapies are generally poor overall.

[0006] There is still a need for improved targeted therapies for airway inflammation and related conditions. Summary of the Invention

[0007] In one aspect, antibodies capable of binding to Notch4 and antigen - binding fragments thereof (also referred to herein as Notch4 antibodies and Notch4 - binding fragments, respectively) are provided. For example, in some embodiments, an antibody or an antigen - binding fragment thereof capable of binding to Notch4 is provided, which comprises:

[0008] (a)(i) A heavy - chain variable domain (V H ), which comprises complementarity - determining regions:

[0009] CDR - H1, which comprises the amino acid sequence of SEQ ID NO:2, or a sequence that differs from this sequence by 1 or 2 amino acids,

[0010] CDR - H2, which comprises the amino acid sequence of SEQ ID NO:3, or a sequence that differs from this sequence by 1 or 2 amino acids, and

[0011] CDR-H3, which comprises the amino acid sequence of SEQ ID NO:4, or a sequence that differs from this sequence by 1 or 2 amino acids; and

[0012] (ii) a variable light chain (VK), which comprises complementarity determining regions:

[0013] CDR-L1, which comprises the amino acid sequence of SEQ ID NO:6, or a sequence that differs from this sequence by 1 or 2 amino acids,

[0014] CDR-L2, which comprises the amino acid sequence of SEQ ID NO:7, or a sequence that differs from this sequence by 1 or 2 amino acids, and

[0015] CDR-L3, which comprises the amino acid sequence of SEQ ID NO:8, or a sequence that differs from this sequence by 1 or 2 amino acids;

[0016] (b)(i)V H , which comprises complementarity determining regions:

[0017] CDR-H1, which comprises the amino acid sequence of SEQ ID NO:10, or a sequence that differs from this sequence by 1 or 2 amino acids,

[0018] CDR-H2, which comprises the amino acid sequence of SEQ ID NO:11, or a sequence that differs from this sequence by 1 or 2 amino acids, and

[0019] CDR-H3, which comprises the amino acid sequence of SEQ ID NO:12, or a sequence that differs from this sequence by 1 or 2 amino acids; and

[0020] (ii)V K , which comprises complementarity determining regions:

[0021] CDR-L1, which comprises the amino acid sequence of SEQ ID NO:14, or a sequence that differs from this sequence by 1 or 2 amino acids,

[0022] CDR-L2, which comprises the amino acid sequence of SEQ ID NO:15, or a sequence that differs from this sequence by 1 or 2 amino acids, and

[0023] CDR-L3, which comprises the amino acid sequence of SEQ ID NO:16, or a sequence that differs from this sequence by 1 or 2 amino acids;

[0024] (c)(i)V H , which comprises complementarity determining regions:

[0025] CDR-H1, which comprises the amino acid sequence of SEQ ID NO:18, or a sequence that differs from this sequence by 1 or 2 amino acids,

[0026] CDR-H2, which comprises the amino acid sequence of SEQ ID NO:19, or a sequence that differs from this sequence by 1 or 2 amino acids, and

[0027] CDR-H3, which comprises the amino acid sequence of SEQ ID NO:20, or a sequence that differs from this sequence by 1 or 2 amino acids; and

[0028] (ii)V K , which comprises complementarity-determining regions:

[0029] CDR-L1, which comprises the amino acid sequence of SEQ ID NO:22, or a sequence that differs from this sequence by 1 or 2 amino acids,

[0030] CDR-L2, which comprises the amino acid sequence of SEQ ID NO:23, or a sequence that differs from this sequence by 1 or 2 amino acids, and

[0031] CDR-L3, which comprises the amino acid sequence of SEQ ID NO:24, or a sequence that differs from this sequence by 1 or 2 amino acids;

[0032] (d)(i)V H , which comprises complementarity-determining regions:

[0033] CDR-H1, which comprises the amino acid sequence of SEQ ID NO:26, or a sequence that differs from this sequence by 1 or 2 amino acids,

[0034] CDR-H2, which comprises the amino acid sequence of SEQ ID NO:27, or a sequence that differs from this sequence by 1 or 2 amino acids, and

[0035] CDR-H3, which comprises the amino acid sequence of SEQ ID NO:28, or a sequence that differs from this sequence by 1 or 2 amino acids; and

[0036] (ii)V K , which comprises complementarity-determining regions:

[0037] CDR-L1, which comprises the amino acid sequence of SEQ ID NO:30, or a sequence that differs from this sequence by 1 or 2 amino acids,

[0038] CDR-L2, which comprises the amino acid sequence of SEQ ID NO:31, or a sequence that differs from this sequence by 1 or 2 amino acids, and

[0039] CDR-L3, which comprises the amino acid sequence of SEQ ID NO:32, or a sequence that differs from this sequence by 1 or 2 amino acids;

[0040] (e)(i)V H , which comprises complementarity-determining regions:

[0041] CDR-H1, which comprises the amino acid sequence of SEQ ID NO:34, or a sequence that differs from this sequence by 1 or 2 amino acids,

[0042] CDR-H2, which comprises the amino acid sequence of SEQ ID NO:35, or a sequence that differs from this sequence by 1 or 2 amino acids, and

[0043] CDR-H3, which comprises the amino acid sequence of SEQ ID NO:36, or a sequence that differs from this sequence by 1 or 2 amino acids; and

[0044] (ii)V K , which comprises complementarity-determining regions:

[0045] CDR-L1, which comprises the amino acid sequence of SEQ ID NO:38, or a sequence that differs from this sequence by 1 or 2 amino acids,

[0046] CDR-L2, which comprises the amino acid sequence of SEQ ID NO:39, or a sequence that differs from this sequence by 1 or 2 amino acids, and

[0047] CDR-L3, which comprises the amino acid sequence of SEQ ID NO:40, or a sequence that differs from this sequence by 1 or 2 amino acids;

[0048] (f)(i)V H , which comprises complementarity-determining regions:

[0049] CDR-H1, which comprises the amino acid sequence of SEQ ID NO:42, or a sequence that differs from this sequence by 1 or 2 amino acids,

[0050] CDR-H2, which comprises the amino acid sequence of SEQ ID NO:43, or a sequence that differs from this sequence by 1 or 2 amino acids, and

[0051] CDR-H3, which comprises the amino acid sequence of SEQ ID NO:44, or a sequence that differs from this sequence by 1 or 2 amino acids; and

[0052] (ii)V K , which comprises complementarity-determining regions:

[0053] CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 46, or a sequence that differs from this sequence by one or two amino acids,

[0054] CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 47, or a sequence that differs from this sequence by one or two amino acids, and

[0055] CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 48, or a sequence that differs from this sequence by one or two amino acids;

[0056] (g)(i)V H , which comprises complementarity-determining regions:

[0057] CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 50, or a sequence that differs from this sequence by one or two amino acids,

[0058] CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 51, or a sequence that differs from this sequence by one or two amino acids, and

[0059] CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 52, or a sequence that differs from this sequence by one or two amino acids; and

[0060] (ii)V K , which comprises complementarity-determining regions:

[0061] CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 54, or a sequence that differs from this sequence by one or two amino acids,

[0062] CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 55, or a sequence that differs from this sequence by one or two amino acids, and

[0063] CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 56, or a sequence that differs from this sequence by one or two amino acids;

[0064] (h)(i)V H , which comprises complementarity-determining regions:

[0065] CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 58, or a sequence that differs from this sequence by one or two amino acids,

[0066] CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 59, or a sequence that differs from this sequence by one or two amino acids, and

[0067] CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 60, or a sequence that differs from this sequence by 1 or 2 amino acids; and

[0068] (ii)V K , which comprises complementarity-determining regions:

[0069] CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 62, or a sequence that differs from this sequence by 1 or 2 amino acids,

[0070] CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 63, or a sequence that differs from this sequence by 1 or 2 amino acids, and

[0071] CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 64, or a sequence that differs from this sequence by 1 or 2 amino acids;

[0072] (i)(i) which comprises complementarity-determining regions:

[0073] CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 66, or a sequence that differs from this sequence by 1 or 2 amino acids,

[0074] CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 67, or a sequence that differs from this sequence by 1 or 2 amino acids, and

[0075] CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 68, or a sequence that differs from this sequence by 1 or 2 amino acids; and

[0076] (ii)V K , which comprises complementarity-determining regions:

[0077] CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 70, or a sequence that differs from this sequence by 1 or 2 amino acids,

[0078] CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 71, or a sequence that differs from this sequence by 1 or 2 amino acids, and

[0079] CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 72, or a sequence that differs from this sequence by 1 or 2 amino acids;

[0080] (j)(i)V H , which comprises complementarity-determining regions:

[0081] CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 74, or a sequence that differs from this sequence by 1 or 2 amino acids,

[0082] CDR-H2, which comprises the amino acid sequence of SEQ ID NO:75, or a sequence that differs from this sequence by one or two amino acids, and

[0083] CDR-H3, which comprises the amino acid sequence of SEQ ID NO:76, or a sequence that differs from this sequence by one or two amino acids; and

[0084] (ii)V K , which comprises complementarity-determining regions:

[0085] CDR-L1, which comprises the amino acid sequence of SEQ ID NO:78, or a sequence that differs from this sequence by one or two amino acids,

[0086] CDR-L2, which comprises the amino acid sequence of SEQ ID NO:79, or a sequence that differs from this sequence by one or two amino acids, and

[0087] CDR-L3, which comprises the amino acid sequence of SEQ ID NO:80, or a sequence that differs from this sequence by one or two amino acids;

[0088] (k)(i)V H , which comprises complementarity-determining regions:

[0089] CDR-H1, which comprises the amino acid sequence of SEQ ID NO:82, or a sequence that differs from this sequence by one or two amino acids,

[0090] CDR-H2, which comprises the amino acid sequence of SEQ ID NO:83, or a sequence that differs from this sequence by one or two amino acids, and

[0091] CDR-H3, which comprises the amino acid sequence of SEQ ID NO:84, or a sequence that differs from this sequence by one or two amino acids; and

[0092] (ii)V K , which comprises complementarity-determining regions:

[0093] CDR-L1, which comprises the amino acid sequence of SEQ ID NO:86, or a sequence that differs from this sequence by one or two amino acids,

[0094] CDR-L2, which comprises the amino acid sequence of SEQ ID NO:87, or a sequence that differs from this sequence by one or two amino acids, and

[0095] CDR-L3, which comprises the amino acid sequence of SEQ ID NO:88, or a sequence that differs from this sequence by one or two amino acids;

[0096] (l)(i)V H , which comprises complementarity determining regions:

[0097] CDR-H1, which comprises the amino acid sequence of SEQ ID NO:90, or a sequence differing from this sequence by 1 or 2 amino acids,

[0098] CDR-H2, which comprises the amino acid sequence of SEQ ID NO:91, or a sequence differing from this sequence by 1 or 2 amino acids, and

[0099] CDR-H3, which comprises the amino acid sequence of SEQ ID NO:92, or a sequence differing from this sequence by 1 or 2 amino acids; and

[0100] (ii)V K , which comprises complementarity determining regions:

[0101] CDR-L1, which comprises the amino acid sequence of SEQ ID NO:94, or a sequence differing from this sequence by 1 or 2 amino acids,

[0102] CDR-L2, which comprises the amino acid sequence of SEQ ID NO:95, or a sequence differing from this sequence by 1 or 2 amino acids, and

[0103] CDR-L3, which comprises the amino acid sequence of SEQ ID NO:96, or a sequence differing from this sequence by 1 or 2 amino acids;

[0104] (m)(i)V H , which comprises complementarity determining regions:

[0105] CDR-H1, which comprises the amino acid sequence of SEQ ID NO:98, or a sequence differing from this sequence by 1 or 2 amino acids,

[0106] CDR-H2, which comprises the amino acid sequence of SEQ ID NO:99, or a sequence differing from this sequence by 1 or 2 amino acids, and

[0107] CDR-H3, which comprises the amino acid sequence of SEQ ID NO:100, or a sequence differing from this sequence by 1 or 2 amino acids; and

[0108] (ii)V K , which comprises complementarity determining regions:

[0109] CDR-L1, which comprises the amino acid sequence of SEQ ID NO:102, or a sequence differing from this sequence by 1 or 2 amino acids,

[0110] CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 103, or a sequence that differs from this sequence by 1 or 2 amino acids, and

[0111] CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 104, or a sequence that differs from this sequence by 1 or 2 amino acids;

[0112] (n)(i)V H , which comprises complementarity-determining regions:

[0113] CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 106, or a sequence that differs from this sequence by 1 or 2 amino acids,

[0114] CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 107, or a sequence that differs from this sequence by 1 or 2 amino acids, and

[0115] CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 108, or a sequence that differs from this sequence by 1 or 2 amino acids; and

[0116] (ii)V K , which comprises complementarity-determining regions:

[0117] CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 110, or a sequence that differs from this sequence by 1 or 2 amino acids,

[0118] CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 111, or a sequence that differs from this sequence by 1 or 2 amino acids, and

[0119] CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 112, or a sequence that differs from this sequence by 1 or 2 amino acids;

[0120] (o)(i)V H , which comprises complementarity-determining regions:

[0121] CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 114, or a sequence that differs from this sequence by 1 or 2 amino acids,

[0122] CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 115, or a sequence that differs from this sequence by 1 or 2 amino acids, and

[0123] CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 116, or a sequence that differs from this sequence by 1 or 2 amino acids; and

[0124] (ii)V K , which comprises complementarity determining regions:

[0125] CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 118, or a sequence that differs from this sequence by 1 or 2 amino acids,

[0126] CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 119, or a sequence that differs from this sequence by 1 or 2 amino acids, and

[0127] CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 120, or a sequence that differs from this sequence by 1 or 2 amino acids;

[0128] (p)(i)V H , which comprises complementarity determining regions:

[0129] CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 122, or a sequence that differs from this sequence by 1 or 2 amino acids,

[0130] CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 123, or a sequence that differs from this sequence by 1 or 2 amino acids, and

[0131] CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 124, or a sequence that differs from this sequence by 1 or 2 amino acids; and

[0132] (ii)V K , which comprises complementarity determining regions:

[0133] CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 126, or a sequence that differs from this sequence by 1 or 2 amino acids,

[0134] CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 127, or a sequence that differs from this sequence by 1 or 2 amino acids, and

[0135] CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 128, or a sequence that differs from this sequence by 1 or 2 amino acids; or

[0136] (q)(i)V H , which comprises complementarity determining regions:

[0137] CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 130, or a sequence that differs from this sequence by 1 or 2 amino acids,

[0138] CDR-H2, which comprises the amino acid sequence of SEQ ID NO:131, or a sequence that differs from this sequence by one or two amino acids, and

[0139] CDR-H3, which comprises the amino acid sequence of SEQ ID NO:132, or a sequence that differs from this sequence by one or two amino acids; and

[0140] (ii)V K , which comprises complementarity-determining regions:

[0141] CDR-L1, which comprises the amino acid sequence of SEQ ID NO:134, or a sequence that differs from this sequence by one or two amino acids,

[0142] CDR-L2, which comprises the amino acid sequence of SEQ ID NO:135, or a sequence that differs from this sequence by one or two amino acids, and

[0143] CDR-L3, which comprises the amino acid sequence of SEQ ID NO:136, or a sequence that differs from this sequence by one or two amino acids.

[0144] In some embodiments, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 comprise amino acid sequences that, taken together, differ by no more than two amino acid residues from the sequences of:

[0145] (a) SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively;

[0146] (b) SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16, respectively;

[0147] (c) SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24, respectively;

[0148] (d) SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32, respectively;

[0149] (e) are SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, respectively;

[0150] (f) are SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, respectively;

[0151] (g) are SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, respectively;

[0152] (h) are SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, respectively;

[0153] (i) are SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, respectively;

[0154] (j) are SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, respectively;

[0155] (k) are SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, respectively;

[0156] (l) are SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, respectively;

[0157] (m) are SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, respectively;

[0158] (n) are SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112;

[0159] (o) are SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120;

[0160] (p) are SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128; or

[0161] (q) are SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136.

[0162] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0163] (a)(i)V H , which comprises complementarity-determining regions:

[0164] CDR-H1, which comprises the amino acid sequence of SEQ ID NO:2,

[0165] CDR-H2, which comprises the amino acid sequence of SEQ ID NO:3, and

[0166] CDR-H3, which comprises the amino acid sequence of SEQ ID NO:4; and

[0167] (ii)V K , which comprises complementarity-determining regions:

[0168] CDR-L1, which comprises the amino acid sequence of SEQ ID NO:6,

[0169] CDR-L2, which comprises the amino acid sequence of SEQ ID NO:7, and

[0170] CDR-L3, which comprises the amino acid sequence of SEQ ID NO:8;

[0171] (b)(i)V H , which comprises complementarity-determining regions:

[0172] CDR-H1, which comprises the amino acid sequence of SEQ ID NO:10,

[0173] CDR-H2, which comprises the amino acid sequence of SEQ ID NO:11, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:12; and

[0174] (ii)V K , which comprises complementarity determining regions:

[0175] CDR-L1, which comprises the amino acid sequence of SEQ ID NO:14,

[0176] CDR-L2, which comprises the amino acid sequence of SEQ ID NO:15, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:16;

[0177] (c)(i)V H , which comprises complementarity determining regions:

[0178] CDR-H1, which comprises the amino acid sequence of SEQ ID NO:18,

[0179] CDR-H2, which comprises the amino acid sequence of SEQ ID NO:19, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:20; and

[0180] (ii)V K , which comprises complementarity determining regions:

[0181] CDR-L1, which comprises the amino acid sequence of SEQ ID NO:22,

[0182] CDR-L2, which comprises the amino acid sequence of SEQ ID NO:23, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:24;

[0183] (d)(i)V H , which comprises complementarity determining regions:

[0184] CDR-H1, which comprises the amino acid sequence of SEQ ID NO:26,

[0185] CDR-H2, which comprises the amino acid sequence of SEQ ID NO:27, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:28; and

[0186] (ii)V K , which comprises complementarity determining regions:

[0187] CDR-L1, which comprises the amino acid sequence of SEQ ID NO:30,

[0188] CDR-L2, which comprises the amino acid sequence of SEQ ID NO:31, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:32;

[0189] (e)(i)V H , which comprises complementarity determining regions:

[0190] CDR-H1, which comprises the amino acid sequence of SEQ ID NO:34,

[0191] CDR-H2, which comprises the amino acid sequence of SEQ ID NO:35, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:36; and

[0192] (ii)V K , which comprises complementarity determining regions:

[0193] CDR-L1, which comprises the amino acid sequence of SEQ ID NO:38,

[0194] CDR-L2, which comprises the amino acid sequence of SEQ ID NO:39, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:40;

[0195] (f)(i)V H , which comprises complementarity determining regions:

[0196] CDR-H1, which comprises the amino acid sequence of SEQ ID NO:42,

[0197] CDR-H2, which comprises the amino acid sequence of SEQ ID NO:43, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:44; and

[0198] (ii)V K , which comprises complementarity determining regions:

[0199] CDR-L1, which comprises the amino acid sequence of SEQ ID NO:46,

[0200] CDR-L2, which comprises the amino acid sequence of SEQ ID NO:47, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:48;

[0201] (g)(i)V H , which comprises complementarity determining regions:

[0202] CDR-H1, which comprises the amino acid sequence of SEQ ID NO:50,

[0203] CDR-H2, which comprises the amino acid sequence of SEQ ID NO:51, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:52; and

[0204] (ii)V K , which comprises complementarity determining regions:

[0205] CDR-L1, which comprises the amino acid sequence of SEQ ID NO:54,

[0206] CDR-L2, which comprises the amino acid sequence of SEQ ID NO:55, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:56;

[0207] (h)(i)V H , which comprises complementarity determining regions:

[0208] CDR-H1, which comprises the amino acid sequence of SEQ ID NO:58,

[0209] CDR-H2, which comprises the amino acid sequence of SEQ ID NO:59, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:60; and

[0210] (ii)V K , which comprises complementarity determining regions:

[0211] CDR-L1, which comprises the amino acid sequence of SEQ ID NO:62,

[0212] CDR-L2, which comprises the amino acid sequence of SEQ ID NO:63, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:64;

[0213] (i)(i)V H , which comprises complementarity determining regions:

[0214] CDR-H1, which comprises the amino acid sequence of SEQ ID NO:66,

[0215] CDR-H2, which comprises the amino acid sequence of SEQ ID NO:67, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:68; and

[0216] (ii)V K , which comprises complementarity determining regions:

[0217] CDR-L1, which comprises the amino acid sequence of SEQ ID NO:70,

[0218] CDR-L2, which comprises the amino acid sequence of SEQ ID NO:71, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:72;

[0219] (j)(i)V H , which comprises complementarity determining regions:

[0220] CDR-H1, which comprises the amino acid sequence of SEQ ID NO:74,

[0221] CDR-H2, which comprises the amino acid sequence of SEQ ID NO:75, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:76; and

[0222] (ii)V K , which comprises complementarity determining regions:

[0223] CDR-L1, which comprises the amino acid sequence of SEQ ID NO:78,

[0224] CDR-L2, which comprises the amino acid sequence of SEQ ID NO:79, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:80;

[0225] (k)(i)V H , which comprises complementarity determining regions:

[0226] CDR-H1, which comprises the amino acid sequence of SEQ ID NO:82,

[0227] CDR-H2, which comprises the amino acid sequence of SEQ ID NO:83, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:84; and

[0228] (ii)V K , which comprises complementarity determining regions:

[0229] CDR-L1, which comprises the amino acid sequence of SEQ ID NO:86,

[0230] CDR-L2, which comprises the amino acid sequence of SEQ ID NO:87, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:88;

[0231] (l)(i)V H , which comprises complementarity determining regions:

[0232] CDR-H1, which comprises the amino acid sequence of SEQ ID NO:90,

[0233] CDR-H2, which comprises the amino acid sequence of SEQ ID NO:91, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:92; and

[0234] (ii)V K , which comprises complementarity determining regions:

[0235] CDR-L1, which comprises the amino acid sequence of SEQ ID NO:94,

[0236] CDR-L2, which comprises the amino acid sequence of SEQ ID NO:95, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:96;

[0237] (m)(i)V H , which comprises complementarity determining regions:

[0238] CDR-H1, which comprises the amino acid sequence of SEQ ID NO:98,

[0239] CDR-H2, which comprises the amino acid sequence of SEQ ID NO:99, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:100; and

[0240] (ii)V K , which comprises complementarity determining regions:

[0241] CDR-L1, which comprises the amino acid sequence of SEQ ID NO:102,

[0242] CDR-L2, which comprises the amino acid sequence of SEQ ID NO:103, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:104;

[0243] (n)(i)V H , which comprises complementarity determining regions:

[0244] CDR-H1, which comprises the amino acid sequence of SEQ ID NO:106,

[0245] CDR-H2, which comprises the amino acid sequence of SEQ ID NO:107, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:108; and

[0246] (ii)V K , which comprises complementarity determining regions:

[0247] CDR-L1, which comprises the amino acid sequence of SEQ ID NO:110,

[0248] CDR-L2, which comprises the amino acid sequence of SEQ ID NO:111, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:112;

[0249] (o)(i)V H , which comprises complementarity-determining regions:

[0250] CDR-H1, which comprises the amino acid sequence of SEQ ID NO:114,

[0251] CDR-H2, which comprises the amino acid sequence of SEQ ID NO:115, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:116; and

[0252] (ii)V K , which comprises complementarity-determining regions:

[0253] CDR-L1, which comprises the amino acid sequence of SEQ ID NO:118,

[0254] CDR-L2, which comprises the amino acid sequence of SEQ ID NO:119, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:120;

[0255] (p)(i)V H , which comprises complementarity-determining regions:

[0256] CDR-H1, which comprises the amino acid sequence of SEQ ID NO:122,

[0257] CDR-H2, which comprises the amino acid sequence of SEQ ID NO:123, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:124; and

[0258] (ii)V K , which comprises complementarity-determining regions:

[0259] CDR-L1, which comprises the amino acid sequence of SEQ ID NO:126,

[0260] CDR-L2, which comprises the amino acid sequence of SEQ ID NO:127, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:128; or (q)(i)V H , which comprises complementarity-determining regions:

[0261] CDR-H1, which comprises the amino acid sequence of SEQ ID NO:130,

[0262] CDR-H2, which comprises the amino acid sequence of SEQ ID NO:131, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:132; and

[0263] (ii)V K , which comprises complementarity determining regions:

[0264] CDR-L1, which comprises the amino acid sequence of SEQ ID NO:134,

[0265] CDR-L2, which comprises the amino acid sequence of SEQ ID NO:135, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:136. In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0266] (i)V H , which comprises complementarity determining regions:

[0267] CDR-H1, which comprises the amino acid sequence of SEQ ID NO:2;

[0268] CDR-H2, which comprises the amino acid sequence of SEQ ID NO:3; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:4; and

[0269] (ii)V K , which comprises complementarity determining regions:

[0270] CDR-L1, which comprises the amino acid sequence of SEQ ID NO:6;

[0271] CDR-L2, which comprises the amino acid sequence of SEQ ID NO:7; and

[0272] CDR-L3, which comprises the amino acid sequence of SEQ ID NO:8.

[0273] In some embodiments, V H comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:1; and V K comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:5.

[0274] In some embodiments, V H comprises the amino acid sequence of SEQ ID NO:1, and V K comprises the amino acid sequence of SEQ ID NO:5.

[0275] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0276] (i)V H , which comprises complementarity determining regions:

[0277] CDR-H1, which comprises the amino acid sequence of SEQ ID NO:10;

[0278] CDR-H2, which comprises the amino acid sequence of SEQ ID NO:11; and

[0279] CDR-H3, which comprises the amino acid sequence of SEQ ID NO:12; and

[0280] (ii)V K , which comprises complementarity determining regions:

[0281] CDR-L1, which comprises the amino acid sequence of SEQ ID NO:14;

[0282] CDR-L2, which comprises the amino acid sequence of SEQ ID NO:15; and

[0283] CDR-L3, which comprises the amino acid sequence of SEQ ID NO:16.

[0284] In some embodiments, V H comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:9; and V K comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:13.

[0285] In some embodiments, V H comprises the amino acid sequence of SEQ ID NO:9, and V K comprises the amino acid sequence of SEQ ID NO:13.

[0286] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0287] (i)V H , which comprises complementarity determining regions:

[0288] CDR-H1, which comprises the amino acid sequence of SEQ ID NO:18;

[0289] CDR-H2, which comprises the amino acid sequence of SEQ ID NO:19; and

[0290] CDR-H3, which comprises the amino acid sequence of SEQ ID NO:20; and

[0291] (ii)V K , which comprises complementarity determining regions:

[0292] CDR-L1, which comprises the amino acid sequence of SEQ ID NO:22;

[0293] CDR-L2, which comprises the amino acid sequence of SEQ ID NO:23; and

[0294] CDR-L3, which comprises the amino acid sequence of SEQ ID NO:24.

[0295] In some embodiments, V H comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:17; and V K comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:21.

[0296] In some embodiments, V H comprises the amino acid sequence of SEQ ID NO:17, and V K comprises the amino acid sequence of SEQ ID NO:21.

[0297] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0298] (i)V H , which comprises complementarity-determining regions:

[0299] CDR-H1, which comprises the amino acid sequence of SEQ ID NO:34;

[0300] CDR-H2, which comprises the amino acid sequence of SEQ ID NO:35; and

[0301] CDR-H3, which comprises the amino acid sequence of SEQ ID NO:36; and

[0302] (ii)V K , which comprises complementarity-determining regions:

[0303] CDR-L1, which comprises the amino acid sequence of SEQ ID NO:38;

[0304] CDR-L2, which comprises the amino acid sequence of SEQ ID NO:39; and

[0305] CDR-L3, which comprises the amino acid sequence of SEQ ID NO:40.

[0306] In some embodiments, V H comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:33; and V KComprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:37.

[0307] In some embodiments, V H Comprises the amino acid sequence of SEQ ID NO:33, and V K Comprises the amino acid sequence of SEQ ID NO:37.

[0308] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0309] (i) V H , which comprises complementarity-determining regions:

[0310] CDR-H1, which comprises the amino acid sequence of SEQ ID NO:98;

[0311] CDR-H2, which comprises the amino acid sequence of SEQ ID NO:99; and

[0312] CDR-H3, which comprises the amino acid sequence of SEQ ID NO:100; and

[0313] (ii) V K , which comprises complementarity-determining regions:

[0314] CDR-L1, which comprises the amino acid sequence of SEQ ID NO:102;

[0315] CDR-L2, which comprises the amino acid sequence of SEQ ID NO:103; and

[0316] CDR-L3, which comprises the amino acid sequence of SEQ ID NO:104.

[0317] In some embodiments, V H Comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:97; and V K Comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:101.

[0318] In some embodiments, V H Comprises the amino acid sequence of SEQ ID NO:97, and V K Comprises the amino acid sequence of SEQ ID NO:101.

[0319] In some embodiments,

[0320] (a) V Hcomprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:1, and V K comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:5;

[0321] (b)V H comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:9, and V K comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:13;

[0322] (c)V H comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:17, and V K comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:21;

[0323] (d)V H comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25, and V K comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:29;

[0324] (e)V H comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:33, and V K comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:37;

[0325] (f)V H comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:41, and V K comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:45;

[0326] (g)V H comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:49, and VK comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:53;

[0327] (h)V H comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:57, and V K comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:61;

[0328] (i)V H comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:65, and V K comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:69;

[0329] (j)V H comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:73, and V K comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:77;

[0330] (k)V H comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:81, and V K comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:85;

[0331] (l)V H comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:89, and V K comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:93;

[0332] (m)V H comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:97, and V Kcomprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 101;

[0333] (n)V H comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 105, and V K comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 109;

[0334] (o)V H comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 113, and V K comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 117;

[0335] (p)V H comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 121, and V K comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 125; or

[0336] (q)V H comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 129, and V K comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 133.

[0337] In some embodiments,

[0338] (a)V H comprising the amino acid sequence of SEQ ID NO: 1, and V K comprising the amino acid sequence of SEQ ID NO: 5;

[0339] (b)V H comprising the amino acid sequence of SEQ ID NO: 9, and V K comprising the amino acid sequence of SEQ ID NO: 13;

[0340] (c)V H comprising the amino acid sequence of SEQ ID NO: 17, and V Kcomprising the amino acid sequence of SEQ ID NO:21;

[0341] (d)V H comprising the amino acid sequence of SEQ ID NO:25, and V K comprising the amino acid sequence of SEQ ID NO:29;

[0342] (e)V H comprising the amino acid sequence of SEQ ID NO:33, and V K comprising the amino acid sequence of SEQ ID NO:37;

[0343] (f)V H comprising the amino acid sequence of SEQ ID NO:41, and V K comprising the amino acid sequence of SEQ ID NO:45;

[0344] (g)V H comprising the amino acid sequence of SEQ ID NO:49, and V K comprising the amino acid sequence of SEQ ID NO:53;

[0345] (h)V H comprising the amino acid sequence of SEQ ID NO:57, and V K comprising the amino acid sequence of SEQ ID NO:61;

[0346] (i)V H comprising the amino acid sequence of SEQ ID NO:65, and V K comprising the amino acid sequence of SEQ ID NO:69;

[0347] (j)V H comprising the amino acid sequence of SEQ ID NO:73, and V K comprising the amino acid sequence of SEQ ID NO:77;

[0348] (k)V H comprising the amino acid sequence of SEQ ID NO:81, and V K comprising the amino acid sequence of SEQ ID NO:85;

[0349] (l)V H comprising the amino acid sequence of SEQ ID NO:89, and V K comprising the amino acid sequence of SEQ ID NO:93;

[0350] (m)V H comprising the amino acid sequence of SEQ ID NO:97, and VK Comprising the amino acid sequence of SEQ ID NO: 101;

[0351] (n)V H Comprising the amino acid sequence of SEQ ID NO: 105, and V K Comprising the amino acid sequence of SEQ ID NO: 109;

[0352] (o)V H Comprising the amino acid sequence of SEQ ID NO: 113, and V K Comprising the amino acid sequence of SEQ ID NO: 117;

[0353] (p)V H Comprising the amino acid sequence of SEQ ID NO: 121, and V K Comprising the amino acid sequence of SEQ ID NO: 125; or

[0354] (q)V H Comprising the amino acid sequence of SEQ ID NO: 129, and V K Comprising the amino acid sequence of SEQ ID NO: 133.

[0355] In some embodiments, the antibody or its antigen-binding fragment can bind to human Notch4 with a K D value of 10 nM or less.

[0356] In some embodiments, the antibody is a human antibody.

[0357] In some embodiments, the antibody or its antigen-binding fragment comprises an antibody heavy chain constant region.

[0358] In some embodiments, the heavy chain constant region is a human IgG heavy chain constant region, for example, an IgG4 heavy chain constant region.

[0359] Also provided are isolated nucleic acids encoding the heavy chain variable domain and / or the light chain variable domain of the antibody or its antigen-binding fragment disclosed herein, expression vectors comprising such isolated nucleic acids, and host cells comprising such isolated nucleic acids or expression vectors.

[0360] Also provided are compositions comprising the Notch4 antibody or its Notch-binding fragment disclosed herein and a pharmaceutically acceptable carrier.

[0361] In one aspect, provided is a method of treating, ameliorating, or preventing airway inflammation in a subject, which comprises the step of administering to the subject an effective amount of the Notch4 antibody or its Notch4-binding fragment disclosed herein.

[0362] In some embodiments, the subject is a mammal, such as a human.

[0363] In some embodiments, the subject is diagnosed with or at risk of developing an airway inflammation-related disorder, e.g., the airway inflammation-related disorder is selected from the group consisting of asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), or bronchopulmonary dysplasia (BPD).

[0364] In some embodiments, the administering step comprises administering by a systemic route (e.g., a systemic route such as intravenous, intramuscular, or subcutaneous).

[0365] In one aspect, provided is a method for detecting the presence of Notch4 or a fragment thereof in a sample, comprising contacting the sample with a Notch4 antibody or Notch4 binding fragment disclosed herein and detecting the presence of a complex between the antibody or antigen-binding fragment and Notch4, wherein the detection of the complex indicates the presence of Notch4. Brief Description of the Drawings

[0367] Figure 1 Shows airway hyperresponsiveness from a methacholine challenge test in PBMC-humanized mice sensitized to house dust mite allergen. Airway hyperresponsiveness (“AHR”) was determined by relative peak airway resistance at the highest dose of methacholine (40 mg / mL) tested after treatment with novel Notch4-specific antibodies (C08-Lib3, iggrefmat 6-28, iggrefmat 3-11, H07-Lib3, and iggrefmat 6-16), 4H1 (previously described Notch4 antibody), or isotype control. * indicates p<0.05, ** indicates p<0.01.

[0368] Figure 2A Shows representative IL-4 and IL-13 flow cytometry plots of Teff cells from bronchoalveolar lavage fluid of individual PBMC-humanized mice sensitized to house dust mite allergen and treated with novel Notch4-specific antibodies (C08-Lib3, iggrefmat 6-28, iggrefmat 3-11, H07-Lib3, and iggrefmat 6-16), 4H1, or isotype control. Figure 2B and 2C Respectively show the mean percentages of IL13-positive and IL4-positive cells in Teff cells of each group. * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001.

[0369] Figure 3AShows representative flow cytometry plots of BAL fluid Teff cells stained for CD4 and CCR6 from individual PBMC humanized mice sensitized to house dust mite allergen and treated with novel Notch4-specific antibodies (C08-Lib3, iggrefmat 6-28, iggrefmat 3-11, H07-Lib3, and iggrefmat 6-16), 4H1, or isotype control. Figure 3B Shows the mean percentage of CCR6-positive cells in Teff cells for each treatment group.

[0370] Figure 4A Shows representative flow cytometry plots of Teff cells from bronchoalveolar lavage fluid of individual PBMC humanized mice sensitized to house dust mite allergen and treated with novel Notch4-specific antibodies (C08-Lib3, iggrefmat 6-28, iggrefmat 3-11, H07-Lib3, and iggrefmat 6-16), 4H1, or isotype control. Figure 4B Shows the mean percentage of Notch4-positive cells in Treg cells for each mouse group. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001. Figure 4C Shows the mean percentage of Notch4-positive cells in effector T (Teff) cells for the same treatment groups.

[0371] Figure 5A Shows the mean absorbance at 450 nm in samples with and without Notch4-specific antibody H07-Lib3 in an ELISA-based assay to evaluate binding to Notch4 or Notch3. Binding to BSA was also evaluated as a control. Figure 5B Shows the mean absorbance at 450 nm in a binding competition assay experiment between H07-Lib3 and a commercial mouse monoclonal Notch4 antibody against Notch4 (antigen).

[0372] Figure 6A Shows the normalized reactivity of Notch4-specific antibody H07-Lib3 and other commercial therapeutic antibodies (bococizumab, efalizumab, gantenerumab, ebalizumab, lenzilumab, and panitumumab) against cardiolipin. Figure 6BShows the normalized reactivity of H07-Lib3 and other commercial therapeutic antibodies (bococizumab, efalizumab, gantenerumab, ebalizumab, lenzilumab, and panitumumab) against keyhole limpet hemocyanin (KLH). Figure 6C Shows the normalized reactivity of H07-Lib3 and other commercial therapeutic antibodies (bococizumab, efalizumab, gantenerumab, ebalizumab, lenzilumab, and panitumumab) against lipopolysaccharide (LPS). Figure 6D Shows the normalized reactivity of H07-Lib3 and other commercial therapeutic antibodies (bococizumab, efalizumab, gantenerumab, ebalizumab, lenzilumab, and panitumumab) against single-stranded DNA (ssDNA). Figure 6E Shows the normalized reactivity of H07-Lib3 and other commercial therapeutic antibodies (bococizumab, efalizumab, gantenerumab, ebalizumab, lenzilumab, and panitumumab) against double-stranded DNA (dsDNA). Figure 6F Shows the normalized reactivity of H07-Lib3 and other commercial therapeutic antibodies (bococizumab, efalizumab, gantenerumab, ebalizumab, lenzilumab, and panitumumab) against insulin. Figure 6G Shows the normalized reactivity of H07-Lib3 and other commercial therapeutic antibodies (bococizumab, efalizumab, gantenerumab, ebalizumab, lenzilumab, and panitumumab) against bovine serum albumin (BSA).

[0373] Detailed description of certain embodiments of the invention

[0374] Notch4 (the amino acid sequence of which is shown below as SEQ ID NO:137) is expressed on Treg cells, and its signaling in Treg cells drives allergic inflammation induced by allergens or particulate pollutants (see, for example, International Patent Publication No. WO 2019 / 178488). Novel therapeutic Notch4 antibodies and their compositions, as well as related methods for treating, ameliorating, or preventing airway inflammation, including airway inflammation associated with certain conditions, are disclosed herein. The Notch4 antibodies disclosed herein are fully human antibodies and are specific for Notch4 because they essentially do not bind to other Notch proteins such as Notch1, Notch2, and Notch3.

[0375] SEQ ID NO:137 (human Notch4 protein sequence)

[0376]

[0377] Definition

[0378] As used herein, when used in reference to a value, the terms "about", "approximately", and "substantially equivalent to" refer to a value that is similar to the recited value in the context of that recited value. In general, one of ordinary skill in the art familiar with the context will understand the relevant degree of difference covered by "about", "approximately", and "substantially equivalent to" in that context. For example, in some embodiments, the terms "about", "approximately", and "substantially equivalent to" may cover a range of values within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the recited value.

[0379] As used herein, when used in reference to an antibody or an antigen-binding fragment thereof, the terms "antagonistic", "neutralizing", or "blocking" are intended to refer to an antibody or fragment thereof whose binding to its target results in inhibition of at least some biological activity of the target.

[0380] As used herein, "antibody" refers to a polypeptide whose amino acid sequence comprises an immunoglobulin and fragments thereof that specifically bind to a designated antigen or a fragment thereof. Antibodies according to the invention can be of any type (e.g., IgA, IgD, IgE, IgG, or IgM) or subtype (e.g., IgA1, IgA2, IgG1, IgG2, IgG3, or IgG4). One of ordinary skill in the art will understand that the characteristic sequence or portion of an antibody can include amino acids found in one or more regions of the antibody (e.g., variable region, hypervariable region, constant region, heavy chain, light chain, and combinations thereof). In addition, one of ordinary skill in the art will understand that the characteristic sequence or portion of an antibody can include one or more polypeptide chains and can include sequence elements found in the same polypeptide chain or different polypeptide chains.

[0381] An "antigen-binding fragment" of an antibody, or "antibody fragment", comprises a portion of a full antibody that is still capable of binding to a target antigen. In some embodiments, the antibody has a function other than antigen-binding function, and the antigen-binding fragment retains that function. Generally, the antigen-binding fragment comprises the variable region of the antibody. Papain digestion of an antibody yields two identical antigen-binding fragments (referred to as "Fab" fragments) and a residual "Fc" fragment, the name reflecting the ability to crystallize readily. The Fab fragment consists of the entire light chain together with the variable region of the heavy chain (V H ) and the first constant domain of one heavy chain (C H1) Composition. Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen binding site. Pepsin treatment of an antibody produces a single large F(ab')2 fragment, which roughly corresponds to two disulfide-linked Fab fragments with different antigen binding activities, and this single large F(ab')2 fragment is still able to cross-link antigens. The Fab' fragment differs from the Fab fragment in having some additional residues at the carboxyl terminus of the C H domain, including one or more cysteines from the antibody hinge region. Fab'-SH designates a Fab' in which the cysteine residue of the constant domain carries a free thiol group. The F(ab')2 antibody fragment is initially produced as a pair of Fab' fragments with hinge cysteines in between. Other chemical conjugations of antibody fragments are also known.

[0382] A "complementary determining region" (abbreviated as "CDR") is a hypervariable region that is scattered within more conserved regions called "framework regions" (abbreviated as "FR"). In some embodiments, the sequence of the framework region is the same as the framework region in the human germline sequence. In some embodiments, the sequence of the framework region is modified relative to the human germline sequence.

[0383] As used herein, the expression "control sequence" refers to a DNA sequence that is necessary or advantageous for expressing an operably linked coding sequence in a particular host organism. For example, control sequences that are commonly applicable to prokaryotes include a promoter, an optional operator sequence, and a ribosome binding site. It is known that eukaryotic cells utilize promoters, polyadenylation signals, and enhancers.

[0384] As used herein, "antibody effector function" refers to those biological activities that can be attributed to the Fc region of an antibody (native sequence Fc region or amino acid sequence variant Fc region), and which generally vary with the antibody isotype. Examples of antibody effector functions include, but are not limited to, Clq binding and complement-dependent cytotoxicity, Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis, downregulation of cell surface receptors (such as B cell receptors), and B cell activation.

[0385] As used herein, the term "epitope" is an antigenic determinant that interacts with a specific antigen binding site (called a paratope and which is composed of six complementary determining regions of the antibody) in the variable region of an antibody molecule. A single antigen can have more than one epitope. Epitopes can be conformational or linear. A conformational epitope contains spatially juxtaposed amino acids from different segments of a linear polypeptide chain. A linear epitope contains adjacent amino acid residues in a polypeptide chain.

[0386] The Fc fragment contains the carboxy-terminal portions of two heavy chains held together by a disulfide bond. The effector functions of an antibody are determined by sequences in the Fc region, which is also recognized by Fc receptors (FcRs) found on certain types of cells.

[0387] As used herein, when used in reference to an antibody, the term "humanized" refers to a form of a chimeric non-human (e.g., murine) antibody. A "humanized antibody" contains minimal sequences derived from a non-human immunoglobulin. Ordinarily, a humanized antibody is a human immunoglobulin (recipient antibody or acceptor antibody) in which residues from the hypervariable regions of the recipient are replaced by residues from the hypervariable regions of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. In some instances, residues from the framework regions (FRs) of the human immunoglobulin are replaced by corresponding non-human residues. Moreover, a humanized antibody may contain residues not found in the recipient antibody or donor antibody. These modifications are made to further improve antibody performance, such as binding affinity. Ordinarily, a humanized antibody will contain substantially all of at least one, and usually two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the framework regions are those of a human immunoglobulin sequence, although the framework regions may contain one or more amino acid substitutions that improve binding affinity. In some embodiments, no more than six amino acid substitutions are made in the heavy chain and no more than three amino acid substitutions are made in the light chain in the framework regions. Optionally, a humanized antibody will also contain at least a portion of an immunoglobulin constant region (Fc), usually containing at least a portion of a human immunoglobulin constant region.

[0388] As used herein, an "isolated" nucleic acid molecule is a nucleic acid molecule identified and separated from at least one contaminant molecule with which it is ordinarily associated in the natural source of the antibody nucleic acid. The isolated nucleic acid molecule is in a form or environment different from that in which it is naturally found. Thus, an isolated nucleic acid molecule is distinct from a nucleic acid molecule that exists in natural cells.

[0389] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., each antibody in the population is identical except for possible naturally occurring mutations that may be present in minor amounts. A monoclonal antibody is highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies can be prepared by the hybridoma method (such as the method first described by Kohler et al., Nature 256:495 (1975)), or can be prepared by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries, e.g., using the techniques described in Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991).

[0390] As used herein, a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader that is expressed as a preprotein participating in the secretion of a polypeptide is operably linked to DNA for the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to promote translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking can be accomplished, for example, by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers can be used in accordance with conventional practice.

[0391] As used herein, "polypeptide" refers to a string of at least two amino acids attached to one another by peptide bonds. In some embodiments, the polypeptide can comprise at least 3-5 amino acids, each attached to other amino acids by at least one peptide bond. Those of ordinary skill in the art will understand that a polypeptide can include one or more "non-natural" amino acids or other entities that are nonetheless capable of being incorporated into a polypeptide chain. In some embodiments, the polypeptide can be glycosylated; for example, the polypeptide can contain one or more covalently linked sugar moieties. In some embodiments, a single "polypeptide" (e.g., an antibody polypeptide) can comprise two or more separate polypeptide chains that can be linked to one another in some instances, e.g., by one or more disulfide bonds or other means.

[0392] As used herein, the phrase "reference level" generally refers to a level that is considered "normal" for purposes of comparison, e.g., the level of a suitable control. For example, in the context of airway inflammation, the "reference level" can refer to the level of airway resistance in a subject having a condition associated with airway inflammation and not receiving the therapeutic agent of interest or in a subject receiving a treatment other than the therapeutic agent of interest (e.g., the current standard of care). The reference level can be determined contemporaneously or can be predetermined, e.g., known or inferred from past observations.

[0393] As used herein, the phrases "therapeutically effective amount" and "effective amount" are used interchangeably and refer to an amount that is effective to achieve a desired therapeutic result at a necessary dosage and for a period of time. The therapeutically effective amount can vary depending on factors such as the type of disease (e.g., an airway inflammation-associated disorder such as asthma), the disease state, the age, sex, and / or weight of the individual, and the ability of the Notch4 antibody (or its pharmaceutical composition) to elicit a desired response in the individual. The effective amount can also be an amount at which the therapeutic beneficial effects of the foregoing component or its pharmaceutical composition exceed any toxic or detrimental effects.

[0394] As used herein, "treating" a condition or "treatment" of a condition (such as, e.g., a condition described herein, such as an airway inflammation-associated disorder such as asthma) is a method for obtaining a beneficial or desired result, such as a clinical result. Beneficial or desired results can include, but are not limited to, alleviating or ameliorating one or more symptoms or conditions; reducing the severity of a disease, disorder, or condition; stabilizing the state of a disease, disorder, or condition (i.e., not worsening); preventing the spread of a disease, disorder, or condition; delaying or slowing the progression of a disease, disorder, or condition; improving or palliating a disease, disorder, or condition; and remission (whether partial or total), whether detectable or undetectable. "Palliating" a disease, disorder, or condition means that the severity of the disease, disorder, or condition and / or the time course of the progression of the undesired clinical manifestations are reduced and / or the time course of the progression is slowed or extended as compared to the degree or time course in the absence of treatment.

[0395] Antibodies and antigen-binding fragments thereof

[0396] In one aspect, antibodies and antigen-binding fragments thereof that are capable of binding Notch4 are provided. In some embodiments, the antibody or antigen-binding fragment is a monoclonal antibody or an antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment is a human antibody or an antigen-binding fragment thereof, such as a human monoclonal antibody or an antigen-binding fragment thereof.

[0397] In some embodiments, antigen-binding fragments are provided. For example, the fragment can be, for example, scFv, Fab, scFab (single-chain Fab).

[0398] As used herein, according to its common usage in the art, the term "scFv" is used to refer to a single chain in which the V H domain from an antibody and the V L or V K domains are typically linked via a linker.

[0399] As used herein, the term "Fab fragment" is used according to its common usage in the art. The Fab fragment typically comprises the entire light chain (V L domain or V K domain and C L 1 domain), the variable region of the heavy chain (V H ), and the first constant domain of one heavy chain (C H 1).

[0400] In some embodiments, immunoconjugates are provided that comprise an antibody or antigen-binding fragment as disclosed herein, the conjugate being labeled and / or conjugated to a cytotoxic agent such as a toxin or a radioisotope.

[0401] Exemplary antibodies

[0402] In some embodiments, the provided antibody or antigen-binding fragment comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises CDR-H1, CDR-H2, and CDR-H3, and the light chain variable domain comprises CDR-L1, CDR-L2, and CDR-L3, wherein CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are those of the antibody described in Table 1 below. In some embodiments, an antibody or antigen-binding fragment of a variant of the antibody shown in Table 1 is provided, because such an antibody or antigen-binding fragment has a CDR sequence that differs from the CDR sequence of the antibody described in Table 1 by no more than two amino acid residues per CDR (e.g., two or one amino acid residue). In some embodiments, an antibody or antigen-binding fragment of a variant of the antibody shown in Table 1 is provided, wherein such an antibody or antigen-binding fragment has a set of six CDRs whose sequences, taken together, differ from the CDRs of the antibody described in Table 1 by no more than two amino acid residues (e.g., two or one amino acid residue).

[0403] In some embodiments, the provided antibody or antigen-binding fragment comprises a heavy chain variable domain and a light chain variable domain that comprise the heavy chain variable domain and the light chain variable sequences of the antibody described in Table 1. In some embodiments, an antibody or antigen-binding fragment of a variant of the antibody shown in Table 1 is provided, because such an antibody or antigen-binding fragment has (1) a heavy chain domain that comprises an amino acid sequence that is at least 85%, at least 87.5%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of the heavy chain variable domain of the antibody described in Table 1; and (2) a light chain domain that comprises an amino acid sequence that is at least 85%, at least 87.5%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of the light chain variable domain of the same antibody described in Table 1.

[0404] Table 1. Exemplary heavy chain variable domain, light chain variable domain, and complementarity determining region sequences of Notch4 antibodies

[0405]

[0406]

[0407]

[0408]

[0409]

[0410] Constant region of the antibody

[0411] In some embodiments, the provided Notch4 antibody or its Notch4-binding fragment comprises one or more constant regions, such as a heavy chain constant region and / or a light chain constant region.

[0412] In some embodiments, the provided Notch4 antibody or its Notch4-binding fragment comprises an IgG4 heavy chain constant region, e.g., having at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the IgG4 heavy chain constant region of SEQ ID NO:138, or an IgG4 heavy chain constant region identical thereto. In some embodiments, the provided Notch4 antibody or its Notch4-binding fragment comprises an Igκ light chain constant region, e.g., having at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or identity to the Igκ light chain constant region of SEQ ID NO:142, or an Igκ light chain constant region identical thereto.

[0413] SEQ ID NO:138 Human IgG4 heavy chain constant region sequence

[0414] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0415] SEQ ID NO:139 Human IgG1 heavy chain constant region sequence

[0416] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0417] SEQ ID NO:140 Human IgG2 heavy chain constant region sequence

[0418] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0419] SEQ ID NO:141 Human IgG3 heavy chain constant region sequence

[0420] ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK

[0421] SEQ ID NO:142 Human Igκ light chain constant region sequence

[0422] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0423] SEQ ID NO:143 Human Igλ1 light chain constant region sequence

[0424] GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0425] SEQ ID NO:144 Human Igλ2 light chain constant region sequence

[0426] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0427] SEQ ID NO:145 Human Igλ3 light chain constant region sequence

[0428] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTECS

[0429] SEQ ID NO:146 Human Igλ6 light chain constant region sequence

[0430] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVKVAWKADGSPVNTGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPAECS

[0431] SEQ ID NO:147 Human Igλ7 light chain constant region sequence

[0432] GQPKAAPSVTLFPPSSEELQANKATLVCLVSDFNPGAVTVAWKADGSPVKVGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCRVTHEGSTVEKTVAPAECS

[0433] Antibody fragment

[0434] In certain embodiments, antibody fragments are provided rather than whole antibodies.

[0435] A variety of techniques have been developed for generating antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv, and ScFv antibody fragments can all be expressed in and secreted from Escherichia coli, thus allowing for the large-scale production of these fragments. Antibody fragments can be isolated from, for example, antibody phage libraries. Alternatively or additionally, Fab′-SH fragments can be directly recovered from Escherichia coli and chemically conjugated to form F(ab′)2 fragments (Carter et al., Bio / Technology 10:163-167 (1992)). According to another method, F(ab')2 fragments can be directly isolated from recombinant host cell cultures. Fab and F(ab')2 fragments with increased in vivo half-life containing salvage receptor binding epitope residues are described, for example, in U.S. Patent No. 5,869,046. Other techniques for generating antibody fragments will be apparent to those skilled in the art.

[0436] In some embodiments, the antigen-binding fragment is a single-chain Fv fragment (scFv). See, e.g., WO 93 / 16185; U.S. Patent No. 5,571,894; and U.S. Patent No. 5,587,458. Fv and scFv are species having a complete combining site without constant regions; thus, these fragments may be suitable for reduced non-specific binding during in vivo use. An scFv fusion protein can be constructed to produce a fusion of an effector protein at the amino terminus or alternatively the carboxy terminus of the scFv. See, e.g., Antibody Engineering, edited by Borrebaeck. Antigen-binding antibody fragments can also be "linear antibodies", e.g., as described in U.S. Patent No. 5,641,870.

[0437] Amino acid sequence modifications

[0438] Amino acid sequence modifications of the antibodies or antigen-binding fragments disclosed herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of an antibody or antigen-binding fragment. Amino acid sequence variants can be prepared, for example, by introducing appropriate nucleotide changes into the nucleic acid sequence encoding the antibody or antigen-binding fragment, or by peptide synthesis. Such modifications can include, for example, deletions from, and / or insertions into, and / or substitutions of residues within the amino acid sequence of the antibody or antigen-binding fragment. Any combination of deletions, insertions and substitutions can be made, provided that the antibody or antigen-binding fragment has the desired characteristics. In some embodiments, amino acid changes are introduced to alter post-translational processes, such as changing the number or location of glycosylation sites.

[0439] A useful method for identifying certain residues or regions as preferred sites for mutagenesis is called "alanine-scanning mutagenesis", e.g., as described by Cunningham and Wells in Science, 244:1081-1085 (1989). In this method, target residues or a group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced with a neutral or negatively charged amino acid, most preferably alanine or polyalanine, to affect the interaction of the amino acid with the antigen. Those amino acid positions that prove to be functionally sensitive to the substitution are then refined by introducing further or other variants at or for the substitution site. Thus, while the sites for introducing amino acid sequence variation are predetermined, the nature of the mutations themselves need not be predetermined. For example, to analyze the performance of a mutation at a given site, alanine-scanning or random mutagenesis can be performed at the target codon or region, and the expressed variants can be screened for the desired activity.

[0440] Examples of amino acid sequence insertions include, but are not limited to, amino- and / or carboxyl-terminal fusions of polypeptides ranging in length from one residue to polypeptides containing one hundred or more residues, and intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include, but are not limited to, an N-terminal methionyl residue.

[0441] In some embodiments, the antibody or antigen-binding fragment is fused at one terminus to another polypeptide, such as a cytotoxic polypeptide, an enzyme, or a polypeptide that increases the serum half-life of the antibody or antigen-binding fragment.

[0442] Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue in the antibody molecule or antigen-binding fragment molecule replaced by a different residue. The sites of substitution mutagenesis of greatest interest are typically the hypervariable regions, although framework region alterations are also considered. Examples of conservative substitutions are shown under the heading "Preferred Substitutions" in Table 2. More substantial changes, such as those shown under the heading "Exemplary Substitutions" in Table 2, or changes further described below with reference to amino acid classes, can be introduced, and the resulting antibodies or antigen-binding fragments are screened.

[0443] Table 2: Exemplary amino acid substitutions

[0444]

[0445] Substantial modification of the biological properties of an antibody can be achieved by selecting substitutions that are significantly different in their effect on maintaining (a) the structure of the polypeptide backbone in the substituted region, such as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are generally divided into several classes based on common side-chain properties:

[0446] (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

[0447] (2) Neutral hydrophilic: Cys, Ser, Thr;

[0448] (3) Acidic: Asp, Glu;

[0449] (4) Basic: Asn, Gln, His, Lys, Arg;

[0450] (5) Residues that affect chain orientation: Gly, Pro; and

[0451] (6) Aromatic: Trp, Tyr, Phe.

[0452] Non-conservative substitutions may involve replacing a member of one of these classes with a member of another class.

[0453] Additionally or alternatively, cysteine residues that do not participate in maintaining the proper conformation of the antibody or antigen-binding fragment can be replaced, typically with serine, to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine bonds can be added to the antibody to improve its stability (especially when the antibody is an antibody fragment such as an Fv fragment).

[0454] In some embodiments, substitution variants contain substitutions within one or more hypervariable region residues of a parental antibody (e.g., a humanized antibody or a human antibody). Typically, the resulting variants that have improved biological properties relative to the parental antibody from which they are derived are selected for further development.

[0455] Methods for generating such substituted variants involve the use of phage display affinity maturation. In one instance of such methods, several hypervariable regions (e.g., 6 - 7 regions) are mutated to generate all possible amino acid substitutions at each site. The resulting antibody variants are displayed monovalently, for example, fused to the gene III product of M13 packaged within each filamentous phage particle. The phage-displayed variants are then screened for biological activity (e.g., binding affinity).

[0456] To identify candidate hypervariable regions for modification, alanine-scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively or additionally, it may be beneficial to analyze the crystal structure of the antigen-antibody complex to identify the contact points between the antibody or antigen-binding fragment and the antigen. Such contact residues and adjacent residues are candidates for substitution according to the techniques detailed herein. Once such variants are generated, the set of variants is screened, and antibodies with excellent properties in one or more relevant assays can be selected for further development.

[0457] In some embodiments, the original glycosylation pattern of the parental antibody is altered. Such alterations can include the deletion of one or more carbohydrate moieties found in the antibody, and / or the addition of one or more glycosylation sites not present in the antibody.

[0458] Antibody glycosylation is generally N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid other than proline) are recognition sequences for the enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide creates potential glycosylation sites. O-linked glycosylation refers to the attachment of one of galactosamine, galactose, or xylose to a hydroxy amino acid (most commonly serine or threonine), although 5-hydroxyproline or 5-hydroxylysine can also be used.

[0459] Addition of glycosylation sites to an antibody can be achieved by altering the amino acid sequence of the antibody or antigen-binding fragment such that it contains one or more of the above tripeptide sequences (for N-linked glycosylation sites). Alterations can also be made by adding or substituting one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).

[0460] Nucleic acid molecules encoding amino acid sequence variants of antibodies or antigen-binding fragments can be prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from natural sources (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of early prepared variants or non-variant forms of the antibody or its antigen-binding fragment.

[0461] In some embodiments, modifications that increase the serum half-life of the antibody or antigen-binding fragment are used. For example, salvage receptor binding epitopes can be incorporated into the antibody (especially antibody fragments), as described, for example, in U.S. Patent No. 5,739,277. As used herein, the term "salvage receptor binding epitope" refers to an epitope of the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.

[0462] Antibody Characteristics

[0463] In certain embodiments, the disclosed Notch4 antibodies or their Notch4-binding fragments selectively bind to Notch4, as they show detectable binding to Notch4 in a binding assay but do not show detectable binding to other Notch proteins (e.g., Notch1, Notch2, or Notch3) in a similar assay. In some embodiments, the Notch4 antibody or its Notch4-binding fragment is antagonistic.

[0464] In some embodiments, the Notch4 antibody binds to Notch4 with a dissociation constant (K D ) of ≤10 nM, ≤9 nM, ≤8 nM, ≤7 nM, ≤6 nM, ≤5 nM, ≤4 nM, ≤3 nM, ≤2 nM, or ≤1 nM. In some embodiments, the Notch4 antibody or its Notch4-binding fragment has a dissociation constant (K D ) between 300 pM and 10 nM (including the endpoints).

[0465] In one embodiment, K D is measured by a radiolabeled antigen-binding assay (radioimmunoassay, RIA) using the Fab version of the antibody or its antigen-binding fragment of interest and its antigen.

[0466] According to another embodiment, K D is measured using a surface plasmon resonance assay with immobilized antigen.

[0467] Vectors, Host Cells, and Recombinant Methods

[0468] Also provided are isolated nucleic acids encoding antibodies and antigen-binding fragments, vectors and host cells comprising such nucleic acids, and recombinant techniques for producing such antibodies.

[0469] For recombinant production of an antibody or antigen-binding fragment, a nucleic acid encoding the antibody or antigen-binding fragment can be isolated and inserted into a replicable vector for further cloning (amplifying the DNA) or for expression. DNA encoding a monoclonal antibody can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to the genes encoding the heavy and light chains of the antibody). A variety of vectors are available. Vector components typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, enhancer elements, a promoter, and a transcription termination sequence.

[0470] For large-scale production of antibodies, plasmids from yeast clones that produce the desired Notch4 antibody can be transfected into suitable host cells (such as insect host cells or mammalian host cells). Non-limiting examples of mammalian host cell lines that can be suitable for antibody production include NS0 murine myeloma cells, PER.C human cells, Chinese hamster ovary (CHO) cells, Sp2 / 0, and HEK293 cells.

[0471] Pharmaceutical compositions

[0472] In certain embodiments, the provided Notch4 antibody is incorporated, together with one or more pharmaceutically acceptable carriers, into a pharmaceutical composition suitable for administration to a subject. As used herein, "pharmaceutically acceptable carrier" refers to any of a variety of physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. Examples of pharmaceutically acceptable carriers include, but are not limited to, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof.

[0473] In some embodiments, the pharmaceutical composition comprises one or more tonicity agents or stabilizers. Non-limiting examples of such tonicity agents or stabilizers include sugars (e.g., sucrose), polyols (e.g., mannitol or sorbitol), and sodium chloride.

[0474] In some embodiments, the pharmaceutical composition comprises one or more bulking agents and / or lyoprotectants (e.g., mannitol or glycine), buffers (e.g., phosphate, acetate, or histidine buffers), surfactants (e.g., polysorbates), antioxidants (e.g., methionine), and / or metal ions or chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)).

[0475] In some embodiments, the pharmaceutical composition comprises one or more auxiliary substances such as wetting agents or emulsifiers, preservatives (such as benzyl alcohol) or buffering agents, which may enhance the shelf life and / or effectiveness of the Notch4 antibodies disclosed herein.

[0476] The pharmaceutical composition can be provided in any of a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The suitability of certain forms may depend on the intended mode of administration and therapeutic application.

[0477] In some embodiments, the pharmaceutical composition is in the form of an injectable or infusible solution.

[0478] The pharmaceutical composition is generally sterile and stable under the conditions of manufacture, transport and storage. The pharmaceutical composition can be formulated, for example, as a solution, microemulsion, dispersion, liposome or other ordered structure. In some embodiments, the pharmaceutical composition is formulated into a structure particularly suitable for high drug concentrations. For example, a sterile injectable solution can be prepared by incorporating a therapeutic agent (e.g., a Notch4 antibody) in a required amount into a suitable solvent having one or a combination of the ingredients listed herein, optionally followed by sterilization (e.g., filtration sterilization). Generally, a dispersion can be prepared by incorporating a therapeutic agent (e.g., a Notch4) antibody into a sterile vehicle containing a base dispersion medium and other ingredients (such as those additional ingredients mentioned herein). In the case of sterile powders for the preparation of sterile injectable solutions, examples of the preparation methods include vacuum drying and freeze drying to produce, for example, a powder of the therapeutic agent and any additional desired ingredients from its previously sterile filtered solution.

[0479] The proper fluidity of the solution can be maintained, for example, by using coatings such as lecithin, by maintaining certain particle sizes (e.g., in the case of dispersions) and / or by using surfactants. Prolonged absorption of the injectable composition can be achieved, for example, by including an agent that delays absorption (e.g., monostearate and / or gelatin) in the composition.

[0480] Production of human Notch4 antibodies

[0481] As further described in the Examples, the antibodies disclosed by the present invention are fully human antibodies identified using phage display technology (McCafferty et al., Nature 348:552-553

[1990] ), which can be used to generate human antibodies and antibody fragments in vitro from immunoglobulin variable (V) domain gene libraries from unimmunized donors. According to this technology, antibody V domain genes are cloned in-frame into the gene of the major or minor coat protein of filamentous phages such as M13 or fd, and are displayed as functional antibody fragments on the surface of phage particles. Since the filamentous particles contain single-stranded DNA copies of the phage genome, selection based on the functional properties of the antibody also results in the selection of the genes encoding the antibodies that display those properties. Thus, phages mimic some of the properties of B cells. Phage display can be carried out in various forms, which are reviewed, for example, in Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). Several sources of V gene segments can be used for phage display. Clackson et al., Nature, 352:624-628 (1991) isolated a variety of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. Libraries of V genes from unimmunized human donors can be constructed, and antibodies against a variety of antigens, including autoantigens, can be isolated substantially according to the techniques described by Marks et al., J. Mol. Biol. 222:581-597 (1991), or Griffith et al., EMBO J. 12:725-734 (1993). See also U.S. Patent Nos. 5,565,332 and 5,573,905.

[0482] For larger scale production of antibodies, plasmids from yeast clones that produce the desired Notch4 antibody can be transfected into suitable host cells (such as the host cells further described herein). (See the "Vectors, Host Cells, and Recombinant Methods" subsection.)

[0483] Therapeutic methods

[0484] The methods for treating airway inflammation and / or related conditions disclosed herein generally include administering a therapeutically effective amount of a Notch4 antibody or a Notch4-binding fragment thereof (or a pharmaceutical composition thereof) of the present disclosure to a mammalian subject (e.g., a human subject) in need thereof. In some embodiments, the airway inflammation is chronic airway inflammation. In some embodiments, the subject has or is at risk of having an airway inflammation-associated disorder such as asthma (e.g., severe eosinophilic asthma), chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), or bronchopulmonary dysplasia (BPD).

[0485] In some embodiments, the administering step includes systemic administration of a therapeutically effective amount, such as via an intravenous route, a subcutaneous route, or an intramuscular route.

[0486] The therapeutically effective amount can be administered via a single dose or via multiple doses (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten doses). When administered via multiple doses, any of a variety of suitable therapeutic regimens can be used, including administration at regular intervals (e.g., once every other day, once every three days, once every four days, once every five days, three times a week, twice a week, once a week, once every two weeks, once every three weeks, etc.).

[0487] The dosage regimen effective in the treatment method (e.g., the amount of each therapeutic agent, the relative timing of the therapies, etc.) can depend on the severity of the disease or disorder and the weight and general condition of the subject. For example, the therapeutically effective amount of a particular composition containing a therapeutic agent applied to a mammal (e.g., a human) can be determined by a person of ordinary skill in the art considering the individual differences in the age, weight, and condition of the mammal. The therapeutically effective amount and / or the optimal amount can also be determined empirically by those skilled in the art. In some embodiments, a dose between 0.4 mg / kg every 3 days and 20 mg / kg every 3 days is administered to the subject. The Notch4 antibody and its pharmaceutical composition can be administered via any of a variety of suitable routes, including but not limited to systemic routes such as parenteral (e.g., intranasal, intravenous, or subcutaneous) or enteral routes.

[0488] In some embodiments, administration of the antibody or its pharmaceutical composition results in improvement of one or more symptoms or signs of airway inflammation or associated disorders. For example, airway hyperreactivity can be improved due to administration of the antibody or pharmaceutical composition. For example, in a methacholine challenge test, the airway hyperreactivity measured from the peak calculated airway resistance can be 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, or 30% or less of the reference level airway hyperreactivity.

[0489] As another example, other indicators of inflammation, such as the presence of eosinophils, CD4+ Th2 cells, or neutrophils in bronchoalveolar lavage fluid; allergen-specific IgE responses or Th cell cytokine expression (e.g., IL-4 and / or IL-13) can be reduced due to administration of the antibody or pharmaceutical composition. The measurement of any one or any combination of these inflammation indicators can be reduced such that they are 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, or 30% or less of the measurement of the reference level. Examples

[0490] Example 1. Identification and generation of a humanized antibody specific for Notch4

[0491] Notch4 shares sequence homology (approximately 50% amino acid sequence identity) with each of Notch1, Notch2, and Notch3. This example describes the generation of a humanized Notch4 antibody specific for human Notch4 using phage display technology.

[0492] Recombinant human Notch4 was used as the antigen for positive selection, and recombinant human Notch1, Notch2, and Notch3 were used as antigens for negative selection of candidate antibodies. The recombinant Notch4 protein was biotinylated, and biotinylation was evaluated using SDS-PAGE electrophoresis and size exclusion chromatography (HPLC-SEC).

[0493] Expose the following phage libraries to biotinylated human Notch4: abrilumab (Lib1), mepolizumab (Lib2A), crenezumab (Lib3), necitumumab (Lib4), evolocumab (Lib6), and adalimumab (Lib8). Use a polyclonal phage ELISA assay to test the final selection output using the biotinylated Notch4 antigen and a control off-target protein. Subclone the final selection output into a yeast display vector and analyze it using flow cytometry to detect and sort the population that binds to human Notch4. Then, deplete the enriched Notch4-binding population against clones that bind to and / or compete with other Notch proteins (Notch1, Notch2, and Notch3) to retain clones that show specific binding activity to Notch4.

[0494] Then, perform an epitope competition assay on the enriched Notch4-binding population with Notch4 antibodies (4H1 or 3B11; see International Patent Publication No. WO 2022 / 081971) known to interfere with the binding of Notch4 to the Notch ligand Jagged1. Briefly, perform a "sandwich" assay using yeast displaying anti-Notch4 antibodies to capture Notch4; subsequently, incubate the yeast with 4H1 or 3B11 and detect 4H1 or 3B11 with a fluorescently labeled secondary reagent. Detection of the 4H1 or 3B11 binding signal leads to the identification and sorting of yeast-displayed clones that do not compete for the same epitope of either of the two monoclonal antibodies (4H1 or 3B11). Lack of detection of the binding signal on the same population incubated with 4H1 or 3B11 leads to the identification and sorting of yeast-displayed clones that compete for the same epitope of either of the two monoclonal antibodies.

[0495] Select the clones that successfully compete with 4H1 or 3B11 (these clones should therefore also be able to inhibit the binding of Notch4 to Jagged1) for further analysis as described in Example 2.

[0496] Example 2. Characterization of Notch4 Antibodies

[0497] Further characterize the candidate Notch4-specific antibodies identified in Example 1 to determine their sequences and binding parameters.

[0498] Materials and Methods

[0499] Sequencing.A combined parallel sequencing method was implemented, including Sanger-based screening and next-generation sequencing (NGS). For Sanger-based screening, up to 4x96 individual clones were sequenced, and unique clones were identified.

[0500] PacBio sequencing was performed on the yeast selection output DNA of each candidate antibody. Sequence clonotypes were identified by analyzing the PacBio output data. Sanger sequencing data was used to identify unique clusters of positive clones to ensure that not all Sanger clones belong to a single cluster.

[0501] Binding kinetics measurement. After expression and purification, surface plasmon resonance (SPR) was used to characterize the clones to determine binding kinetics and epitope binding activity. The HC30M chip (a polycarboxylate hydrogel surface with medium charge density and 30 nm coating thickness) was coated with anti-human Fc antibody (unlabeled goat anti-human IgG Fc from Southern Biotech) to capture the candidate antibodies. The candidate antibodies were diluted to 10 μg / mL in HEPES buffered saline Tris-EDTA (HBSTE) and exposed to the surface for 15 minutes. After stabilizing the baseline using HBSTE, Notch4 analyte was sequentially injected from the lowest concentration of 0.41 nM to the highest concentration of 300 nM. (Notch4 was diluted in HBSTE + 0.5 mg / mL bovine serum albumin (BSA) to obtain a 3-fold dilution series) To evaluate specificity, Notch2 was also used as an analyte and experiments were performed with a single injection of 100 nM in HBSTE + 0.5 mg / mL.

[0502] Results

[0503] The sequences and binding characteristics of nineteen candidate Notch4 antibodies were obtained. Table 1 of the present disclosure lists the V H 、V K and CDR sequences of representative candidate clones.

[0504] Table 3 (below) summarizes the binding parameters of the candidate clones from this experiment.

[0505] Table 3: Binding parameters of candidate Notch4 antibodies determined by surface plasmon resonance

[0506]

[0507] Example 3. Therapeutic effects of Notch4 antibodies in a humanized in vivo mouse model In a humanized mouse model of allergen-induced inflammation, the in vivo effects of five Notch4-specific antibodies identified in Example 1 and characterized in Example 2 were further analyzed.

[0508] Materials and methods:

[0509] Humanized mouse allergen-induced inflammation model

[0510] Peripheral blood mononuclear cell (PBMC)-humanized mice were obtained from “NSG” TM mice that carry the scid (severe combined immunodeficiency) mutation and a complete null allele of the interleukin-2 receptor common gamma chain (IL2rg 空 ), and are severely immunodeficient. Female NSG mice were injected with human peripheral blood mononuclear cells (hu-PBMC). Prior to allergen sensitization, the mice were maintained under specific pathogen-free (SPF) conditions for one week. TM To sensitize the mice, on days 1, 2, and 3 of the experiment, 50 μL of a solution of house dust mite (HDM) extract (available from Stallergenes Greer) (5 μg HDM per 100 μL) in phosphate-buffered saline was administered intranasally to the mice. On days 15, 16, and 17, the mice were challenged again by intranasal administration of the same concentration and volume of HDM.

[0511] At the end of the experiment, on day 18, the mice were euthanized with bronchoalveolar lavage (BAL) fluid, and lung tissues were collected for subsequent analysis.

[0512]

[0513] Antibody therapy

[0514] One of the test Notch4 antibody clones (C08-Lib3, iggrefmat 6-28, iggrefmat 3-11, H07-Lib3, or iggrefmat 6-16) was administered to the allergen-sensitized mice by intraperitoneal (i.p.) injection. A 100 μL solution of 10 μg / mL antibody was administered on days 1, 2, 3, 15, and 16 of the experiment. For comparison, a group of mice was administered 4H1, the previously described Notch4 antibody (see International Patent Publication No. WO 2022 / 081971). A control group was administered an isotype (IgG) control.

[0515] Methacholine challenge and airway hyperresponsiveness

[0516] On day 18 of the experiment, the mice were anesthetized and exposed to escalating concentrations (10 mg / mL, 20 mg / mL, and 40 mg / mL) of nebulized methacholine acetyl-β (Sigma-Aldrich) using a Buxco small animal ventilator (Data Sciences International). The relative peak airway resistance normalized to the saline baseline was calculated for each methacholine dose.

[0517] FACS analysis ​

[0518] Bronchoalveolar lavage fluid was analyzed by flow cytometry. By gating on CD3 + CD4 + Foxp3 - populations and assessing (1) intracellular expression of IL-4 and IL-13 and (2) CCR6 expression, the effector T (Teff) cell population was analyzed. By gating on CD3 + CD4 + Foxp3 + populations, Notch4 expression in regulatory T cells (Tregs) was analyzed.

[0519] Therapeutic effect of Notch4 antibody in an airway hyperreactivity model

[0520] To evaluate the ability of a novel Notch4-specific antibody to improve airway inflammation, humanized mice sensitized to house dust mite allergen were subjected to a methacholine challenge test. Figure 1 Airway hyperreactivity, as determined by relative peak airway resistance calculated at the highest dose of methacholine (40 mg / mL) tested, is shown for each treatment group. As Figure 1 can be seen, treatment with iggrefmat 6-28, H07-Lib3, or iggrefmat6-16 resulted in a significant reduction in airway hyperreactivity relative to that observed in the isotype control, and this reduction was comparable to or better than that observed with the 4H1 antibody.

[0521] Antibodies were generally well tolerated in mice, with most mouse groups showing no excess mortality after administration of the fully human Notch4 antibody. However, mice treated with C08-Lib3 showed a slightly reduced survival (60% survival at 20 days after antibody administration) relative to the other groups. (Data not shown.)

[0522] Effect of Notch4 antibody on inflammation

[0523] To evaluate the effect of treatment with a novel Notch4-specific antibody on inflammation, intracellular IL-4 and IL-13 expression was evaluated in T effector cells from bronchoalveolar lavage fluid (BAL). In response to asthma inflammation, IL-4 and IL-13 are expressed in Th2 cells. CCR6 (a chemokine receptor expressed on Th2 cells).

[0524] Figure 2A Representative flow cytometry plots of BAL fluid Teff cells stained for IL-4 and IL-13 from individual mice in each treatment group are shown; andFigure 2B and 2C separately show the average percentages of IL13-positive and IL4-positive cells among the Teff cells of each group. As Figure 2A shown, administration of the test antibodies appears to result in a decrease in the proportion of IL-4 + IL13 + cells, with H07-Lib3 showing a similar level of double-positive Teff cells as the 4H1 antibody. Treatment with three of the Notch4 antibodies (iggrefmat 3-11, H07-Lib3, and iggrefmat 6-16) resulted in a significant decrease in the proportion of IL-13+ cells relative to the isotype control ( Figure 2B ), and treatment with two of the Notch4 antibodies (H07-Lib3 and iggrefmat 6-16) resulted in a significant decrease in the proportion of IL-13+ cells relative to the isotype control ( Figure 2C ).

[0525] Figure 3A shows representative flow cytometry plots of BAL fluid Teff cells stained for CD4 and CCR6 from individual mice in each treatment group; and Figure 3B shows the average percentages of CCR6-positive cells among the Teff cells of each group. Treatment with three of the Notch4 antibodies (iggrefmat 3-11, H07-Lib3, and iggrefmat 6-16) resulted in a significant decrease in the proportion of CCR6+ cells relative to the isotype control ( Figure 2B ).

[0526] These results indicate that treatment with some of the Notch4 antibodies results in less inflammation and less attraction of Th2 cells to the lung tissue.

[0527] In vivo inhibition of Notch4 expression on Treg cells by Notch4 antibodies

[0528] Notch4 is expressed on Treg cells, and its signaling in Treg cells drives allergic inflammation induced by allergens or particulate pollutants (see, for example, International Patent Publication No. WO 2019 / 178488). To evaluate whether the novel Notch4-specific antibodies can block Notch4 expression on Treg cells, Notch4 expression on Treg cells from antibody-treated mice was evaluated by flow cytometry.

[0529] Figure 4A shows representative flow cytometry plots of individual mice in each treatment group; and Figure 4Bshows the mean percentage of Notch4-positive cells among the various groups of Treg cells. As Figure 4A and 4B shown, administration of any one of the five test antibodies reduced the percentage of Notch4-positive Treg cells relative to the isotype control, and the level of Notch4-positive Treg cells appeared comparable to that observed in mice treated with the 4H1 antibody. Figure 4C shows the mean percentage of Notch4-positive cells among the effector T (Teff) cells of the same treatment groups. In Teff cells, the percentage of Notch4-positive cells was very low and appeared similar in all groups.

[0530] These results demonstrate that three of the Notch4-specific antibodies (iggrefmat 6-28, H07-Lib3, and iggrefmat 6-16) showed in vivo therapeutic effects in a humanized mouse allergen model.

[0531] Example 4. In Vitro Evaluation of the Binding Specificity of H07-Lib3 to Notch4

[0532] The specificity of the Notch4-specific antibody clone H07-Lib3 was further evaluated using in vitro assays.

[0533] Materials and Methods:

[0534] Binding specificity measurement. The binding specificity of H07-Lib3 to Notch4 was evaluated by enzyme-linked immunosorbent assay (ELISA). Samples containing Notch3 or Notch4 protein or bovine serum albumin (BSA, control) were assayed by ELISA. Antibody-antigen binding was measured by absorbance at 450 nm light.

[0535] Epitope binning assay. The epitope specificity of H07-Lib3 against a commercial Notch4 monoclonal mouse antibody (MHN4-2, available from BioLegend) was evaluated by performing a competitive ELISA assay. The assay was performed in samples containing Notch4 (antigen) in the presence or absence of MHN4-2. Antibody-antigen binding was measured by absorbance at 450 nm.

[0536] Binding kinetics measurement.Surface plasmon resonance (SPR) was used to determine the binding kinetics of H07-Lib3. A CM5 chip was coated with anti-human polyclonal Fc antibody (Southern Biotech) to capture the candidate antibody. The candidate antibody was diluted in HEPES buffered saline with 0.005% surfactant P20 (HBS-P) and exposed to the surface for 20 minutes. The Notch4 analyte was injected at a concentration of 300 nM to determine the affinity of the candidate antibody. To evaluate specificity, Notch3 was also used as an analyte at 300 nM for the experiment.

[0537] Specificity of H07-Lib3 for Notch4

[0538] To determine the specificity of the Notch4-specific antibody clone H07-Lib3 for Notch4, an ELISA experiment was conducted. Figure 5A Binding of H07-Lib3 to Notch4, Notch3, and bovine serum albumin as measured by the average absorbance at 450 nm is shown. As Figure 5A can be seen.

[0539] Figure 5B Epitope classification of H07-Lib3 and a commercial Notch4 antibody (mouse Ab) is shown. Binding of H07-Lib3 to Notch4 was observed in both the absence and presence of the mouse Ab, as indicated by the increased average absorbance at 450 nm compared to samples without antibody.

[0540] These results demonstrate that H07-Lib3 binds to Notch4 but not to Notch3, and that the mouse antibody MHN4-2 does not compete with H07-Lib3 for binding to Notch4.

[0541] Binding kinetics of H07-Lib3 to Notch4

[0542] Table 4 (below) summarizes the binding parameters of H07-Lib3 as determined by surface plasmon resonance (SPR).

[0543] Table 4: Binding parameters of candidate Notch4 antibody H07-Lib3 determined by surface plasmon resonance

[0544]

[0545] Example 5. Characterization of H07-Lib3 stability

[0546] To evaluate the developability of the candidate Notch4-specific antibody H07-Lib3, the stability of H07-Lib3 was evaluated.

[0547] Materials and methods

[0548] Melting temperature measurement. The melting temperatures of H07-Lib3 and two control monoclonal antibodies (control mAb1 and control mAb2) were determined by measuring the intrinsic fluorescence emission of certain amino acids (such as tryptophan, tyrosine, and phenylalanine) in each sample after excitation with a 266-nm laser. The second inflection point of the plotted melting curve was determined to find the melting temperature (Tm2).

[0549] Aggregation temperature measurement. The aggregation temperature (the temperature at which protein aggregation begins) of H07-Lib3 and two control monoclonal antibodies (control mAb1 and control mAb2) was determined by measuring the intrinsic fluorescence of aromatic amino acids and static light scattering (SLS). For lower concentrations and smaller aggregate formation, the intrinsic fluorescence of 266-nm light (Tagg 266 ) was measured, and for higher concentrations and larger aggregate formation, the SLS of 473-nm light (Tagg 473 ) was measured.

[0550] Results

[0551] The melting temperatures and aggregation temperatures of H07-Lib3, control mAb1, and control mAb2 were obtained. Table 5 (below) summarizes the temperature parameters of the antibodies from this experiment.

[0552] Table 5: Melting temperature and aggregation temperature of H07-Lib3 and control antibodies

[0553] Antibody cloning <![CDATA[Tm2]]> <![CDATA[Tagg 266 > <![CDATA[Tagg 473 > H07-Lib3 80.0±0.2℃ 83.7±0.6℃ 81.9±2.1℃ Control mAb1 69.9±0.2℃ 71.1±0.2℃ 69.3±3.5℃ Control mAb2 68.2±0.2℃ 71.2±0.2℃ 70.9±0.7℃

[0554] These results indicate that the Notch4-specific antibody H07-Lib3 has high thermal stability and an elevated aggregation temperature compared to the control antibodies.

[0555] Example 6. Evaluation of the polyreactivity of H07-Lib3

[0556] To further evaluate the developability of the candidate Notch4-specific antibody H07-Lib3, the non-specific polyreactivity of H07-Lib3 was further evaluated.

[0557] Materials and methods:

[0558] Polyreactivity assay.The non-specific reactivity of H07-Lib3 against cardiolipin, keyhole limpet hemocyanin (KLH), lipopolysaccharide (LPS), single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), insulin, and bovine serum albumin (BSA) was evaluated. Additional therapeutic antibodies (bococizumab, efalizumab, gantenerumab, ibalizumab, lenzilumab, and panitumumab) were included in the experiment for comparison. The percentage reactivity of each antibody was normalized to the highest reactivity value for each probe. For all probes, an acceptable polyreactivity threshold was set at 30%.

[0559] Results

[0560] The polyreactivity of H07-Lib3 against cardiolipin, KLH, LPS, ssDNA, dsDNA, insulin, and BSA was determined. Figure 6A-6G The mean percentage reactivity of H07-Lib3 and commercial antibodies against cardiolipin, KLH, LPS, ssDNA, dsDNA, insulin, and BSA, respectively, is shown. As Figure 6A-6G shown, H07-Lib3 showed a reactivity of less than 30% against each of the above antigens.

[0561] These results demonstrate that H07-Lib3 does not react with any of the seven evaluated probes (cardiolipin, KLH, LPS, dsDNA, ssDNA, insulin, and BSA).

[0562] Equivalents

[0563] For all purposes, the entire disclosure of each of the patent documents and scientific articles mentioned herein is incorporated by reference.

[0564] Without departing from the spirit or essential characteristics of the present disclosure, the present disclosure may be embodied in other specific forms. Accordingly, the foregoing embodiments are to be considered in all respects illustrative rather than limiting of the disclosure described herein. The various structural elements of the different embodiments and the various disclosed method steps may be utilized in various combinations and permutations, and all such variations will be regarded as being within the form of the present disclosure. Accordingly, the scope of the present disclosure is indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

1. An antibody or antigen-binding fragment thereof that can bind to Notch4, comprising: (a)(i) Heavy chain variable domain (V H ), which contains complementarity determining regions: CDR-H1, which comprises the amino acid sequence of SEQ ID NO:2, or a sequence that differs from this sequence by 1 or 2 amino acids, CDR-H2, which comprises the amino acid sequence of SEQ ID NO:3, or a sequence that differs from this sequence by 1 or 2 amino acids, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:4, or a sequence that differs from this sequence by 1 or 2 amino acids; and (ii) a light chain variable domain (VK), which comprises complementarity-determining regions: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:6, or a sequence that differs from this sequence by 1 or 2 amino acids, CDR-L2, which comprises the amino acid sequence of SEQ ID NO:7, or a sequence that differs from this sequence by 1 or 2 amino acids, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:8, or a sequence that differs from this sequence by 1 or 2 amino acids; (b)(i)V H , which comprises complementarity determining regions: CDR-H1, which comprises the amino acid sequence of SEQ ID NO:10, or a sequence that differs from this sequence by 1 or 2 amino acids, CDR-H2, which comprises the amino acid sequence of SEQ ID NO:11, or a sequence that differs from this sequence by 1 or 2 amino acids, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:12, or a sequence that differs from this sequence by 1 or 2 amino acids; and (ii)V K , which comprises complementarity determining regions: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:14, or a sequence that differs from this sequence by 1 or 2 amino acids, CDR-L2, which comprises the amino acid sequence of SEQ ID NO:15, or a sequence that differs from this sequence by 1 or 2 amino acids, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:16, or a sequence that differs from this sequence by 1 or 2 amino acids; (c)(i)V H , which comprises complementarity determining regions: CDR-H1, which comprises the amino acid sequence of SEQ ID NO:18, or a sequence that differs from this sequence by 1 or 2 amino acids, CDR-H2, which comprises the amino acid sequence of SEQ ID NO:19, or a sequence that differs from this sequence by 1 or 2 amino acids, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:20, or a sequence that differs from this sequence by 1 or 2 amino acids; and (ii)V K , which comprises complementarity-determining regions: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:22, or a sequence that differs from this sequence by 1 or 2 amino acids, CDR-L2, which comprises the amino acid sequence of SEQ ID NO:23, or a sequence that differs from this sequence by 1 or 2 amino acids, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:24, or a sequence that differs from this sequence by 1 or 2 amino acids; (d)(i)V H , which comprises complementarity determining regions: CDR-H1, which comprises the amino acid sequence of SEQ ID NO:26, or a sequence that differs from this sequence by 1 or 2 amino acids, CDR-H2, which comprises the amino acid sequence of SEQ ID NO:27, or a sequence that differs from this sequence by 1 or 2 amino acids, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:28, or a sequence that differs from this sequence by 1 or 2 amino acids; and (ii)V K , which comprises complementarity determining regions: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:30, or a sequence that differs from this sequence by 1 or 2 amino acids, CDR-L2, which comprises the amino acid sequence of SEQ ID NO:31, or a sequence that differs from this sequence by 1 or 2 amino acids, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:32, or a sequence that differs from this sequence by 1 or 2 amino acids; (e)(i)V H , which comprises complementarity determining regions: CDR-H1, which comprises the amino acid sequence of SEQ ID NO:34, or a sequence that differs from this sequence by 1 or 2 amino acids, CDR-H2, which comprises the amino acid sequence of SEQ ID NO:35, or a sequence that differs from this sequence by 1 or 2 amino acids, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:36, or a sequence that differs from this sequence by 1 or 2 amino acids; and (ii)V K , which comprises complementarity determining regions: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:38, or a sequence that differs from this sequence by 1 or 2 amino acids, CDR-L2, which comprises the amino acid sequence of SEQ ID NO:39, or a sequence that differs from this sequence by 1 or 2 amino acids, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:40, or a sequence that differs from this sequence by 1 or 2 amino acids; (f)(i)V H , which comprises complementarity determining regions: CDR-H1, which comprises the amino acid sequence of SEQ ID NO:42, or a sequence that differs from this sequence by 1 or 2 amino acids, CDR-H2, which comprises the amino acid sequence of SEQ ID NO:43, or a sequence that differs from this sequence by 1 or 2 amino acids, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:44, or a sequence that differs from this sequence by 1 or 2 amino acids; and (ii)V K , which comprises complementarity-determining regions: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:46, or a sequence that differs from this sequence by 1 or 2 amino acids, CDR-L2, which comprises the amino acid sequence of SEQ ID NO:47, or a sequence that differs from this sequence by 1 or 2 amino acids, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:48, or a sequence that differs from this sequence by 1 or 2 amino acids; (g)(i)V H , which comprises complementarity determining regions: CDR-H1, which comprises the amino acid sequence of SEQ ID NO:50, or a sequence that differs from this sequence by 1 or 2 amino acids, CDR-H2, which comprises the amino acid sequence of SEQ ID NO:51, or a sequence that differs from this sequence by 1 or 2 amino acids, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:52, or a sequence that differs from this sequence by 1 or 2 amino acids; and (ii)V K , which comprises complementarity determining regions: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:54, or a sequence that differs from this sequence by 1 or 2 amino acids, CDR-L2, which comprises the amino acid sequence of SEQ ID NO:55, or a sequence that differs from this sequence by 1 or 2 amino acids, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:56, or a sequence that differs from this sequence by 1 or 2 amino acids; (h)(i)V H , which comprises complementarity determining regions: CDR-H1, which comprises the amino acid sequence of SEQ ID NO:58, or a sequence that differs from this sequence by 1 or 2 amino acids, CDR-H2, which comprises the amino acid sequence of SEQ ID NO:59, or a sequence that differs from this sequence by 1 or 2 amino acids, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:60, or a sequence that differs from this sequence by 1 or 2 amino acids; and (ii)V K , which comprises complementarity determining regions: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:62, or a sequence that differs from this sequence by 1 or 2 amino acids, CDR-L2, which comprises the amino acid sequence of SEQ ID NO:63, or a sequence that differs from this sequence by 1 or 2 amino acids, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:64, or a sequence that differs from this sequence by 1 or 2 amino acids; (i)(i) which comprises complementarity-determining regions: CDR-H1, which comprises the amino acid sequence of SEQ ID NO:66, or a sequence that differs from this sequence by 1 or 2 amino acids, CDR-H2, which comprises the amino acid sequence of SEQ ID NO:67, or a sequence that differs from this sequence by 1 or 2 amino acids, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:68, or a sequence that differs from this sequence by 1 or 2 amino acids; and (ii)V K , which comprises complementarity determining regions: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:70, or a sequence that differs from this sequence by 1 or 2 amino acids, CDR-L2, which comprises the amino acid sequence of SEQ ID NO:71, or a sequence that differs from this sequence by 1 or 2 amino acids, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:72, or a sequence that differs from this sequence by 1 or 2 amino acids; (j)(i)V H , which comprises complementarity determining regions: CDR-H1, which comprises the amino acid sequence of SEQ ID NO:74, or a sequence that differs from this sequence by 1 or 2 amino acids, CDR-H2, which comprises the amino acid sequence of SEQ ID NO:75, or a sequence that differs from this sequence by 1 or 2 amino acids, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:76, or a sequence that differs from this sequence by 1 or 2 amino acids; and (ii)V K , which comprises complementarity determining regions: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:78, or a sequence that differs from this sequence by 1 or 2 amino acids, CDR-L2, which comprises the amino acid sequence of SEQ ID NO:79, or a sequence that differs from this sequence by 1 or 2 amino acids, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:80, or a sequence that differs from this sequence by 1 or 2 amino acids; (k)(i)V H , which comprises complementarity determining regions: CDR-H1, which comprises the amino acid sequence of SEQ ID NO:82, or a sequence that differs from this sequence by 1 or 2 amino acids, CDR-H2, which comprises the amino acid sequence of SEQ ID NO:83, or a sequence that differs from this sequence by 1 or 2 amino acids, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:84, or a sequence that differs from this sequence by 1 or 2 amino acids; and (ii)V K , which comprises complementarity determining regions: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:86, or a sequence that differs from this sequence by 1 or 2 amino acids, CDR-L2, which comprises the amino acid sequence of SEQ ID NO:87, or a sequence that differs from this sequence by 1 or 2 amino acids, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:88, or a sequence that differs from this sequence by 1 or 2 amino acids; (l)(i)V H , which comprises complementarity determining regions: CDR-H1, which comprises the amino acid sequence of SEQ ID NO:90, or a sequence that differs from this sequence by 1 or 2 amino acids, CDR-H2, which comprises the amino acid sequence of SEQ ID NO:91, or a sequence that differs from this sequence by 1 or 2 amino acids, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:92, or a sequence that differs from this sequence by 1 or 2 amino acids; and (ii)V K , which comprises complementarity-determining regions: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:94, or a sequence that differs from this sequence by 1 or 2 amino acids, CDR-L2, which comprises the amino acid sequence of SEQ ID NO:95, or a sequence that differs from this sequence by 1 or 2 amino acids, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:96, or a sequence that differs from this sequence by 1 or 2 amino acids; (m)(i)V H , which comprises complementarity determining regions: CDR-H1, which comprises the amino acid sequence of SEQ ID NO:98, or a sequence that differs from this sequence by 1 or 2 amino acids, CDR-H2, which comprises the amino acid sequence of SEQ ID NO:99, or a sequence that differs from this sequence by 1 or 2 amino acids, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:100, or a sequence that differs from this sequence by 1 or 2 amino acids; and (ii)V K , which comprises complementarity determining regions: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:102, or a sequence that differs from this sequence by 1 or 2 amino acids, CDR-L2, which comprises the amino acid sequence of SEQ ID NO:103, or a sequence that differs from this sequence by 1 or 2 amino acids, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:104, or a sequence that differs from this sequence by 1 or 2 amino acids; (n)(i)V H , which comprises complementarity determining regions: CDR-H1, which comprises the amino acid sequence of SEQ ID NO:106, or a sequence that differs from this sequence by 1 or 2 amino acids, CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 107, or a sequence that differs from this sequence by 1 or 2 amino acids, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 108, or a sequence that differs from this sequence by 1 or 2 amino acids; and (ii)V K , which comprises complementarity determining regions: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 110, or a sequence that differs from this sequence by 1 or 2 amino acids, CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 111, or a sequence that differs from this sequence by 1 or 2 amino acids, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 112, or a sequence that differs from this sequence by 1 or 2 amino acids; (o)(i)V H , which comprises complementarity determining regions: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 114, or a sequence that differs from this sequence by 1 or 2 amino acids, CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 115, or a sequence that differs from this sequence by 1 or 2 amino acids, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 116, or a sequence that differs from this sequence by 1 or 2 amino acids; and (ii)V K , which comprises complementarity determining regions: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 118, or a sequence that differs from this sequence by 1 or 2 amino acids, CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 119, or a sequence that differs from this sequence by 1 or 2 amino acids, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 120, or a sequence that differs from this sequence by 1 or 2 amino acids; (p)(i)V H , which comprises complementarity determining regions: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 122, or a sequence that differs from this sequence by 1 or 2 amino acids, CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 123, or a sequence that differs from this sequence by 1 or 2 amino acids, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 124, or a sequence that differs from this sequence by 1 or 2 amino acids; and (ii)V K , which comprises complementarity determining regions: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 126, or a sequence that differs from this sequence by 1 or 2 amino acids, CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 127, or a sequence that differs from this sequence by 1 or 2 amino acids, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 128, or a sequence that differs from this sequence by 1 or 2 amino acids; or (q)(i)V H , which comprises complementarity determining regions: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 130, or a sequence that differs from this sequence by 1 or 2 amino acids, CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 131, or a sequence that differs from this sequence by 1 or 2 amino acids, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 132, or a sequence that differs from this sequence by 1 or 2 amino acids; and (ii)V K , which comprises complementarity determining regions: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:134, or a sequence that differs from this sequence by 1 or 2 amino acids, CDR-L2, which comprises the amino acid sequence of SEQ ID NO:135, or a sequence that differs from this sequence by 1 or 2 amino acids, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:136, or a sequence that differs from this sequence by 1 or 2 amino acids.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprise amino acid sequences that, taken together, differ from the following sequences by no more than two amino acid residues: (a) SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, respectively; (b) SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, respectively; (c) SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, respectively; (d) SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, respectively; (e) SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, respectively; (f) SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, respectively; (g) SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, respectively; (h) SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, respectively; (i) SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, respectively; (j) are SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, respectively; (k) are SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, respectively; (l) are SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, respectively; (m) are SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, respectively; (n) are SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, respectively; (o) are SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, respectively; (p) are SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, respectively; or (q) are SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, respectively.

3. The antibody or antigen-binding fragment thereof according to claim 2, comprising: (a)(i)V H , which comprises complementarity determining regions: CDR-H1, which comprises the amino acid sequence of SEQ ID NO:2, CDR-H2, which comprises the amino acid sequence of SEQ ID NO:3, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:4; and (ii)V K , which comprises complementarity determining regions: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:6, CDR-L2, which comprises the amino acid sequence of SEQ ID NO:7, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:8; (b)(i)V H , which comprises complementarity determining regions: CDR-H1, which comprises the amino acid sequence of SEQ ID NO:10, CDR-H2, which comprises the amino acid sequence of SEQ ID NO:11, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:12; and (ii)V K , which comprises complementarity determining regions: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:14, CDR-L2, which comprises the amino acid sequence of SEQ ID NO:15, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:16; (c)(i)V H , which comprises complementarity determining regions: CDR-H1, which comprises the amino acid sequence of SEQ ID NO:18, CDR-H2, which comprises the amino acid sequence of SEQ ID NO:19, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:20; and (ii)V K , which comprises complementarity determining regions: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:22, CDR-L2, which comprises the amino acid sequence of SEQ ID NO:23, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:24; (d)(i)V H , which comprises complementarity determining regions: CDR-H1, which comprises the amino acid sequence of SEQ ID NO:26, CDR-H2, which comprises the amino acid sequence of SEQ ID NO:27, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:28; and (ii)V K , which comprises complementarity-determining regions: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:30, CDR-L2, which comprises the amino acid sequence of SEQ ID NO:31, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:32; (e)(i)V H , which comprises complementarity-determining regions: CDR-H1, which comprises the amino acid sequence of SEQ ID NO:34, CDR-H2, which comprises the amino acid sequence of SEQ ID NO:35, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:36; and (ii)V K , which comprises complementarity determining regions: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:38, CDR-L2, which comprises the amino acid sequence of SEQ ID NO:39, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:40; (f)(i)V H , which comprises complementarity determining regions: CDR-H1, which comprises the amino acid sequence of SEQ ID NO:42, CDR-H2, which comprises the amino acid sequence of SEQ ID NO:43, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:44; and (ii)V K , which comprises complementarity determining regions: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:46, CDR-L2, which comprises the amino acid sequence of SEQ ID NO:47, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:48; (g)(i)V H , which comprises complementarity determining regions: CDR-H1, which comprises the amino acid sequence of SEQ ID NO:50, CDR-H2, which comprises the amino acid sequence of SEQ ID NO:51, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:52; and (ii)V K , which comprises complementarity-determining regions: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:54, CDR-L2, which comprises the amino acid sequence of SEQ ID NO:55, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:56; (h)(i)V H , which comprises complementarity determining regions: CDR-H1, which comprises the amino acid sequence of SEQ ID NO:58, CDR-H2, which comprises the amino acid sequence of SEQ ID NO:59, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:60; and (ii)V K , which contains complementarity determining regions: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:62, CDR-L2, which comprises the amino acid sequence of SEQ ID NO:63, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:64; (i)(i)V H , which comprises complementarity determining regions: CDR-H1, which comprises the amino acid sequence of SEQ ID NO:66, CDR-H2, which comprises the amino acid sequence of SEQ ID NO:67, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:68; and (ii)V K , which comprises complementarity determining regions: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:70, CDR-L2, which comprises the amino acid sequence of SEQ ID NO:71, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:72; (j)(i)V H , which comprises complementarity determining regions: CDR-H1, which comprises the amino acid sequence of SEQ ID NO:74, CDR-H2, which comprises the amino acid sequence of SEQ ID NO:75, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:76; and (ii)V K , which comprises complementarity determining regions: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:78, CDR-L2, which comprises the amino acid sequence of SEQ ID NO:79, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:80; (k)(i)V H , which comprises complementarity determining regions: CDR-H1, which comprises the amino acid sequence of SEQ ID NO:82, CDR-H2, which comprises the amino acid sequence of SEQ ID NO:83, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:84; and (ii)V K , which comprises complementarity determining regions: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:86, CDR-L2, which comprises the amino acid sequence of SEQ ID NO:87, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:88; (l)(i)V H , which comprises complementarity determining regions: CDR-H1, which comprises the amino acid sequence of SEQ ID NO:90, CDR-H2, which comprises the amino acid sequence of SEQ ID NO:91, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:92; and (ii)V K , which comprises complementarity determining regions: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:94, CDR-L2, which comprises the amino acid sequence of SEQ ID NO:95, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:96; (m)(i)V H , which comprises complementarity determining regions: CDR-H1, which comprises the amino acid sequence of SEQ ID NO:98, CDR-H2, which comprises the amino acid sequence of SEQ ID NO:99, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:100; and (ii)V K , which comprises complementarity determining regions: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:102, CDR-L2, which comprises the amino acid sequence of SEQ ID NO:103, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:104; (n)(i)V H , which comprises complementarity determining regions: CDR-H1, which comprises the amino acid sequence of SEQ ID NO:106, CDR-H2, which comprises the amino acid sequence of SEQ ID NO:107, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:108; and (ii)V K , which contains complementarity determining regions: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:110, CDR-L2, which comprises the amino acid sequence of SEQ ID NO:111, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:112; (o)(i)V H , which comprises complementarity determining regions: CDR-H1, which comprises the amino acid sequence of SEQ ID NO:114, CDR-H2, which comprises the amino acid sequence of SEQ ID NO:115, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:116; and (ii)V K , which comprises complementarity determining regions: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:118, CDR-L2, which comprises the amino acid sequence of SEQ ID NO:119, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:120; (p)(i)V H , which comprises complementarity determining regions: CDR-H1, which comprises the amino acid sequence of SEQ ID NO:122, CDR-H2, which comprises the amino acid sequence of SEQ ID NO:123, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:124; and (ii)V K , which comprises complementarity determining regions: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:126, CDR-L2, which comprises the amino acid sequence of SEQ ID NO:127, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 128; or (q)(i) V H , which comprises complementarity determining regions: CDR-H1, which comprises the amino acid sequence of SEQ ID NO:130, CDR-H2, which comprises the amino acid sequence of SEQ ID NO:131, and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:132; and (ii)V K , which comprises complementarity-determining regions: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:134, CDR-L2, which comprises the amino acid sequence of SEQ ID NO:135, and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:

136.

4. The antibody or antigen-binding fragment thereof according to claim 3, which comprises: (i)V H , which comprises complementarity determining regions: CDR-H1, which comprises the amino acid sequence of SEQ ID NO:2; CDR-H2, which comprises the amino acid sequence of SEQ ID NO:3; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:4; and (ii)V K , which comprises complementarity determining regions: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:6; CDR-L2, which comprises the amino acid sequence of SEQ ID NO:7; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:

8.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein: Said V H comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:1; and The V K comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:

5.

6. The antibody or antigen-binding fragment thereof according to claim 5, wherein: The said V H comprises the amino acid sequence of SEQ ID NO:1; and The said V K contains the amino acid sequence of SEQ ID NO:

5.

7. The antibody or antigen-binding fragment thereof according to claim 3, comprising: (i)V H , which comprises complementarity determining regions: CDR-H1, which comprises the amino acid sequence of SEQ ID NO:10; CDR-H2, which comprises the amino acid sequence of SEQ ID NO:11; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:12; and (ii)V K , which comprises complementarity-determining regions: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:14; CDR-L2, which comprises the amino acid sequence of SEQ ID NO:15; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:

16.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein: Said V H comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:9; and The V K contains an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:

13.

9. The antibody or antigen-binding fragment thereof according to claim 8, wherein: The said V H comprises the amino acid sequence of SEQ ID NO:9; and The said V K comprises the amino acid sequence of SEQ ID NO:

13.

10. The antibody or antigen-binding fragment thereof according to claim 3, comprising: (i)V H , which comprises complementarity-determining regions: CDR-H1, which comprises the amino acid sequence of SEQ ID NO:18; CDR-H2, which comprises the amino acid sequence of SEQ ID NO:19; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:20; and (ii)V K , which comprises complementarity determining regions: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:22; CDR-L2, which comprises the amino acid sequence of SEQ ID NO:23; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:

24.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein: The V H comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:17; and The V K comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:

21.

12. The antibody or antigen-binding fragment thereof according to claim 11, wherein: The said V H comprises the amino acid sequence of SEQ ID NO: 17; and The V K comprises the amino acid sequence of SEQ ID NO:

21.

13. The antibody or antigen-binding fragment thereof according to claim 3, comprising: (i)V H , which comprises complementarity determining regions: CDR-H1, which comprises the amino acid sequence of SEQ ID NO:34; CDR-H2, which comprises the amino acid sequence of SEQ ID NO:35; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:36; and (ii)V K , which comprises complementarity determining regions: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:38; CDR-L2, which comprises the amino acid sequence of SEQ ID NO:39; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:

40.

14. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein: The V H comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 33; and The V K comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:

37.

15. The antibody or antigen-binding fragment thereof according to claim 14, wherein: The V H comprises the amino acid sequence of SEQ ID NO: 33; and The V K contains the amino acid sequence of SEQ ID NO:

37.

16. The antibody or antigen-binding fragment thereof according to claim 3, comprising: (i)V H , which comprises complementarity-determining regions: CDR-H1, which comprises the amino acid sequence of SEQ ID NO:98; CDR-H2, which comprises the amino acid sequence of SEQ ID NO:99; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO:100; and (ii)V K , which comprises complementarity determining regions: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:102; CDR-L2, which comprises the amino acid sequence of SEQ ID NO:103; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:

104.

17. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein: The V H comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:97; and The V K comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:

101.

18. The antibody or antigen-binding fragment thereof according to claim 16, wherein: The V H comprises the amino acid sequence of SEQ ID NO:97; and The said V K comprises the amino acid sequence of SEQ ID NO:

101.

19. The antibody or antigen-binding fragment thereof according to claim 1, wherein (a) said V H comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1, and said V K comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 5; (b) said V H comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:9, and said V K comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:13; (c) said V H comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 17, and said V K comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 21; (d) said V H comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 25, and said V K comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 29; (e) said V H comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 33, and said V K comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 37; (f) said V H comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 41, and said V K comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 45; (g) said V H comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 49, and said V K comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 53; (h) said V H comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 57, and said V K comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 61; (i) said V H comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 65, and said V K comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 69; (j) said V H comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:73, and said V K comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:77; (k) said V H comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 81, and said V K comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 85; (l) said V H comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:89, and said V K comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:93; (m) said V H comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 97, and said V K comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 101; (n) said V H comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 105, and said V K comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 109; (o) said V H comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 113, and said V K comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 117; (p) said V H comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 121, and said V K comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 125; or (q) said V H comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 129, and said V K comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

133.

20. The antibody or antigen-binding fragment thereof according to claim 1, wherein (a) said V H comprises the amino acid sequence of SEQ ID NO:1, and said V K comprises the amino acid sequence of SEQ ID NO:5; (b) said V H comprises the amino acid sequence of SEQ ID NO:9, and said V K comprises the amino acid sequence of SEQ ID NO:13; (c) said V H comprises the amino acid sequence of SEQ ID NO: 17, and said V K comprises the amino acid sequence of SEQ ID NO: 21; (d) said V H comprises the amino acid sequence of SEQ ID NO: 25, and said V K comprises the amino acid sequence of SEQ ID NO: 29; (e) said V H comprises the amino acid sequence of SEQ ID NO: 33, and said V K comprises the amino acid sequence of SEQ ID NO: 37; (f) said V H comprises the amino acid sequence of SEQ ID NO: 41, and said V K comprises the amino acid sequence of SEQ ID NO: 45; (g) said V H comprises the amino acid sequence of SEQ ID NO: 49, and said V K comprises the amino acid sequence of SEQ ID NO: 53; (h) said V H comprises the amino acid sequence of SEQ ID NO:57, and said V K comprises the amino acid sequence of SEQ ID NO:61; (i) said V H comprises the amino acid sequence of SEQ ID NO: 65, and said V K comprises the amino acid sequence of SEQ ID NO: 69; (j) said V H comprises the amino acid sequence of SEQ ID NO:73, and said V K comprises the amino acid sequence of SEQ ID NO:77; (k) said V H comprises the amino acid sequence of SEQ ID NO:81, and said V K comprises the amino acid sequence of SEQ ID NO:85; (l) said V H comprises the amino acid sequence of SEQ ID NO:89, and said V K comprises the amino acid sequence of SEQ ID NO:93; (m) said V H comprises the amino acid sequence of SEQ ID NO: 97, and said V K comprises the amino acid sequence of SEQ ID NO: 101; (n) the V H comprises the amino acid sequence of SEQ ID NO: 105, and the V K comprises the amino acid sequence of SEQ ID NO: 109; (o) said V H comprises the amino acid sequence of SEQ ID NO: 113, and said V K comprises the amino acid sequence of SEQ ID NO: 117; (p) said V H comprises the amino acid sequence of SEQ ID NO: 121, and said V K comprises the amino acid sequence of SEQ ID NO: 125; or (q) said V H comprises the amino acid sequence of SEQ ID NO: 129, and said V K comprises the amino acid sequence of SEQ ID NO:

133.

21. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof is capable of binding to human Notch4 with a K D value of 10 nM or less.

22. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody is a human antibody.

23. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen-binding fragment comprises an antibody heavy chain constant region.

24. The antibody or antigen-binding fragment thereof according to claim 22, wherein the heavy chain constant region is a human IgG heavy chain constant region.

25. The antibody or antigen-binding fragment thereof according to claim 23, wherein the antibody heavy chain constant region is a human IgG4 heavy chain constant region.

26. An isolated nucleic acid encoding the heavy chain variable domain and / or the light chain variable domain of the antibody or antigen-binding fragment thereof according to any one of the preceding claims.

27. An expression vector comprising the isolated nucleic acid according to claim 25.

28. A host cell comprising the molecule of the isolated nucleic acid according to claim 25 or the expression vector according to claim 26.

29. A composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-24 and a pharmaceutically acceptable carrier.

30. A method for treating, ameliorating or preventing airway inflammation in a subject, which comprises the step of administering to the subject an effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1-24.

31. The method according to claim 29, wherein the subject is a mammal.

32. The method according to claim 30, wherein the subject is a human.

33. The method according to any one of claims 29-32, wherein the subject is diagnosed with an airway inflammation-related disorder or is at risk of developing an airway inflammation-related disorder.

34. The method according to claim 32, wherein the airway inflammation-related disorder is selected from the group consisting of asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF) or bronchopulmonary dysplasia (BPD).

35. The method according to any one of claims 29-33, wherein the step of administering comprises administering by a systemic route.

36. The method according to claim 34, wherein the systemic route is selected from intravenous, intramuscular or subcutaneous.

37. A method for detecting the presence of Notch4 or a fragment thereof in a sample, which comprises contacting the sample with the antibody or antigen-binding fragment thereof according to any one of claims 1-24 and detecting the presence of a complex between the antibody or antigen-binding fragment and Notch4, wherein the detection of the complex indicates the presence of Notch4.

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