NOTCH4 antibodies, compositions and methods for treating airway inflammation

Abstract

Antibodies specific for Notch4 and related methods for treating conditions such as airway inflammation. Also provided are isolated nucleic acids encoding the heavy chain variable domain and / or light chain variable domain of the antibody or its antigen-binding fragment disclosed herein, as well as expression vectors comprising such isolated nucleic acids, and host cells comprising such isolated nucleic acids or expression vectors. Also provided are compositions comprising the Notch4 antibody or its Notch-binding fragment disclosed herein and a pharmaceutically acceptable carrier.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 378,479 (filed October 5, 2022), the contents of which are incorporated herein by reference in their entirety and for all purposes.

[0002] (Sequence Listing) This application references a Sequence Listing which has been provided electronically in XML format and is incorporated herein by reference in its entirety. The XML file (created October 4, 2023) is named ALC-050WO_ST26.xml and is 133kb in size. [Background technology]

[0003] (background) Chronic airway inflammation is a common feature of many conditions, including asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), and bronchopulmonary dysplasia (BPD). Current strategies for treating these chronic conditions largely focus on systemically inhibiting pro-inflammatory pathways. One of the challenges of such strategies is effectively halting part of the immune response without rendering patients vulnerable to opportunistic pathogens or crippling them with side effects. However, due to the diverse profiles of inflammatory responses, no single strategy has proven to be broadly effective, and response rates for existing therapies are generally unsatisfactory. Summary of the Invention [Means for solving the problem]

[0004] There remains a need for improved targeted therapies for airway inflammation and related disorders.

[0005] (overview) In one aspect, an antibody or antigen-binding fragment thereof capable of binding to Notch4 (also referred to herein as a Notch4 antibody and a Notch4-binding fragment thereof, respectively) is provided. For example, in some embodiments, an antibody or antigen-binding fragment thereof capable of binding to Notch4 is provided, wherein the antibody or antigen-binding fragment thereof comprises:

[0006] (a)(i) a complementarity-determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 2 or a sequence which differs therefrom by one or two amino acids; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 3 or a sequence that differs therefrom by one or two amino acids, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 4 or a sequence that differs therefrom by one or two amino acids. a heavy chain variable domain (V H ); and (ii) the complementarity-determining regions of: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 6 or a sequence which differs therefrom by one or two amino acids; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 7 or a sequence that differs therefrom by one or two amino acids, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8 or a sequence that differs therefrom by one or two amino acids. a light chain variable domain (V K ); (b)(i) a complementarity-determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 10 or a sequence which differs therefrom by one or two amino acids; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 11 or a sequence that differs therefrom by one or two amino acids, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 12 or a sequence that differs therefrom by one or two amino acids. a heavy chain variable domain (V H ); and (ii) the complementarity-determining regions of: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 14 or a sequence which differs therefrom by one or two amino acids; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 15 or a sequence that differs therefrom by one or two amino acids, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 16 or a sequence that differs therefrom by one or two amino acids. a light chain variable domain (V K );

[0007] (c)(i) a complementarity-determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 18 or a sequence which differs therefrom by one or two amino acids; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 19 or a sequence that differs therefrom by one or two amino acids, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 20 or a sequence that differs therefrom by one or two amino acids. a heavy chain variable domain (V H ); and (ii) the following complementarity-determining regions: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22 or a sequence which differs therefrom by one or two amino acids; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23 or a sequence that differs therefrom by one or two amino acids, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 24 or a sequence that differs therefrom by one or two amino acids. a light chain variable domain (V K );

[0008] (d)(i) a complementarity-determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 26 or a sequence which differs therefrom by one or two amino acids; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 27 or a sequence that differs therefrom by one or two amino acids, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 28 or a sequence that differs therefrom by one or two amino acids. a heavy chain variable domain (V H ); and (ii) the complementarity-determining regions of: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 30 or a sequence which differs therefrom by one or two amino acids; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 31 or a sequence that differs therefrom by one or two amino acids, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 32 or a sequence that differs therefrom by one or two amino acids. a light chain variable domain (V K );

[0009] (e)(i) a complementarity-determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34 or a sequence which differs therefrom by one or two amino acids; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35 or a sequence that differs therefrom by one or two amino acids, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36 or a sequence that differs therefrom by one or two amino acids. a heavy chain variable domain (V H ); and (ii) the complementarity-determining regions of: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 38 or a sequence which differs therefrom by one or two amino acids; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 39 or a sequence that differs therefrom by one or two amino acids, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 40 or a sequence that differs therefrom by one or two amino acids. a light chain variable domain (V K );

[0010] (f)(i) a complementarity-determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 42 or a sequence which differs therefrom by one or two amino acids; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 43 or a sequence that differs therefrom by one or two amino acids, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 44 or a sequence that differs therefrom by one or two amino acids. a heavy chain variable domain (V H ); and (ii) the complementarity-determining regions of: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 46 or a sequence which differs therefrom by one or two amino acids; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 47 or a sequence that differs therefrom by one or two amino acids, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 48 or a sequence that differs therefrom by one or two amino acids. a light chain variable domain (V K );

[0011] (g)(i) the complementarity-determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 50 or a sequence which differs therefrom by one or two amino acids; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 51 or a sequence that differs therefrom by one or two amino acids, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 52 or a sequence that differs therefrom by one or two amino acids. a heavy chain variable domain (V H ); and (ii) the complementarity-determining regions of: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 54 or a sequence which differs therefrom by one or two amino acids; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 55 or a sequence which differs therefrom by one or two amino acids, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 56 or a sequence that differs therefrom by one or two amino acids. a light chain variable domain (V K );

[0012] (h)(i) a complementarity-determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 58 or a sequence which differs therefrom by one or two amino acids; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 59 or a sequence that differs therefrom by one or two amino acids, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 60 or a sequence that differs therefrom by one or two amino acids. a heavy chain variable domain (V H ); and (ii) the complementarity-determining regions of: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 62 or a sequence which differs therefrom by one or two amino acids; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 63 or a sequence that differs therefrom by one or two amino acids, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 64 or a sequence that differs therefrom by one or two amino acids. a light chain variable domain (V K );

[0013] (i) a complementarity-determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 66 or a sequence which differs therefrom by one or two amino acids; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 67 or a sequence that differs therefrom by one or two amino acids, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 68 or a sequence that differs therefrom by one or two amino acids. a heavy chain variable domain (V H ); and (ii) the complementarity-determining regions of: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 70 or a sequence which differs therefrom by one or two amino acids; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 71 or a sequence that differs therefrom by one or two amino acids, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 72 or a sequence that differs therefrom by one or two amino acids. a light chain variable domain (V K );

[0014] (j)(i) the complementarity-determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 74 or a sequence which differs therefrom by one or two amino acids; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 75 or a sequence that differs therefrom by one or two amino acids, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 76 or a sequence that differs therefrom by one or two amino acids. a heavy chain variable domain (V H ); and (ii) the complementarity-determining regions of: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 78 or a sequence which differs therefrom by one or two amino acids; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 79 or a sequence that differs therefrom by one or two amino acids, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 80 or a sequence that differs therefrom by one or two amino acids. a light chain variable domain (V K );

[0015] (k)(i) a complementarity-determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 82 or a sequence which differs therefrom by one or two amino acids; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 83 or a sequence that differs therefrom by one or two amino acids, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 84 or a sequence that differs therefrom by one or two amino acids. a heavy chain variable domain (V H); and (ii) the complementarity-determining regions of: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 86 or a sequence which differs therefrom by one or two amino acids; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 87 or a sequence that differs therefrom by one or two amino acids, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 88 or a sequence that differs therefrom by one or two amino acids. a light chain variable domain (V K );

[0016] (l)(i) a complementarity-determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 90 or a sequence which differs therefrom by one or two amino acids; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 91 or a sequence that differs therefrom by one or two amino acids, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 92 or a sequence that differs therefrom by one or two amino acids. a heavy chain variable domain (V H ); and (ii) the complementarity-determining regions of: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 94 or a sequence which differs therefrom by one or two amino acids; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 95 or a sequence which differs therefrom by one or two amino acids, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 96 or a sequence that differs therefrom by one or two amino acids. a light chain variable domain (V K );

[0017] (m)(i) a complementarity-determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 98 or a sequence which differs therefrom by one or two amino acids; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 99 or a sequence that differs therefrom by one or two amino acids, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 100 or a sequence that differs therefrom by one or two amino acids. a heavy chain variable domain (V H ); and (ii) the complementarity-determining regions of: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 102 or a sequence which differs therefrom by one or two amino acids; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 103 or a sequence that differs therefrom by one or two amino acids, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 104 or a sequence that differs therefrom by one or two amino acids. a light chain variable domain (V K );

[0018] (n)(i) a complementarity-determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 106 or a sequence which differs therefrom by one or two amino acids; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 107 or a sequence that differs therefrom by one or two amino acids, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 108 or a sequence that differs therefrom by one or two amino acids. a heavy chain variable domain (V H ); and (ii) the complementarity-determining regions of: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 110 or a sequence which differs therefrom by one or two amino acids; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 111 or a sequence that differs therefrom by one or two amino acids, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 112 or a sequence that differs therefrom by one or two amino acids. a light chain variable domain (V K );

[0019] (o)(i) a complementarity-determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 114 or a sequence which differs therefrom by one or two amino acids; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 115 or a sequence that differs therefrom by one or two amino acids, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 116 or a sequence that differs therefrom by one or two amino acids. a heavy chain variable domain (V H ); and (ii) the following complementarity-determining regions: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 118 or a sequence which differs therefrom by one or two amino acids; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 119 or a sequence that differs therefrom by one or two amino acids, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 120 or a sequence that differs therefrom by one or two amino acids. a light chain variable domain (V K );

[0020] (p)(i) a complementarity-determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 122 or a sequence which differs therefrom by one or two amino acids; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 123 or a sequence that differs therefrom by one or two amino acids, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 124 or a sequence that differs therefrom by one or two amino acids. a heavy chain variable domain (V H ); and (ii) the following complementarity-determining regions: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 126 or a sequence which differs therefrom by one or two amino acids; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 127 or a sequence that differs therefrom by one or two amino acids, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 128 or a sequence that differs therefrom by one or two amino acids. a light chain variable domain (V K );or

[0021] (q)(i) a complementarity-determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 130 or a sequence which differs therefrom by one or two amino acids; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 131 or a sequence that differs therefrom by one or two amino acids, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 132 or a sequence that differs therefrom by one or two amino acids. a heavy chain variable domain (V H ); and (ii) the complementarity-determining regions of: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 134 or a sequence which differs therefrom by one or two amino acids; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 135 or a sequence which differs therefrom by one or two amino acids, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 136 or a sequence that differs therefrom by one or two amino acids. a light chain variable domain (V K ).

[0022] In some embodiments, the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 have the following sequences: (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, and 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, and 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, and 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, and 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, and 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, and 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, and SEQ ID NO:72, respectively; (j) SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:79, and SEQ ID NO:80, respectively; (k) SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:87, and SEQ ID NO:88, respectively; (l) SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:95, and SEQ ID NO:96, respectively; (m) SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:103, and SEQ ID NO:104, respectively; (n) SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:111, and SEQ ID NO:112, respectively; (o) SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:119, and SEQ ID NO:120, respectively; (p) SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:127, and SEQ ID NO:128, respectively; or (q) SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 135, and SEQ ID NO: 136, respectively. Collectively, these include amino acid sequences that differ by no more than two amino acid residues.

[0023] In some embodiments, the antibody or antigen-binding fragment thereof comprises: (a)(i) a complementarity-determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 2; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 3, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 4 Including V H ; and (ii) the complementarity-determining regions of: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 6; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 7, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8 Including V K ;

[0024] (b)(i) a complementarity-determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 10; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 11, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 12 Including V H ; and (ii) the complementarity-determining regions of: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 14; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 15, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 16 Including V K ;

[0025] (c)(i) the complementarity-determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 18; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 20 Including V H ; and (ii) the complementarity-determining regions of: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 24 Including V K ;

[0026] (d)(i) a complementarity-determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 26; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 27, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 28 Including V H ; and (ii) the complementarity-determining regions of: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 30; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 31, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 32 Including V K ;

[0027] (e)(i) a complementarity-determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36 Including V H ; and (ii) the complementarity-determining regions of: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 38; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 39, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 40 Including V K ;

[0028] (f)(i) a complementarity-determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 42; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 43, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 44 Including V H ; and (ii) the complementarity-determining regions of: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 46; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 47, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 48 Including V K ;

[0029] (g)(i) the complementarity-determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 50; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 51, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 52 Including V H ; and (ii) the complementarity-determining regions of: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 54; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 55, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 56 Including V K ;

[0030] (h)(i) a complementarity-determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 58; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 59, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 60 Including V H ; and (ii) the complementarity-determining regions of: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 62; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 63, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 64 Including V K ;

[0031] (i) a complementarity-determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 66; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 67, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 68 Including V H ; and (ii) the complementarity-determining regions of: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 70; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 71, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 72 Including V K ;

[0032] (j)(i) the complementarity-determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 74; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 75, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 76 Including V H ; and (ii) the complementarity-determining regions of: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 78; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 79, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 80 Including V K ;

[0033] (k)(i) a complementarity-determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 82; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 83, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 84 Including V H ; and (ii) the complementarity-determining regions of: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 86; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 87, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 88 Including V K ;

[0034] (l)(i) a complementarity-determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 90; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 91, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 92 Including V H ; and (ii) the complementarity-determining regions of: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 94; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 95, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 96 Including V K ;

[0035] (m)(i) a complementarity-determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 98; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 99, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 100 Including V H ; and (ii) the complementarity-determining regions of: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 102; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 103, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 104 Including V K ;

[0036] (n)(i) a complementarity-determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 106; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 107, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 108 Including V H ; and (ii) the following complementarity-determining regions: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 110; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 111, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 112 Including V K ;

[0037] (o)(i) a complementarity-determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 114; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 115, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 116 Including V H ; and (ii) the following complementarity-determining regions: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 118; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 119, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 120 Including V K ;

[0038] (p)(i) a complementarity-determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 122; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 123, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 124 Including V H ; and (ii) the following complementarity-determining regions: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 126; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 127, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 128 Including V K ;or

[0039] (q)(i) a complementarity-determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 130; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 131, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 132 Including V H ; and (ii) the following complementarity-determining regions: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 134; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 135, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 136 Including V K .

[0040] In some embodiments, the antibody or antigen-binding fragment thereof (i) the complementarity-determining regions of: CDR-H1 comprising the amino acid sequence of SEQ ID NO:2; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 3; and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 4 Including V H ; and (ii) the following complementarity-determining regions: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 6; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 7; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8 Including V K Includes.

[0041] In some embodiments, the V H comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 1; K comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:5.

[0042] In some embodiments, the V H comprises the amino acid sequence of SEQ ID NO: 1; K comprises the amino acid sequence of SEQ ID NO:5.

[0043] In some embodiments, the antibody or antigen-binding fragment thereof (i) the complementarity-determining regions of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 10; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 11; and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 12 Including V H ; and (ii) the complementarity-determining regions of: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 14; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 15; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 16 Including V K Includes.

[0044] In some embodiments, the V H comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 9; K comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:13.

[0045] In some embodiments, the V H comprises the amino acid sequence of SEQ ID NO: 9; K comprises the amino acid sequence of SEQ ID NO:13.

[0046] In some embodiments, the antibody or antigen-binding fragment thereof (i) the complementarity-determining regions of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 18; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 19; and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 20 Including V H ; and (ii) the complementarity-determining regions of: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 24 Including V K Includes.

[0047] In some embodiments, the V H comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 17; K comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:21.

[0048] In some embodiments, the V H comprises the amino acid sequence of SEQ ID NO: 17; K comprises the amino acid sequence of SEQ ID NO:21.

[0049] In some embodiments, the antibody or antigen-binding fragment thereof (i) the complementarity-determining regions of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35; and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36 Including V H ; and (ii) the complementarity-determining regions of: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 38; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 39; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 40 Including V K Includes.

[0050] In some embodiments, the V H comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 33; K comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:37.

[0051] In some embodiments, the V H comprises the amino acid sequence of SEQ ID NO: 33; K comprises the amino acid sequence of SEQ ID NO:37.

[0052] In some embodiments, the antibody or antigen-binding fragment thereof (i) the complementarity-determining regions of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 98; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 99; and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 100 Including V H ; and (ii) the following complementarity-determining regions: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 102; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 103; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 104 Including V K Includes.

[0053] In some embodiments, the V H comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 97; K comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:101.

[0054] In some embodiments, the V H comprises the amino acid sequence of SEQ ID NO: 97; K comprises the amino acid sequence of SEQ ID NO:101.

[0055] In some embodiments, (a) That 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 K comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:5; (b) That 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 K comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13; (c) That 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 K comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 21; (d) That 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 K comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:29; (e) That 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 K comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 37; (f) That 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 K comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 45; (g) That V Hcomprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 49, and K comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 53; (h) That 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 K comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 61; (i) That 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 K comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 69; (j) That 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 K comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 77; (k) That 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 K comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 85; (l) That 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 K comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 93; (m) That V Hcomprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 97, and K comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 101; (n) that 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 K comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 109; (o)That 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 K comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 117; (p)That 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 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) That 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 K comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:133.

[0056] In some embodiments, (a) That V H comprises the amino acid sequence of SEQ ID NO: 1, K comprises the amino acid sequence of SEQ ID NO:5; (b) That V H comprises the amino acid sequence of SEQ ID NO: 9, K comprises the amino acid sequence of SEQ ID NO: 13; (c) That V H comprises the amino acid sequence of SEQ ID NO: 17, K comprises the amino acid sequence of SEQ ID NO: 21; (d) That V H comprises the amino acid sequence of SEQ ID NO: 25, K comprises the amino acid sequence of SEQ ID NO: 29; (e) That V H comprises the amino acid sequence of SEQ ID NO: 33, K comprises the amino acid sequence of SEQ ID NO: 37; (f) That V H comprises the amino acid sequence of SEQ ID NO: 41, K comprises the amino acid sequence of SEQ ID NO: 45; (g) That V H comprises the amino acid sequence of SEQ ID NO: 49, K comprises the amino acid sequence of SEQ ID NO: 53; (h) That V H comprises the amino acid sequence of SEQ ID NO: 57, K comprises the amino acid sequence of SEQ ID NO: 61; (i) That V H comprises the amino acid sequence of SEQ ID NO: 65, K comprises the amino acid sequence of SEQ ID NO: 69; (j) That V H comprises the amino acid sequence of SEQ ID NO: 73, K comprises the amino acid sequence of SEQ ID NO: 77; (k) That V H comprises the amino acid sequence of SEQ ID NO: 81, K comprises the amino acid sequence of SEQ ID NO: 85; (l) That V H comprises the amino acid sequence of SEQ ID NO: 89, K comprises the amino acid sequence of SEQ ID NO: 93; (m) That V H comprises the amino acid sequence of SEQ ID NO: 97, K comprises the amino acid sequence of SEQ ID NO: 101; (n) that V H comprises the amino acid sequence of SEQ ID NO: 105, K comprises the amino acid sequence of SEQ ID NO: 109; (o)That V H comprises the amino acid sequence of SEQ ID NO: 113, K comprises the amino acid sequence of SEQ ID NO: 117; (p)That V H comprises the amino acid sequence of SEQ ID NO: 121, K comprises the amino acid sequence of SEQ ID NO: 125; or (q) That V H comprises the amino acid sequence of SEQ ID NO: 129, K comprises the amino acid sequence of SEQ ID NO: 133.

[0057] In some embodiments, the antibody or antigen-binding fragment thereof has a K of 10 nM or less for human Notch4. D Can be bound by value.

[0058] In some embodiments, the antibody is a human antibody.

[0059] In some embodiments, the antibody or antigen-binding fragment thereof comprises an antibody heavy chain constant region.

[0060] In some embodiments, the heavy chain constant region is a human IgG heavy chain constant region (eg, an IgG4 heavy chain constant region).

[0061] Also provided are isolated nucleic acids encoding the heavy chain variable domain and / or the light chain variable domain of the antibodies or antigen-binding fragments thereof disclosed herein, as well as expression vectors comprising such isolated nucleic acids, and host cells comprising such isolated nucleic acids or expression vectors.

[0062] Also provided are compositions comprising a Notch4 antibody or Notch-binding fragment thereof disclosed herein and a pharmaceutically acceptable carrier.

[0063] In one aspect, a method for treating, ameliorating, or preventing airway inflammation in a subject is provided, the method comprising administering to the subject an effective amount of a Notch4 antibody or Notch4-binding fragment thereof disclosed herein.

[0064] In some embodiments, the subject is a mammal (eg, a human).

[0065] In some embodiments, the subject is diagnosed with or at risk of having an airway inflammation-associated disorder, for example, the airway inflammation-associated disorder is selected from the group consisting of asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), or bronchopulmonary dysplasia (BPD).

[0066] In some embodiments, the administering step comprises administering by a systemic route (eg, a systemic route such as an intravenous route, an intramuscular route, or a subcutaneous route).

[0067] In one embodiment, a method for detecting the presence of Notch4 or a fragment thereof in a sample is provided, the method 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 detection of the complex indicates the presence of Notch4. [Brief explanation of the drawings]

[0068] [Figure 1]Figure 1 shows airway hyperresponsiveness derived from methacholine challenge in PBMC-humanized mice sensitized to dust mite allergen. The airway hyperresponsiveness ("AHR") was determined by relative peak airway resistance calculated at the highest dose of methacholine tested (40 mg / mL) after treatment with novel Notch4-specific antibodies (C08-Lib3, iggrefmat 6-28, iggrefmat 3-11, H07-Lib3, and iggrefmat 6-16), 4H1 (a previously described Notch4 antibody), or an isotype control. * indicates p<0.05, and ** indicates p<0.01.

[0069] [Figure 2-1] Figure 2A shows representative IL-4 and IL-13 flow cytometry plots for Teff cells derived from bronchoalveolar lavage fluid from individual PBMC-humanized mice sensitized to 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. Figures 2B and 2C show the mean percentages of IL13- and IL4-positive Teff cells, respectively, for the various groups. * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001. [Figure 2-2] Same as above.

[0070] [Figure 3-1] Figure 3A shows representative flow cytometry plots of BAL fluid Teff cells stained for CD4 and CCR6 from individual PBMC-humanized mice sensitized to 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 Teff cells for each treatment group. [Figure 3-2]Same as above.

[0071] [Figure 4-1] Figure 4A shows representative flow cytometry plots of Teff cells derived from bronchoalveolar lavage fluid from individual PBMC-humanized mice sensitized to 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 among 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 among T effector (Teff) cells for the same treatment groups. [Figure 4-2] Same as above.

[0072] [Figure 5] Figure 5A shows the average absorbance at 450 nm for samples with and without the Notch4-specific antibody H07-Lib3 in an ELISA-based assay to assess binding to Notch4 or Notch3. Binding to BSA was also assessed as a control. Figure 5B shows the average absorbance at 450 nm for a binding competition assay experiment between H07-Lib3 and a commercially available mouse monoclonal Notch4 antibody against Notch4 (antigen). [Figure 6-1]Figure 6A shows the normalized reactivity of the Notch4-specific antibody H07-Lib3 and other commercially available therapeutic antibodies (bococizumab, efalizumab, gantenerumab, ibalizumab, lenzilumab, and panitumumab) to cardiolipin. Figure 6B shows the normalized reactivity of H07-Lib3 and other commercially available therapeutic antibodies (bococizumab, efalizumab, gantenerumab, ibalizumab, lenzilumab, and panitumumab) to keyhole limpet hemocyanin (KLH). Figure 6C shows the normalized reactivity of H07-Lib3 and other commercially available therapeutic antibodies (bococizumab, efalizumab, gantenerumab, ibalizumab, lenzilumab, and panitumumab) to lipopolysaccharide (LPS). Figure 6D shows the normalized reactivity of H07-Lib3 and other commercially available therapeutic antibodies (bococizumab, efalizumab, gantenerumab, ibalizumab, lenzilumab, and panitumumab) to single-stranded DNA (ssDNA). Figure 6E shows the normalized reactivity of H07-Lib3 and other commercially available therapeutic antibodies (bococizumab, efalizumab, gantenerumab, ibalizumab, lenzilumab, and panitumumab) to double-stranded DNA (dsDNA). Figure 6F shows the normalized reactivity of H07-Lib3 and other commercially available therapeutic antibodies (bococizumab, efalizumab, gantenerumab, ibalizumab, lenzilumab, and panitumumab) to insulin. Figure 6G shows the normalized reactivity of H07-Lib3 and other commercially available therapeutic antibodies (bococizumab, efalizumab, gantenerumab, ibalizumab, lenzilumab, and panitumumab) to bovine serum albumin (BSA). [Figure 6-2] Same as above. [Figure 6-3] Same as above. [Figure 6-4] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0073] Detailed Description of Specific Embodiments of the Invention Notch4 (whose amino acid sequence is shown below as SEQ ID NO: 137) is expressed on Treg cells, and its signaling in Treg cells drives allergic inflammation induced by either allergens or particulate pollutants (see, e.g., WO 2019 / 178488). Disclosed herein are novel therapeutic Notch4 antibodies and compositions thereof, as well as related methods for treating, ameliorating, or preventing airway inflammation, including airway inflammation associated with certain disorders. The Notch4 antibodies disclosed herein are fully human antibodies, and they are specific for Notch4 in that they do not substantially bind to other Notch proteins (e.g., Notch1, Notch2, and Notch3).

[0074] SEQ ID NO: 137 (human Notch4 protein sequence) [ka]

[0075] (definition) As used herein, the terms "about," "approximately," and "equivalent to," when used herein with respect to a value, refer to a value that is similar to the referenced value in the context of the referenced value. Generally, a person of ordinary skill in the art familiar with the context will understand the appropriate degree of variation encompassed by "about," "approximately," and "equivalent to" in the context. For example, in some embodiments, the terms "about," "approximately," and "equivalent to" can include a range of values ​​that are 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 referenced value.

[0076] As used herein, the terms "antagonistic," "neutralizing," or "blocking," when used in reference to an antibody or antigen-binding fragment thereof, are intended to refer to an antibody or fragment thereof whose binding to its target results in at least partial inhibition of the biological activity of the target.

[0077] As used herein, "antibody" refers to a polypeptide whose amino acid sequence comprises immunoglobulins and fragments thereof that specifically bind to a designated antigen or fragment thereof. Antibodies according to the present 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). Those skilled in the art will understand that a 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). Furthermore, those skilled in the art will understand that a 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 in separate polypeptide chains.

[0078] An "antigen-binding fragment" of an antibody, or "antibody fragment," comprises a portion of an intact antibody that is capable of binding, depending on the target antigen. In some embodiments, the antibody has a function in addition to that of antigen binding, and the antigen-binding fragment retains that function. Typically, an antigen-binding fragment comprises the variable region of the antibody. Papain digestion of an antibody produces two identical antigen-binding fragments (called "Fab" fragments) and a residual "Fc" fragment (a designation reflecting the ability to readily crystallize). The Fab fragment contains the variable region domain of the heavy chain (V H ), and the first constant domain of one heavy chain (C HEach Fab fragment is monovalent with respect to antigen binding; i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab')2 fragment, which roughly corresponds to two disulfide-linked Fab fragments with different antigen-binding activities and is still capable of cross-linking antigen. The Fab' fragment contains its C H F(ab')2 antibody fragments differ from Fab fragments by having a few additional residues at the carboxy terminus of one domain, including one or more cysteines from the antibody hinge region. Fab'-SH indicates Fab' in which the cysteine ​​residues in the constant domains bear free thiol groups. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0079] "Complementarity determining regions" (abbreviated "CDRs") are regions of hypervariability interspersed with more conserved regions called "framework regions" (abbreviated "FRs"). In some embodiments, the sequences of the framework regions are identical to those in human germline sequences. In some embodiments, the sequences of the framework regions are modified relative to the human germline sequences.

[0080] As used herein, expression "control sequence" refers to a DNA sequence that is necessary or advantageous for the expression of an operably linked coding sequence in a specific host organism.Typically, the control sequence suitable for prokaryotes includes, for example, a promoter, optionally an operator sequence, and a ribosome binding site.Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0081] As used herein, antibody "effector function" refers to the biological activities attributable to the Fc region of an antibody (either a native sequence Fc region or an amino acid sequence variant Fc region), which typically vary according to the antibody's isotype. Examples of antibody effector functions include, but are not limited to, C1q binding and complement-dependent cytotoxicity, Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0082] As used herein, the term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site (known as a paratope, which is composed of the six complementarity-determining regions of an antibody) in the variable region of an antibody molecule. A single antigen may have more than one epitope. Epitopes can be conformational or linear. A conformational epitope is composed of spatially adjacent amino acids from separate segments of a linear polypeptide chain. A linear epitope is composed of adjacent amino acid residues in a polypeptide chain.

[0083] The Fc fragment contains the carboxy-terminal portions of both heavy chains held together by disulfides. The effector functions of the antibody are determined by sequences in the Fc region, which is also recognized by Fc receptors (FcRs) found on certain cell types.

[0084] As used herein, the term "humanized," when used with reference to antibodies, refers to chimeric forms of non-human (e.g., murine) antibodies. "Humanized antibodies" contain minimal sequence derived from non-human immunoglobulin. Typically, humanized antibodies are human immunoglobulins (recipient or acceptor antibodies) in which hypervariable region residues of the recipient are replaced with hypervariable region residues from a non-human species (donor antibody) (e.g., mouse, rat, rabbit, or non-human primate) having the desired specificity, affinity, and capacity. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are not found in either the recipient or donor antibody. These modifications are made to further refine antibody performance (e.g., binding affinity). Generally, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, with all or substantially all of the hypervariable loops corresponding to those of a non-human immunoglobulin, and all or substantially all of the framework regions being 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 used in the framework regions in the heavy chain and no more than three amino acid substitutions are used in the light chain. The humanized antibody also optionally comprises an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0085] As used herein, an "isolated" nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminant molecule with which it is ordinarily associated in the natural source of the antibody nucleic acid. An isolated nucleic acid molecule is different from the form or setting in which it is found in nature. Thus, isolated nucleic acid molecules are distinguished from the nucleic acid molecule as it exists in natural cells.

[0086] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies; for example, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which 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 should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies can be made by hybridoma methods (e.g., the hybridoma method first described by Kohler et al., Nature, 256:495 (1975)) or by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991).

[0087] 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 is "operably linked" to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of 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. Enhancers, however, need not be contiguous. Linking can be accomplished, for example, by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers can be used in accordance with conventional practice.

[0088] As used herein, a "polypeptide" refers to a sequence of at least two amino acids linked together by a peptide bond. In some embodiments, a polypeptide may contain at least three to five amino acids, each of which is linked to another amino acid by at least one peptide bond. Those skilled in the art will appreciate that a polypeptide may contain one or more "unnatural" amino acids or other entities that can still be incorporated into the polypeptide chain. In some embodiments, a polypeptide may be glycosylated, e.g., a polypeptide may contain one or more covalently attached sugar moieties. In some embodiments, a single "polypeptide" (e.g., an antibody polypeptide) may contain two or more individual polypeptide chains, which in some cases may be linked together by, for example, one or more disulfide bonds or other means.

[0089] As used herein, the phrase "reference level" generally refers to a level that is considered "normal" for comparison purposes (e.g., the level of an appropriate control).For example, in the context of airway inflammation, "reference level" can refer to the level of airway resistance in a subject with a condition related to airway inflammation who is not receiving a therapeutic agent of interest, or in a subject who is receiving treatment other than a therapeutic agent of interest (e.g., current standard treatment).The reference level can be determined simultaneously or can be predetermined, for example, known or estimated from past findings.

[0090] As used herein, the phrases "therapeutically effective amount" and "effective amount" are used interchangeably, and refer to an amount effective at the dosage and duration necessary to achieve a desired therapeutic result. A therapeutically effective amount can vary depending on factors such as the type of disease (e.g., airway inflammation-related disorder, e.g., asthma), disease state, age, sex, and / or weight of the individual, and the ability of the Notch4 antibody (or pharmaceutical composition thereof) to elicit a desired response in the individual. An effective amount can also be an amount in which any toxic or harmful effects of the antibody or pharmaceutical composition thereof are outweighed by the therapeutically beneficial effects.

[0091] As used herein, "treating" a condition or "treatment" of a condition (e.g., a condition described herein, e.g., an airway inflammation-related disorder, e.g., asthma) is an approach to obtain a beneficial or desired result (e.g., a clinical result). Beneficial or desired results may include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, whether detectable or undetectable; a reduction in the severity of a disease, disorder, or condition; a stabilized (i.e., non-worsening) state of a disease, disorder, or condition; preventing the spread of a disease, disorder, or condition; a delay or slowing of the progression of the disease, disorder, or condition; an improvement or palliation of the disease, disorder, or condition; and remission (whether partial or complete). "Alleviating" a disease, disorder, or condition means that the severity and / or undesired clinical manifestations of the disease, disorder, or condition are attenuated, and / or the time course of its progression is slowed or prolonged, compared to the severity or time course in the absence of treatment.

[0092] (Antibodies and antigen-binding fragments thereof) In one aspect, provided is an antibody or antigen-binding fragment thereof that can bind to Notch4.In some embodiments, the antibody or antigen-binding fragment is a monoclonal antibody or its antigen-binding fragment.In some embodiments, the antibody or antigen-binding fragment is a human antibody or its antigen-binding fragment (for example, a human monoclonal antibody or its antigen-binding fragment).

[0093] In some embodiments, antigen-binding fragments are provided, for example, the fragments can be, for example, scFv, Fab, scFab (single chain Fab).

[0094] As used herein, the term "scFv" refers to a V fragment derived from an antibody. H Domains and V L Domain or VK It is used according to its common usage in the art to refer to a single chain in which the domains are typically connected by a linker.

[0095] As used herein, the term "Fab fragment" is used according to its common usage in the art. A Fab fragment contains an entire light chain (V L Domain or V K domain, and C L 1 domain), the heavy chain variable region domain (V H ), and the first constant domain of one heavy chain (C H 1) is typically included.

[0096] In some embodiments, immunoconjugates are provided that include the antibodies or antigen-binding fragments disclosed herein that are labeled and / or conjugated to a cytotoxic agent (e.g., a toxin or a radioisotope).

[0097] Exemplary Antibodies In some embodiments, the provided antibodies or antigen-binding fragments comprise 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 the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of an antibody set forth in Table 1 below. In some embodiments, antibodies or antigen-binding fragments are provided that are variants of the antibodies set forth in Table 1, wherein such antibodies or antigen-binding fragments have CDR sequences that differ from the CDR sequences of the antibodies set forth in Table 1 by no more than two amino acid residues per CDR (e.g., two amino acid residues or one amino acid residue). In some embodiments, an antibody or antigen-binding fragment is provided that is a variant of an antibody shown in Table 1, and such an antibody or antigen-binding fragment has a set of six CDRs that collectively differ in sequence from the CDRs of the antibody set forth in Table 1 by no more than two amino acid residues (e.g., two amino acid residues or one amino acid residue).

[0098] In some embodiments, the provided antibodies or antigen-binding fragments comprise heavy and light chain variable domains comprising the heavy and light chain variable domain sequences of the antibodies set forth in Table 1. In some embodiments, antibodies or antigen-binding fragments are provided that are variants of the antibodies shown in Table 1, having (1) a heavy chain domain comprising 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 an antibody listed in Table 1; and (2) a light chain domain comprising 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%, or at least 98%, at least 99% identical to the amino acid sequence of the light chain variable domain of the same antibody listed in Table 1. Table 1. Exemplary heavy chain variable domain sequences, light chain variable domain sequences, and complementarity determining region sequences of Notch4 antibodies [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9]

[0099] (antibody constant region) In some embodiments, the provided Notch4 antibodies or Notch4-binding fragments thereof comprise one or more constant regions (eg, a heavy chain constant region and / or a light chain constant region).

[0100] In some embodiments, the provided Notch4 antibodies or Notch4-binding fragments thereof comprise an IgG4 heavy chain constant region (e.g., an IgG4 heavy chain constant region that is at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 138). In some embodiments, the provided Notch4 antibodies or Notch4-binding fragments thereof comprise an Igκ light chain constant region (e.g., an Igκ light chain constant region that is at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 142).

[0101] SEQ ID NO: 138 (human IgG4 heavy chain constant region sequence) [ka]

[0102] SEQ ID NO: 139 (human IgG1 heavy chain constant region sequence) [ka]

[0103] SEQ ID NO: 140 (human IgG2 heavy chain constant region sequence) [ka]

[0104] SEQ ID NO: 141 (human IgG3 heavy chain constant region sequence) [ka]

[0105] SEQ ID NO: 142 (human Igκ light chain constant region sequence) [ka]

[0106] SEQ ID NO: 143 (human Igλ1 light chain constant region sequence) [ka]

[0107] SEQ ID NO: 144 (human Ig λ2 light chain constant region sequence) [ka]

[0108] SEQ ID NO: 145 (human Igλ3 light chain constant region sequence) [ka]

[0109] SEQ ID NO: 146 (human Igλ6 light chain constant region sequence) [ka]

[0110] SEQ ID NO: 147 (human Igλ7 light chain constant region sequence) [ka]

[0111] (antibody fragment) In certain embodiments, antibody fragments rather than whole antibodies are provided.

[0112] Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived by 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 E. coli, allowing the production of large amounts of these fragments. Antibody fragments can be isolated, for example, from antibody phage libraries. Alternatively, or additionally, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology, 10:163-167 (1992)). According to another approach, F(ab')2 fragments can be isolated directly from recombinant host cell culture. Fab and F(ab')2 fragments with increased in vivo half-lives that contain salvage receptor-binding epitope residues are described, for example, in U.S. Patent No. 5,869,046. Other techniques for the production of antibody fragments will be apparent to those skilled in the art.

[0113] In some embodiments, the antigen-binding fragment is a single-chain Fv fragment (scFv). See, for example, WO93 / 16185; U.S. Patent No. 5,571,894; and U.S. Patent No. 5,587,458. Fv and scFv are species with intact binding sites that lack constant regions; therefore, these fragments may be suitable for reduced non-specific binding during in vivo use. scFv fusion proteins can be constructed to fuse an effector protein to either the amino or carboxy terminus of scFv. See, for example, Antibody Engineering, edited by Borrebaeck. The antigen-binding antibody fragment can also be a "linear antibody," as described, for example, in U.S. Patent No. 5,641,870.

[0114] (Amino acid sequence modification) 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 the 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 possesses the desired characteristics. In some embodiments, amino acid changes are introduced to alter post-translational processes, for example, to change the number or position of glycosylation sites.

[0115] A useful method for identifying specific residues or regions that are preferred locations for mutagenesis is called "alanine scanning mutagenesis," as described, for example, by Cunningham and Wells in Science, 244:1081-1085 (1989). In this method, a target residue or group (e.g., charged residues such as arg, asp, his, lys, and glu) is identified and substituted with neutral or negatively charged amino acids (most preferably alanine or polyalanine) to affect the interaction of those amino acids with the antigen. Amino acid locations that demonstrate functional sensitivity to these substitutions are then refined by introducing additional or other variants at or for those substitution sites. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation itself need not be. 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.

[0116] Examples of amino acid sequence insertions include, but are not limited to, amino- and / or carboxy-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intersequence insertions of single or multiple amino acid residues. An example of a terminal insertion includes, but is not limited to, an N-terminal methionyl residue.

[0117] In some embodiments, the antibody or antigen-binding fragment is fused at one end to another polypeptide (e.g., a cytotoxic polypeptide, an enzyme, or a polypeptide that increases the serum half-life of the antibody or antigen-binding fragment).

[0118] Another type of variant is amino acid substitution variant.These variants have at least one amino acid residue in the antibody molecule or antigen-binding fragment molecule replaced with another residue.The most interesting site for substitutional mutagenesis is typically the hypervariable region, but changes in framework region are also contemplated.Examples of conservative substitutions are shown in Table 2 under the heading of "preferred substitutions".More substantial changes can be introduced, as listed under the heading "exemplary substitutions" in Table 2, or as further described below for amino acid classes, and the resulting antibody or antigen-binding fragment can be screened. Table 2: Exemplary Amino Acid Substitutions [Table 2]

[0119] Substantial alterations in the biological properties of the antibody can be achieved by selecting substitutions that differ significantly in their effect on (a) the structure of the polypeptide backbone (e.g., sheet or helix conformation) in the region of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) maintaining the bulk of the side chain. Naturally occurring residues are typically divided into groups based on common side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr; (3) Acidic: Asp, Glu; (4) basic: Asn, Gln, His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) Aromatic: Trp, Tyr, Phe.

[0120] Non-conservative substitutions may involve exchanging a member of one of these classes for another class.

[0121] Additionally or alternatively, cysteine ​​residues not involved in maintaining the proper conformation of the antibody or antigen-binding fragment may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine ​​bond(s) may be added to the antibody to improve its stability, particularly where the antibody is an antibody fragment (e.g., an Fv fragment).

[0122] In some embodiments, substitutional variants comprise substitutions within one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variants that have improved biological properties relative to the parent antibody from which they are generated are selected for further development.

[0123] A method for generating such substitutional variants involves affinity maturation using phage display. In an example of such a method, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles, for example, as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for biological activity (e.g., binding affinity).

[0124] To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that significantly contribute to antigen binding. Alternatively, or in addition, it may be beneficial to analyze the crystal structure of the antigen-antibody complex to identify contact points between the antibody or antigen-binding fragment and the antigen. Such contact residues and nearby residues are candidates for substitution according to the techniques described herein. Once such variants are generated, the panel of variants can be screened, and antibodies with superior properties in one or more appropriate assays can be selected for further development.

[0125] In some embodiments, the original glycosylation pattern of the parent antibody is altered. Such alteration may involve deleting one or more carbohydrate moieties found in the antibody and / or adding one or more glycosylation sites that are not present in the antibody.

[0126] Glycosylation of antibodies can typically be either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid (most commonly serine or threonine), although 5-hydroxyproline or 5-hydroxylysine can also be used.

[0127] Addition of glycosylation sites to the antibody can be accomplished by altering the amino acid sequence of the antibody or antigen-binding fragment such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration 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).

[0128] Nucleic acid molecules encoding amino acid sequence variants of an antibody or antigen-binding fragment can be prepared by a variety of methods known in the art, including, but 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 earlier prepared variant or non-variant versions of the antibody or antigen-binding fragment.

[0129] In some embodiments, modifications that increase the serum half-life of the antibody or antigen-binding fragment are used. For example, a salvage receptor-binding epitope can be incorporated into an antibody (especially an antibody fragment), 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 in 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.

[0130] (Antibody characteristics) In certain embodiments, the disclosed Notch4 antibody or Notch4-binding fragment thereof selectively binds to Notch4 in that it exhibits detectable binding to Notch4 in a binding assay, but does not exhibit detectable binding to other Notch proteins (e.g., Notch1, Notch2, or Notch3) in a similar assay. In some embodiments, the Notch4 antibody or Notch4-binding fragment thereof is antagonistic.

[0131] In some embodiments, the Notch4 antibody has a dissociation constant (K D In some embodiments, the Notch4 antibody or Notch4-binding fragment thereof binds to Notch4 with a dissociation constant (K) of between 300 pM and 10 nM, inclusive. D )

[0132] In one embodiment, K D is measured by a radiolabeled antigen binding assay (radioimmunoassay, RIA) performed using a Fab version of the antibody or antigen-binding fragment of interest and its antigen.

[0133] According to another embodiment, K D is measured using a surface plasmon resonance assay with immobilized antigen.

[0134] Vectors, host cells and recombinant methods Also provided are isolated nucleic acids encoding the antibodies and antigen-binding fragments, vectors and host cells containing the nucleic acids, and recombinant techniques for producing the antibodies.

[0135] For recombinant production of the antibody or antigen-binding fragment, the nucleic acid encoding the antibody or antigen-binding fragment can be isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. The DNA encoding the monoclonal antibody can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the antibody heavy and light chains). Many vectors are available. The vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0136] For larger-scale antibody production, plasmids from the yeast clones producing the desired Notch4 antibody can be transfected into suitable host cells (e.g., insect or mammalian host cells). Non-limiting examples of mammalian host cell lines that may be suitable for antibody production include NS0 mouse myeloma cells, PER.C6® human cells, Chinese hamster ovary (CHO) cells, Sp2 / 0, and HEK293 cells.

[0137] (Pharmaceutical composition) In certain embodiments, the provided Notch4 antibody is incorporated into one or more pharmaceutically acceptable carriers in the pharmaceutical composition suitable for administration to subjects.As used herein, " pharmaceutically acceptable carrier " refers to any of physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption retardants, etc. Examples of pharmaceutically acceptable carriers include but are not limited to water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, etc., and their combinations.

[0138] In some embodiments, the pharmaceutical composition comprises one or more tonicity agents or stabilizers, non-limiting examples of which include sugars (e.g., sucrose), polyalcohols (e.g., mannitol or sorbitol), and sodium chloride.

[0139] 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 ion or chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)).

[0140] In some embodiments, the pharmaceutical compositions contain one or more auxiliary substances, such as wetting or emulsifying agents, preservatives (e.g., benzyl alcohol) or buffers, which may enhance the shelf life and / or effectiveness of the Notch4 antibodies disclosed herein.

[0141] Pharmaceutical compositions can be provided in any of a variety of forms, including, for example, liquid, semi-solid, and solid dosage forms, such as liquids (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. The suitability of a particular form can depend on the intended mode of administration and therapeutic application.

[0142] In some embodiments, the pharmaceutical composition is in the form of an injectable or infusible solution.

[0143] Pharmaceutical compositions are typically sterile and stable under manufacturing, transportation, and storage conditions. Pharmaceutical compositions can be formulated, for example, as a solution, microemulsion, dispersion, liposome, or other ordered structure. In some embodiments, pharmaceutical compositions are formulated into structures particularly suitable for high drug concentrations. For example, sterile injectable solutions can be prepared by incorporating a therapeutic agent (e.g., Notch4 antibody) in a desired amount in an appropriate solvent with one or a combination of the ingredients listed herein, and then optionally sterilizing (e.g., filter sterilization). Generally, dispersions can be prepared by incorporating a therapeutic agent (e.g., Notch4 antibody) into a sterile vehicle containing a basic dispersion medium and other ingredients (e.g., additional ingredients described herein). In the case of sterile powders for preparing sterile injectable solutions, exemplary methods include vacuum drying and freeze-drying to produce a powder of the therapeutic agent and any desired additional ingredients from a previously sterile-filtered solution thereof.

[0144] The proper fluidity of a solution can be maintained, for example, by the use of a coating (e.g., lecithin), by the maintenance of a specific particle size (e.g., in the case of dispersions), and / or by the use of surfactants. Prolonged absorption of injectable compositions can be brought about, for example, by including in the composition an agent which delays absorption (e.g., monostearate salts and / or gelatin).

[0145] (Generation of human Notch4 antibody) As further described in the Examples, the antibodies of the present disclosure 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 repertoires from unimmunized donors. According to this technology, antibody V domain genes are cloned in frame into either the major or minor coat protein gene of a filamentous bacteriophage (e.g., M13 or fd) and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. Thus, the phage mimic some of the properties of B cells. Phage display can be performed in a variety of formats, as 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 diverse group of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. Essentially following the techniques described by Marks et al., J. Mol. Biol. 222:581-597 (1991) or Griffith et al., EMBO J., 12:725-734 (1993), repertoires of V genes from unimmunized human donors can be constructed, and antibodies against a diverse group of antigens (including self-antigens) can be isolated. See also U.S. Patent Nos. 5,565,332 and 5,573,905.

[0146] For large-scale production of antibodies, plasmids from the yeast clones producing the desired Notch4 antibody can be transfected into suitable host cells (e.g., host cells further described herein) (see the "Vectors, Host Cells and Recombinant Methods" subsection).

[0147] (Treatment Method) The methods of treating airway inflammation and / or related conditions disclosed herein generally include administering a therapeutically effective amount of a Notch4 antibody or 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 been diagnosed with or is at risk of having an airway inflammation-related disorder (e.g., asthma (e.g., severe eosinophilic asthma), chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), or bronchopulmonary dysplasia (BPD)).

[0148] In some embodiments, the administering step comprises systemic administration (eg, intravenous, subcutaneous, or intramuscular route) of the therapeutically effective amount.

[0149] A therapeutically effective amount can be administered in a single dose or in multiple doses (e.g., at least two doses, at least three doses, at least four doses, at least five doses, at least six doses, at least seven doses, at least eight doses, at least nine doses, or at least ten doses). When administered in multiple doses, any of a variety of suitable treatment regimens can be used, including administration at regular intervals (e.g., once every two days, once every three days, once every four days, once every five days, three times weekly, twice weekly, once weekly, once every two weeks, once every three weeks, etc.).

[0150] The dosage regimen (e.g., the amount of each therapeutic agent, the relative timing of treatment, etc.) effective in the treatment method may depend on the severity of the disease or condition, as well as the subject's weight and the subject's general condition. For example, the therapeutically effective amount of a particular composition containing a therapeutic agent to be administered to a mammal (e.g., a human) can be determined by one skilled in the art, taking into account individual differences in the mammal's age, weight, and condition. The therapeutically effective and / or optimal amount can also be determined empirically by one skilled in the art. In some embodiments, the subject is administered a dose of between 0.4 mg / kg every three days and 20 mg / kg every three days. Notch4 antibodies and pharmaceutical compositions thereof can be administered by any of a variety of appropriate routes, including, but not limited to, systemic routes, such as parenteral (e.g., intranasal, intravenous, or subcutaneous) routes or enteral routes.

[0151] In some embodiments, administration of antibody or pharmaceutical composition thereof leads to improvement of one or more symptoms or signs of airway inflammation or related disorders.For example, administration of the antibody or pharmaceutical composition thereof can improve airway hyperresponsiveness.For example, in methacholine challenge test, the airway hyperresponsiveness measured from calculated peak 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 that of reference level.

[0152] As another example, other indicators of inflammation (e.g., 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)) may be reduced as a result of administration of the antibody or pharmaceutical composition. Metrics for any one or any combination of these indicators of inflammation may be reduced by 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 that of the reference level. [Example]

[0153] (Example) Example 1. Identification and production of humanized antibodies specific to Notch4 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.

[0154] Recombinant human Notch4 was used as an antigen for positive selection, and recombinant human Notch1, Notch2, and Notch3 were used as antigens for negative selection of candidate antibodies. Recombinant Notch4 protein was biotinylated and assessed for biotinylation using SDS-PAGE electrophoresis and size exclusion chromatography (HPLC-SEC).

[0155] Phage libraries based on abrilumab (Lib1), mepolizumab (Lib2A), crenezumab (Lib3), necitumumab (Lib4), evolocumab (Lib6), and adalimumab (Lib8) were exposed to biotinylated human Notch4. The final selection outputs were tested using a polyclonal phage ELISA assay using the biotinylated Notch4 antigen and a control off-target protein. The final selection outputs were subcloned into yeast display vectors and analyzed using flow cytometry to detect and select for human Notch4-binding populations. To retain clones that showed specific binding activity to Notch4, the Notch4-binding enriched population was then deselected from the selection of clones that bound to other Notch proteins (Notch1, Notch2, and Notch3) and / or subjected to competition with other Notch proteins.

[0156] The Notch4-binding enriched population was then subjected to an epitope competition assay using Notch4 antibodies (either 4H1 or 3B11; see WO 2022 / 081971) known to disrupt the binding of Notch4 to Jagged1 (Notch ligand). Briefly, a "sandwich" assay was performed using yeast displaying anti-Notch4 antibodies to capture Notch4; the yeast was then incubated with 4H1 or 3B11, and the latter (4H1 or 3B11) was detected with a fluorescently labeled secondary reagent. Detection of 4H1- or 3B11-binding signals led to the identification and selection of yeast displaying clones that did not compete for the same epitope of either one of the two monoclonal antibodies (4H1 or 3B11). The absence of binding signal detection in the same population incubated with 4H1 or 3B11 led to the identification and selection of yeast displaying clones competing for the same epitope for either one of the two monoclonal antibodies.

[0157] Clones that successfully competed with 4H1 or 3B11 (and therefore should also be able to inhibit Notch4 binding to Jagged1) were selected for further analysis as described in Example 2.

[0158] Example 2. Characterization of Notch4 antibodies The candidate Notch4-specific antibodies identified in Example 1 were further characterized to determine their sequences and binding parameters.

[0159] (material and method) Sequencing: A hybrid parallel sequencing approach was performed, including Sanger-based sequencing and next-generation sequencing (NGS). For Sanger-based sequencing, up to 4x96 individual clones were sequenced to identify unique clones.

[0160] PacBio sequencing was performed on yeast selection output DNA for each candidate antibody. Sequence clonotypes were identified by analyzing the PacBio output data. The Sanger sequencing data was used to identify unique clusters of positive clones to ensure that not all Sanger clones belonged to a single cluster.

[0161] After expression and purification, clones were characterized using surface plasmon resonance (SPR) to determine binding kinetics and epitope binding activity. To capture the candidate antibodies, HC30M chips (polycarboxylate hydrogel surfaces with a medium charge density and a 30-nm coating thickness) were coated with anti-human Fc antibodies (unlabeled goat anti-human IgG Fc obtained from Southern Biotech). 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 analytes were injected sequentially from a minimum concentration of 0.41 nM to a maximum 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 assess specificity, experiments were also performed using Notch2 as the analyte in a single injection at 100 nM in HBSTE+0.5 mg / mL.

[0162] (result) The sequences and binding characteristics of 19 candidate Notch4 antibodies were obtained. Table 1 of the present disclosure lists the V of representative candidate clones. H Array, V K The sequences and CDR sequences are listed.

[0163] Table 3 (below) summarizes the binding parameters of the candidate clones obtained from this experiment. Table 3: Binding parameters of candidate Notch4 antibodies determined by surface plasmon resonance. [Table 3-1] [Table 3-2] [Table 3-3]

[0164] Example 3. Therapeutic Efficacy of Notch4 Antibodies in a Humanized In Vivo Mouse Model Five of the Notch4-specific antibodies identified in Example 1 and characterized in Example 2 were further analyzed for their in vivo efficacy in a humanized mouse model of allergen-induced inflammation.

[0165] (material and method) (Humanized mouse allergen-induced inflammation model) Peripheral blood mononuclear cell (PBMC) humanized mice were obtained from JAX (登録商標) The "NSG" (trademark) mice carry the scid (severe combined immunodeficiency) mutation and a complete null allele of the common gamma chain of the IL2 receptor (IL2rg null Female NSG™ mice were injected with human peripheral blood mononuclear cells (hu-PBMCs). Mice were maintained in specific pathogen-free (SPF) conditions for a week before sensitization to allergens.

[0166] To sensitize the mice, they were administered 50 μL of a solution of house dust mite (HDM) extract (available from Stallergenes Greer) (5 μg HDM per 100 μL phosphate-buffered saline) intranasally on days 1, 2, and 3 of the experiment. On days 15, 16, and 17, the mice were rechallenged by intranasal administration of HDM at the same concentration and volume.

[0167] At the end of the experiment, on day 18, mice were euthanized and bronchoalveolar lavage (BAL) fluid and lung tissue were collected for subsequent analysis.

[0168] (antibody treatment) Allergen-sensitized mice were administered one of the test Notch4 antibody clones (C08-Lib3, iggrefmat 6-28, iggrefmat 3-11, H07-Lib3, or iggrefmat 6-16) by intraperitoneal (ip) injection. 100 μL of a 10 μg / mL antibody solution was administered on days 1, 2, 3, 15, and 16 of the experiment. For comparison, one group of mice received 4H1, a previously described Notch4 antibody (see WO 2022 / 081971). The control group received an isotype (IgG) control.

[0169] (Methacholine challenge and airway hyperresponsiveness) On day 18 of the experiment, mice were anesthetized and exposed to doubling concentrations (10 mg / mL, 20 mg / mL, and 40 mg / mL) of aerosolized acetyl-β-methacholine (Sigma-Aldrich) using a Buxco small animal ventilator (Data Sciences International). Relative peak airway resistance for each methacholine dose, normalized to saline baseline, was calculated.

[0170] (FACS analysis) Bronchoalveolar lavage fluid was analyzed by flow cytometry. CD3 + CD4 + Foxp3 - Effector T (Teff) cell populations were analyzed by gating the populations and assessing (1) intracellular expression of IL-4 and IL-13 and (2) CCR6 expression. + CD4 + Foxp3 + Regulatory T cells (Tregs) were analyzed for Notch4 expression by gating the population.

[0171] Therapeutic effect of Notch4 antibody in a model of airway hyperresponsiveness To evaluate the ability of the above-mentioned novel Notch4-specific antibody to improve airway inflammation, humanized mice sensitized to dust mite allergen were subjected to methacholine challenge. Figure 1 shows the airway hyperresponsiveness in each treatment group, as determined by the relative peak airway resistance calculated at the highest dose of methacholine tested (40 mg / mL). As can be seen in Figure 1, treatment with iggrefmat 6-28, H07-Lib3, or iggrefmat 6-16 resulted in a significant reduction in airway hyperresponsiveness compared to that observed in the isotype control, and this reduction was comparable to or greater than that observed with the 4H1 antibody.

[0172] The antibodies were generally well tolerated in mice, and most mouse groups did not experience excessive mortality after administration of the fully human Notch4 antibodies. However, mice treated with C08-Lib3 showed somewhat reduced survival (60% survival 20 days after antibody administration) compared with other groups (data not shown).

[0173] (Effect of Notch4 antibody on inflammation) To evaluate the effect of treatment with the novel Notch4-specific antibody on inflammation, intracellular IL-4 and IL-13 expression was assessed in T effector cells derived from bronchoalveolar lavage (BAL) fluid. IL-4 and IL-13 are expressed in Th2 cells in response to asthmatic inflammation. CCR6 (a chemokine receptor expressed on Th2 cells)

[0174] Figure 2A shows representative flow cytometry plots of BAL fluid Teff cells stained for IL-4 and IL-13 from individual mice in each of the treatment groups; Figures 2B and 2C show the mean percentages of IL-13- and IL-4-positive cells in Teff cells for the various groups, respectively. As shown in Figure 2A, administration of the tested antibodies reduced the percentage of IL-4 + IL13 +Treatment with three of the Notch4 antibodies (iggrefmat 3-11, H07-Lib3, and iggrefmat 6-16) resulted in a significant decrease in the percentage of IL-13+ cells compared 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 percentage of IL-13+ cells compared to the isotype control (Figure 2C).

[0175] Figure 3A shows representative flow cytometry plots of BAL fluid Teff cells stained for CD4 and CCR6 from individual mice in each treatment group; Figure 3B shows the mean percentage of CCR6-positive Teff cells for the various groups. Treatment with three of the Notch4 antibodies (iggrefmat 3-11, H07-Lib3, and iggrefmat 6-16) resulted in a significant decrease in the percentage of CCR6+ cells compared with the isotype control (Figure 2B).

[0176] These results suggest that treatment with some of the Notch4 antibodies resulted in less inflammation and less attraction of Th2 cells to lung tissue.

[0177] Inhibition of Notch4 expression on Treg cells in vivo by Notch4 antibodies Notch4 is expressed on Treg cells, and its signaling in Treg cells drives allergic inflammation induced by either allergens or particulate pollutants (see, for example, International Publication No. 2019 / 178488). To evaluate whether the above-mentioned novel Notch4-specific antibody can block Notch4 expression on Treg cells, Treg cells from mice treated with the antibody were evaluated for Notch4 expression by flow cytometry.

[0178] Figure 4A shows representative flow cytometry plots from individual mice in each treatment group; Figure 4B shows the average percentage of Notch4-positive cells in Teff cells for the various groups. As shown in Figures 4A and 4B, administration of any one of the five antibodies tested reduced the percentage of Notch4-positive Treg cells compared to the isotype control, and the level of Notch4-positive Treg cells appeared to be equivalent to the level observed in mice treated with the 4H1 antibody. Figure 4C shows the average percentage of Notch4-positive cells in T effector (Teff) cells for the same treatment groups. The percentage of Notch4-positive cells in Teff cells appeared to be very low and similar across all groups.

[0179] These results demonstrate that three of the Notch4-specific antibodies (iggrefmat 6-28, H07-Lib3, and iggrefmat 6-16) demonstrated in vivo therapeutic efficacy in a humanized mouse allergen model.

[0180] Example 4. Evaluation of the binding specificity of H07-Lib3 to Notch4 in vitro The specificity of the Notch4-specific antibody clone, H07-Lib3, was further evaluated using an in vitro assay.

[0181] (material and method) (Binding Specificity Measurement) The binding specificity of H07-Lib3 to Notch4 was evaluated by enzyme-linked immunosorbent assay (ELISA). ELISA was performed on samples containing Notch3 protein, Notch4 protein, or bovine serum albumin (BSA, control). Antibody-antigen binding was measured by absorbance at 450 nm.

[0182] (Epitope binning assay) The epitope specificity of H07-Lib3 was evaluated against a commercially available Notch4 monoclonal mouse antibody (MHN4-2, available from BioLegend) by performing a competitive ELISA assay. The assay was performed on samples containing Notch4 (antigen) with or without MHN4-2. Antibody-antigen binding was measured by absorbance at 450 nm.

[0183] (Binding Kinetics Measurement) Surface plasmon resonance (SPR) was used to determine the binding kinetics of H07-Lib3. To capture candidate antibodies, CM5 chips were coated with anti-human polyclonal Fc antibody (Southern Biotech). Candidate antibodies were diluted in HEPES-buffered saline containing 0.005% surfactant P20 (HBS-P) and exposed to the surface for 20 minutes. Notch4 analyte was injected at 300 nM to determine the affinity of the candidate antibodies. To assess specificity, experiments using Notch3 as the analyte at 300 nM were also performed.

[0184] (Specificity of H07-Lib3 for Notch4) To determine the specificity of H07-Lib3, a Notch4-specific antibody clone, to Notch4, ELISA experiments were performed. Figure 5A shows the binding of H07-Lib3 to Notch4, Notch3, and bovine serum albumin, as measured by the average absorbance at 450 nm. As can be seen in Figure 5A.

[0185] Figure 5B shows epitope binning of H07-Lib3 and a commercially available Notch4 antibody (mouse Ab). Binding of H07-Lib3 to Notch4 was observed both in the absence and presence of the mouse Ab, as seen by the increased mean absorbance at 450 nm compared to samples without any added antibody.

[0186] These results indicate that H07-Lib3 binds to Notch4 but not to Notch3, and that the murine antibody MHN4-2 does not compete with the binding of H07-Lib3 to Notch4.

[0187] (Binding kinetics of H07-Lib3 to Notch4) Table 4 (below) summarizes the binding parameters of H07-Lib3 as determined by surface plasmon resonance (SPR). Table 4: Binding parameters of candidate Notch4 antibody H07-Lib3 determined by surface plasmon resonance. [Table 4]

[0188] Example 5. Stability Characterization of H07-Lib3 To assess the developability of the candidate Notch4-specific antibody H07-Lib3, the stability of H07-Lib3 was evaluated.

[0189] (material and method) (Melt 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 specific amino acids (e.g., tryptophan, tyrosine, and phenylalanine) in each sample after excitation with a 266 nm laser. The second inflection point of the plotted melting curve was identified to determine the melting temperature (Tm2).

[0190] (Aggregation Temperature Measurement) The aggregation temperatures (temperature at which protein aggregation begins) of H07-Lib3 and two control monoclonal antibodies (control mAb1 and control mAb2) were determined by measuring the intrinsic fluorescence of aromatic amino acids and static light scattering (SLS). 266 ) was measured for lower concentrations and smaller aggregate formation, and SLS (Tagg) at 473 nm light 473 ) was measured for higher concentrations and greater aggregate formation.

[0191] (result) The melting and aggregation temperatures of H07-Lib3, control mAb1, and control mAb2 were obtained. Table 5 (below) summarizes the thermal parameters of these antibodies from this experiment. Table 5: Melting and aggregation temperatures of H07-Lib3 and control antibodies [Table 5]

[0192] These results indicate that the Notch4-specific antibody H07-Lib3 has a high temperature stability and an increased aggregation temperature compared to the control antibody.

[0193] Example 6. Evaluation of H07-Lib3 polyreactivity To further evaluate the developability of the candidate Notch4-specific antibody H07-Lib3, H07-Lib3 was further evaluated for non-specific polyreactivity.

[0194] (material and method) (Multiplex Reactivity Assay) The nonspecific reactivity of H07-Lib3 was evaluated against cardiolipin, keyhole limpet hemocyanin (KLH), lipopolysaccharide (LPS), single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), insulin, and bovine serum albumin (BSA). Additional therapeutic antibodies (bococizumab, efalizumab, gantenerumab, ibalizumab, lenzilumab, and panitumumab) were included in the experiment for comparison. The reactivity percentage of each antibody was normalized to the highest reactivity value for each probe. The threshold for acceptable multiple reactivity was set at 30% for all probes.

[0195] (result) The multiplex reactivity of H07-Lib3 with cardiolipin, KLH, LPS, ssDNA, dsDNA, insulin, and BSA was determined. Figures 6A-6G show the average reactivity percentages of H07-Lib3 and commercial antibodies with cardiolipin, KLH, LPS, ssDNA, dsDNA, insulin, and BSA, respectively. As shown in Figures 6A-6G, H07-Lib3 showed less than 30% reactivity with each of the above antigens.

[0196] These results show that H07-Lib3 is not reactive with any of the seven probes evaluated (cardiolipin, KLH, LPS, dsDNA, ssDNA, insulin, and BSA).

[0197] (Equivalent) The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.

[0198] The present disclosure may be embodied in other specific forms without departing from its spirit or essential characteristics. Accordingly, the above-described embodiments should be considered in all respects as illustrative rather than limiting of the disclosure described herein. The various components of the various embodiments and steps of the various disclosed methods may be utilized in various combinations and permutations, and all such variations should be considered aspects of the present disclosure. Therefore, the scope of the present disclosure is indicated by the appended claims, rather than by the above 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 capable of binding to Notch4, (a)(i) a complementarity determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 2 or a sequence which differs therefrom by one or two amino acids; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 3 or a sequence that differs therefrom by one or two amino acids, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 4 or a sequence that differs therefrom by one or two amino acids. a heavy chain variable domain (V Ｈ ); and (ii) the following complementarity determining regions: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 6 or a sequence which differs therefrom by one or two amino acids; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 7 or a sequence that differs therefrom by one or two amino acids, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8 or a sequence that differs therefrom by one or two amino acids. a light chain variable domain (V Ｋ ); (b)(i) the following complementarity determining regions: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 10 or a sequence which differs therefrom by one or two amino acids; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 11 or a sequence that differs therefrom by one or two amino acids, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 12 or a sequence that differs therefrom by one or two amino acids. a heavy chain variable domain (V Ｈ ); and (ii) the following complementarity determining regions: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 14 or a sequence which differs therefrom by one or two amino acids; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 15 or a sequence that differs therefrom by one or two amino acids, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 16 or a sequence that differs therefrom by one or two amino acids. a light chain variable domain (V Ｋ ); (c)(i) a complementarity determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 18 or a sequence which differs therefrom by one or two amino acids; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 19 or a sequence that differs therefrom by one or two amino acids, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 20 or a sequence that differs therefrom by one or two amino acids. a heavy chain variable domain (V Ｈ ); and (ii) the following complementarity determining regions: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22 or a sequence which differs therefrom by one or two amino acids; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23 or a sequence that differs therefrom by one or two amino acids, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 24 or a sequence that differs therefrom by one or two amino acids. a light chain variable domain (V Ｋ ); (d)(i) the following complementarity determining regions: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 26 or a sequence which differs therefrom by one or two amino acids; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 27 or a sequence that differs therefrom by one or two amino acids, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 28 or a sequence that differs therefrom by one or two amino acids. a heavy chain variable domain (V Ｈ ); and (ii) the following complementarity determining regions: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 30 or a sequence which differs therefrom by one or two amino acids; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 31 or a sequence that differs therefrom by one or two amino acids, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 32 or a sequence that differs therefrom by one or two amino acids. a light chain variable domain (V Ｋ ); (e)(i) a complementarity determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34 or a sequence which differs therefrom by one or two amino acids; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35 or a sequence that differs therefrom by one or two amino acids, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36 or a sequence that differs therefrom by one or two amino acids. a heavy chain variable domain (V Ｈ ); and (ii) the following complementarity determining regions: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 38 or a sequence which differs therefrom by one or two amino acids; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 39 or a sequence that differs therefrom by one or two amino acids, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 40 or a sequence that differs therefrom by one or two amino acids. a light chain variable domain (V Ｋ ); (f)(i) a complementarity determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 42 or a sequence which differs therefrom by one or two amino acids; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 43 or a sequence that differs therefrom by one or two amino acids, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 44 or a sequence that differs therefrom by one or two amino acids. a heavy chain variable domain (V Ｈ ); and (ii) the following complementarity determining regions: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 46 or a sequence which differs therefrom by one or two amino acids; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 47 or a sequence that differs therefrom by one or two amino acids, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 48 or a sequence that differs therefrom by one or two amino acids. a light chain variable domain (V Ｋ ); (g)(i) a complementarity determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 50 or a sequence which differs therefrom by one or two amino acids; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 51 or a sequence that differs therefrom by one or two amino acids, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 52 or a sequence that differs therefrom by one or two amino acids. a heavy chain variable domain (V Ｈ ); and (ii) the following complementarity determining regions: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 54 or a sequence which differs therefrom by one or two amino acids; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 55 or a sequence that differs therefrom by one or two amino acids, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 56 or a sequence that differs therefrom by one or two amino acids. a light chain variable domain (V Ｋ ); (h)(i) a complementarity determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 58 or a sequence which differs therefrom by one or two amino acids; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 59 or a sequence that differs therefrom by one or two amino acids, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 60 or a sequence that differs therefrom by one or two amino acids. a heavy chain variable domain (V Ｈ ); and (ii) the following complementarity determining regions: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 62 or a sequence which differs therefrom by one or two amino acids; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 63 or a sequence that differs therefrom by one or two amino acids, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 64 or a sequence that differs therefrom by one or two amino acids. a light chain variable domain (V Ｋ ); (i) a complementarity determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 66 or a sequence which differs therefrom by one or two amino acids; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 67 or a sequence that differs therefrom by one or two amino acids, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 68 or a sequence that differs therefrom by one or two amino acids. a heavy chain variable domain (V Ｈ ); and (ii) the following complementarity determining regions: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 70 or a sequence which differs therefrom by one or two amino acids; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 71 or a sequence that differs therefrom by one or two amino acids, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 72 or a sequence that differs therefrom by one or two amino acids. a light chain variable domain (V Ｋ ); (j)(i) a complementarity determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 74 or a sequence which differs therefrom by one or two amino acids; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 75 or a sequence which differs therefrom by one or two amino acids, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 76 or a sequence that differs therefrom by one or two amino acids. a heavy chain variable domain (V Ｈ ); and (ii) the following complementarity determining regions: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 78 or a sequence which differs therefrom by one or two amino acids; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 79 or a sequence that differs therefrom by one or two amino acids, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 80 or a sequence that differs therefrom by one or two amino acids. a light chain variable domain (V Ｋ ); (k)(i) a complementarity determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 82 or a sequence which differs therefrom by one or two amino acids; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 83 or a sequence that differs therefrom by one or two amino acids, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 84 or a sequence that differs therefrom by one or two amino acids. a heavy chain variable domain (V Ｈ ); and (ii) the following complementarity determining regions: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 86 or a sequence which differs therefrom by one or two amino acids; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 87 or a sequence that differs therefrom by one or two amino acids, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 88 or a sequence that differs therefrom by one or two amino acids. a light chain variable domain (V Ｋ ); (l)(i) a complementarity determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 90 or a sequence which differs therefrom by one or two amino acids; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 91 or a sequence that differs therefrom by one or two amino acids, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 92 or a sequence that differs therefrom by one or two amino acids. a heavy chain variable domain (V Ｈ ); and (ii) the following complementarity determining regions: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 94 or a sequence which differs therefrom by one or two amino acids; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 95 or a sequence that differs therefrom by one or two amino acids, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 96 or a sequence that differs therefrom by one or two amino acids. a light chain variable domain (V Ｋ ); (m)(i) a complementarity determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 98 or a sequence which differs therefrom by one or two amino acids; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 99 or a sequence which differs therefrom by one or two amino acids, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 100 or a sequence that differs therefrom by one or two amino acids. a heavy chain variable domain (V Ｈ ); and (ii) the following complementarity determining regions: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 102 or a sequence which differs therefrom by one or two amino acids; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 103 or a sequence that differs therefrom by one or two amino acids, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 104 or a sequence that differs therefrom by one or two amino acids. a light chain variable domain (V Ｋ ); (n)(i) a complementarity determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 106 or a sequence which differs therefrom by one or two amino acids; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 107 or a sequence that differs therefrom by one or two amino acids, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 108 or a sequence that differs therefrom by one or two amino acids. a heavy chain variable domain (V Ｈ ); and (ii) the following complementarity determining regions: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 110 or a sequence which differs therefrom by one or two amino acids; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 111 or a sequence that differs therefrom by one or two amino acids, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 112 or a sequence that differs therefrom by one or two amino acids. a light chain variable domain (V Ｋ ); (o)(i) a complementarity determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 114 or a sequence which differs therefrom by one or two amino acids; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 115 or a sequence that differs therefrom by one or two amino acids, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 116 or a sequence that differs therefrom by one or two amino acids. a heavy chain variable domain (V Ｈ ); and (ii) the following complementarity determining regions: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 118 or a sequence which differs therefrom by one or two amino acids; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 119 or a sequence that differs therefrom by one or two amino acids, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 120 or a sequence that differs therefrom by one or two amino acids. a light chain variable domain (V Ｋ ); (p)(i) a complementarity determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 122 or a sequence which differs therefrom by one or two amino acids; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 123 or a sequence that differs therefrom by one or two amino acids, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 124 or a sequence that differs therefrom by one or two amino acids. a heavy chain variable domain (V Ｈ ); and (ii) the following complementarity determining regions: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 126 or a sequence which differs therefrom by one or two amino acids; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 127 or a sequence that differs therefrom by one or two amino acids, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 128 or a sequence that differs therefrom by one or two amino acids. a light chain variable domain (V Ｋ );or (q)(i) a complementarity determining region of: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 130 or a sequence which differs therefrom by one or two amino acids; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 131 or a sequence that differs therefrom by one or two amino acids, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 132 or a sequence that differs therefrom by one or two amino acids. a heavy chain variable domain (V Ｈ ); and (ii) the following complementarity determining regions: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 134 or a sequence which differs therefrom by one or two amino acids; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 135 or a sequence that differs therefrom by one or two amino acids, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 136 or a sequence that differs therefrom by one or two amino acids. a light chain variable domain (V Ｋ ). An antibody or antigen-binding fragment thereof comprising:

2. the CDR-H1, the CDR-H2, the CDR-H3, the CDR-L1, the CDR-L2, and the CDR-L3 have the following sequences: (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, and 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, and 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, and 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, and 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, and 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, and 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, and SEQ ID NO:72, respectively; (j) SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:79, and SEQ ID NO:80, respectively; (k) SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:87, and SEQ ID NO:88, respectively; (l) SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:95, and SEQ ID NO:96, respectively; (m) SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:103, and SEQ ID NO:104, respectively; (n) SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 111, and SEQ ID NO: 112, respectively; (o) SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:119, and SEQ ID NO:120, respectively; (p) SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:127, and SEQ ID NO:128, respectively; or (q) SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 135, and SEQ ID NO: 136, respectively.

2. The antibody or antigen-binding fragment thereof according to claim 1, comprising an amino acid sequence that collectively differs from said antibody or antigen-binding fragment by no more than two amino acid residues.

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

4. (i) the following complementarity determining regions: CDR-H1 comprising the amino acid sequence of SEQ ID NO:2; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 3; and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 4 Including, V Ｈ and (ii) the following complementarity determining regions: CDR-L1 comprising the amino acid sequence of SEQ ID NO:6; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 7; and CDR-L3 comprising the amino acid sequence of SEQ ID NO:8 Including, V Ｋ The antibody or antigen-binding fragment thereof of claim 3, comprising:

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

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

7. (i) the following complementarity determining regions: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 10; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 11; and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 12 Including, V Ｈ and (ii) the following complementarity determining regions: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 14; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 15; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 16 Including, V Ｋ The antibody or antigen-binding fragment thereof of claim 3, comprising:

8. The V Ｈ comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:9; The V Ｋ comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 13; An antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

9. The V Ｈ comprises the amino acid sequence of SEQ ID NO:9; The V Ｋ comprises the amino acid sequence of SEQ ID NO: 13; The antibody or antigen-binding fragment thereof according to claim 8.

10. (i) the following complementarity determining regions: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 18; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 19; and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 20 Including, V Ｈ and (ii) the following complementarity determining regions: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 24 Including, V Ｋ The antibody or antigen-binding fragment thereof of claim 3, comprising:

11. The V Ｈ comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 17; The V Ｋ comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 21; An antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

12. The V Ｈ comprises the amino acid sequence of SEQ ID NO: 17; The V Ｋ comprises the amino acid sequence of SEQ ID NO: 21; The antibody or antigen-binding fragment thereof according to claim 11.

13. (i) the following complementarity determining regions: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35; and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36 Including, V Ｈ and (ii) the following complementarity determining regions: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 38; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 39; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 40 Including, V Ｋ The antibody or antigen-binding fragment thereof of claim 3, comprising:

14. The V Ｈ comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 33; The V Ｋ comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 37; The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

15. The V Ｈ comprises the amino acid sequence of SEQ ID NO: 33; The V Ｋ comprises the amino acid sequence of SEQ ID NO: 37; The antibody or antigen-binding fragment thereof according to claim 14.

16. (i) the following complementarity determining regions: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 98; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 99; and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 100 Including, V Ｈ and (ii) the following complementarity determining regions: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 102; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 103; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 104 Including, V Ｋ The antibody or antigen-binding fragment thereof of claim 3, comprising:

17. The V Ｈ comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:97; The V Ｋ comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 101; The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

18. The V Ｈ comprises the amino acid sequence of SEQ ID NO:97; The V Ｋ comprises the amino acid sequence of SEQ ID NO: 101; 17. The antibody or antigen-binding fragment thereof of claim 16.

19. (a) the V Ｈ comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, Ｋ comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:5; (b) the V Ｈ comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9, 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, Ｋ comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:21; (d) the V Ｈ comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 25, Ｋ comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:29; (e) the V Ｈ comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 33, 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, Ｋ comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:45; (g) the V Ｈ comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 49, Ｋ comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 53; (h) the V Ｈ comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 57, Ｋ comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 61; (i) the V Ｈ comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 65, Ｋ comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 69; (j) the V Ｈ comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 73, Ｋ comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 77; (k) the V Ｈ comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 81, 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, 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, Ｋ comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 101; (n) the V Ｈ comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 105, Ｋ comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 109; (o) the V Ｈ comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 113, Ｋ comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 117; (p) the V Ｈ comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 121, 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) the V Ｈ comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 129, Ｋ comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 133; The antibody or antigen-binding fragment thereof according to claim 1.

20. (a) the V Ｈ comprises the amino acid sequence of SEQ ID NO: 1, Ｋ comprises the amino acid sequence of SEQ ID NO:5; (b) the V Ｈ comprises the amino acid sequence of SEQ ID NO: 9, 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, Ｋ comprises the amino acid sequence of SEQ ID NO:21; (d) the V Ｈ comprises the amino acid sequence of SEQ ID NO: 25, Ｋ comprises the amino acid sequence of SEQ ID NO:29; (e) the V Ｈ comprises the amino acid sequence of SEQ ID NO: 33, 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, Ｋ comprises the amino acid sequence of SEQ ID NO: 45; (g) the V Ｈ comprises the amino acid sequence of SEQ ID NO: 49, Ｋ comprises the amino acid sequence of SEQ ID NO:53; (h) the V Ｈ comprises the amino acid sequence of SEQ ID NO: 57, Ｋ comprises the amino acid sequence of SEQ ID NO: 61; (i) the V Ｈ comprises the amino acid sequence of SEQ ID NO: 65, Ｋ comprises the amino acid sequence of SEQ ID NO:69; (j) the V Ｈ comprises the amino acid sequence of SEQ ID NO: 73, Ｋ comprises the amino acid sequence of SEQ ID NO: 77; (k) the V Ｈ comprises the amino acid sequence of SEQ ID NO: 81, 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, 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, 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, Ｋ comprises the amino acid sequence of SEQ ID NO: 109; (o) the V Ｈ comprises the amino acid sequence of SEQ ID NO: 113, Ｋ comprises the amino acid sequence of SEQ ID NO: 117; (p) the V Ｈ comprises the amino acid sequence of SEQ ID NO: 121, Ｋ comprises the amino acid sequence of SEQ ID NO: 125; or (q) the V Ｈ comprises the amino acid sequence of SEQ ID NO: 129, Ｋ comprises the amino acid sequence of SEQ ID NO: 133; The antibody or antigen-binding fragment thereof according to claim 1.

21. K for human Notch4 of 10 nM or less Ｄ 6. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment is capable of binding at a value of 0.1 to 0.

25.

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

23. An antibody or antigen-binding fragment thereof according to any one of the preceding claims, comprising 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. 26. An expression vector comprising the isolated nucleic acid of claim 25.

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

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

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

31. 30. The method of claim 29, wherein the subject is a mammal.

32. 31. The method of claim 30, wherein the subject is a human.

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

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

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

36. 35. The method of claim 34, wherein the systemic route is selected from an intravenous route, an intramuscular route, or a subcutaneous route.

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

Images