METHODS OF USING IL-33 ANTAGONISTS
20220380450 · 2022-12-01
Inventors
- Rania DAGHER (Gaithersburg, MD, US)
- Kirsty HOUSLAY (San Francisco, CA, US)
- Mahboobe GHAEDI (Gaithersburg, MD, US)
- Sam STRICKSON (San Francisco, CA, US)
- Emma Suzanne COHEN (San Francisco, CA, US)
- Maria BELVISI (Gaithersburg, MD, US)
- Xavier ROMERO ROS (Cambridge, GB)
Cpc classification
C07K2317/76
CHEMISTRY; METALLURGY
C07K2317/51
CHEMISTRY; METALLURGY
International classification
Abstract
The present disclosure relates to an IL-33 antagonist for use in the prevention or treatment of abnormal epithelium physiology or EGFR-mediated diseases, and corresponding methods of prevention or treatment comprising administering an IL-33 antagonist to a patient in need thereof.
Claims
1. An IL-33 antagonist for use in the prevention or treatment of abnormal epithelium physiology by modulating or inhibiting a RAGE-EGFR mediated effect.
2. An IL-33 antagonist for use according to claim 1, wherein the abnormal epithelium physiology is abnormal mucociliary physiology, preferably abnormal mucociliary physiology of respiratory epithelium.
3. An IL-33 antagonist for use according to claim 2, wherein abnormal mucociliary physiology is selected from: abnormal mucus production; abnormal goblet cell differentiation, abnormal goblet cell proliferation; abnormal thickness of the epithelium; abnormal mucus clearance; and/or abnormal mucus composition.
4. An IL-33 antagonist for use according to claim 3, wherein abnormal mucus production comprises abnormal MUC5AC production; and/or wherein abnormal goblet cell differentiation comprises abnormal MUC5AC+goblet cell differentiation; and/or wherein abnormal goblet cell proliferation comprises abnormal MUC5AC+goblet cell proliferation; and/or wherein abnormal thickness of the epithelium comprises an abnormal amount of MUC5AC.sup.+ goblet cells in the total tissue area of the epithelium.
5. An IL-33 antagonist for use according to claim 2, 3 or 4, wherein abnormal mucociliary physiology comprises: increased mucus production; increased goblet cell differentiation; increased goblet cell proliferation; increased thickness of the epithelium; and/or decreased mucus clearance.
6. An IL-33 antagonist for use according to claim 5, wherein increased mucus production comprises increased MUC5AC production; and/or wherein increased goblet cell differentiation comprises increased MUC5AC+goblet cell differentiation; and/or wherein increased goblet cell proliferation comprises increased MUC5AC+goblet cell proliferation; and/or wherein increased thickness of the epithelium comprises an increased amount of MUC5AC.sup.+ goblet cells in the total tissue area of the epithelium.
7. An IL-33 antagonist for use according to claim 3, wherein abnormal mucus composition comprises an increase or a decrease in the ratio of the different mucus compounds contained in mucus; an increase or decrease in one or more mucus compounds; and/or an increase or decrease in the concentration or thickness of mucus.
8. An IL-33 antagonist for use according to claim 7, wherein abnormal mucus composition comprises an increase in the ratio of MUC5AC:MUC5B; and/or wherein abnormal mucus composition comprises an increase in MUC5AC contained in mucus; and/or wherein abnormal mucus composition comprises an increase in thickness of mucus.
9. An IL-33 antagonist for use according to claim 1, wherein the abnormal epithelium physiology is abnormal epithelium remodeling.
10. An IL-33 antagonist for use according to any preceding claim, wherein the abnormal epithelium physiology is of the respiratory tract, preferably abnormal mucociliary physiology of the respiratory tract.
11. An IL-33 antagonist for use according to claim 10, wherein the respiratory tract is the lower respiratory tract, preferably the bronchi.
12. An IL-33 antagonist for use in the prevention or treatment of an EGFR-mediated disease.
13. An IL-33 antagonist for use according to claim 12, wherein the EGFR-mediated disease is a RAGE-EGFR mediated disease.
14. An IL-33 antagonist for use according to claim 12 or 13 wherein the EGFR mediated disease is characterized by aberrant EGFR activity.
15. An IL-33 antagonist for use according to any of claims 12-14 wherein the EGFR mediated disease is characterized by abnormal epithelium physiology.
16. An IL-33 antagonist for use in the prevention or treatment of a disease by improving epithelium physiology.
17. An IL-33 antagonist for use in the prevention or treatment of a disease by inhibiting an EGFR-mediated effect.
18. An IL-33 antagonist for use according to claim 17, wherein the EGFR-mediated effect is EGFR signalling.
19. An IL-33 antagonist for use according to claim 17 or 18, wherein the EGFR-mediated effect is a RAGE-EGFR-mediated effect.
20. An IL-33 antagonist for use according to any of claims 17-19, wherein the EGFR-mediated effect is RAGE-EGFR-mediated signalling.
21. An IL-33 antagonist for use according to any of claims 16-20, wherein the disease is a respiratory disease.
22. An IL-33 antagonist for use according to claim 21 wherein the respiratory disease is characterised by abnormal epithelium physiology and/or aberrant EGFR activity.
23. An IL-33 antagonist for use according to claim 21 or 22, wherein the respiratory disease is a lower respiratory disease, preferably a respiratory disease of the bronchi.
24. An IL-33 antagonist for use according to any of claims 21-23, wherein the respiratory disease is selected from: COPD, bronchitis, emphysema, bronchiectasis, such as CF-bronchiectasis or -CF-bronchiectasis, asthma or asthma and COPD overlap (ACO).
25. An IL-33 antagonist for use according to any of claims 21-24, wherein the respiratory disease is COPD, preferably bronchitic COPD.
26. An IL-33 antagonist for use according to any of claims 21-24, wherein the respiratory disease is asthma, preferably bronchitic asthma.
27. An IL-33 antagonist for use according to any of claims 16-26, wherein the prevention or treatment improves mucus clearance.
28. An IL-33 antagonist for use according to any of claims 16-27, wherein the prevention or treatment inhibits or reduces abnormal mucus production.
29. An IL-33 antagonist for use according to claim 28, wherein the prevention or treatment inhibits or reduces MUC5AC production.
30. An IL-33 antagonist for use according to any of claims 16-29, wherein the prevention or treatment inhibits an abnormal mucus composition.
31. An IL-33 antagonist for use according to claim 30, wherein the prevention or treatment inhibits or reduces the ratio of MUC5AC:MUC5B; and/or wherein the prevention or treatment inhibits or reduces MUC5AC in mucus; and/or wherein the prevention or treatment reduces the thickness of mucus.
32. An IL-33 antagonist for use according to any of claims 16-31, wherein the prevention or treatment inhibits abnormal epithelium remodelling.
33. An IL-33 antagonist for use according to any of claims 16-32, wherein the prevention or treatment inhibits abnormal goblet cell differentiation or proliferation.
34. An IL-33 antagonist for use according to claim 33, wherein the abnormal goblet cell differentiation or proliferation is abnormal MUC5AC.sup.+ goblet cell differentiation or proliferation.
35. An IL-33 antagonist for use according to any of claims 16-34, wherein the prevention or treatment reduces the thickness of the respiratory epithelium.
36. An IL-33 antagonist for use according to claim 35, wherein the prevention or treatment reduces the amount of MUC5AC.sup.+ goblet cells in the total tissue area of the epithelium.
37. An IL-33 antagonist for use according to any preceding claim, wherein the IL-33 antagonist inhibits the activity of oxidised IL-33.
38. An IL-33 antagonist for use according to any preceding claim, wherein the IL-33 antagonist prevents binding of oxidised IL-33 to RAGE, thereby inhibiting RAGE-EGFR signalling.
39. An IL-33 antagonist for use according to any preceding claim, wherein the IL-33 antagonist down-regulates or inhibits RAGE-EGFR dependent signalling and/or RAGE-EGFR mediated effects.
40. An IL-33 antagonist for use according to any preceding claim, wherein the IL-33 antagonist is a binding molecule or fragment thereof which binds to IL-33, preferably which binds to reduced IL-33 or oxidised IL-33, preferably which binds to reduced IL-33.
41. An IL-33 antagonist for use according to any preceding claim, wherein the IL-33 antagonist is an antibody or antigen-binding fragment thereof, preferably an anti-IL-33 antibody or antigen-binding fragment thereof, preferably an anti-reduced-IL-33 antibody or antigen-binding fragment thereof.
42. An IL-33 antagonist for use according to any preceding claim, wherein the IL-33 antagonist is a binding molecule which comprises complementarity determining regions (CDRs) of a variable heavy domain (VH) and a variable light domain (VL) pair selected from Table 1.
43. An IL-33 antagonist for use according to any preceding claim, wherein the IL-33 antagonist is a binding molecule which comprises a variable heavy domain (VH) and variable light domain (VL) pair selected from Table 1.
44. An IL-33 antagonist for use according to any preceding claim, wherein the IL-33 antagonist is a binding molecule which comprises a VHCDR1 having the sequence of SEQ ID NO: 37, a VHCDR2 having the sequence of SEQ ID NO: 38, a VHCDR3 having the sequence of SEQ ID NO: 39, a VLCDR1 having the sequence of SEQ ID NO: 40, a VLCDR2 having the sequence of SEQ ID NO: 41, and a VLCDR3 having the sequence of SEQ ID NO: 42.
45. A method for the prevention or treatment of abnormal epithelium physiology in a subject, by administering a therapeutically effective amount of an IL-33 antagonist to modulate or inhibit a RAGE-EGFR mediated effect.
46. The method of claim 45, wherein the abnormal epithelium physiology is abnormal mucociliary physiology, preferably abnormal mucociliary physiology of respiratory epithelium.
47. The method of claim 46, wherein abnormal mucociliary physiology is selected from: abnormal mucus production; abnormal goblet cell differentiation; abnormal goblet cell proliferation; abnormal thickness of the epithelium; abnormal mucus clearance; and/or abnormal mucus composition.
48. The method of claim 47, wherein abnormal mucus production comprises abnormal MUC5AC production; and/or wherein abnormal goblet cell differentiation comprises abnormal MUC5AC+goblet cell differentiation; and/or wherein abnormal goblet cell proliferation comprises abnormal MUC5AC+goblet cell proliferation; and/or wherein abnormal thickness of the epithelium comprises an abnormal amount of MUC5AC.sup.+ goblet cells in the total tissue area of the epithelium.
49. The method of claim 47 or 48, wherein abnormal mucociliary physiology comprises: increased mucus production; increased goblet cell differentiation; increased goblet cell proliferation; increased thickness of the epithelium; and/or decreased mucus clearance.
50. The method of claim 49, wherein increased mucus production comprises increased MUC5AC production; and/or wherein increased goblet cell differentiation comprises increased MUC5AC+goblet cell differentiation; and/or wherein increased goblet cell proliferation comprises increased MUC5AC+goblet cell proliferation; and/or wherein increased thickness of the epithelium comprises an increased amount of MUC5AC.sup.+ goblet cells in the total tissue area of the epithelium.
51. The method of claim 47, wherein abnormal mucus composition comprises an increase or a decrease in the ratio of the different mucus compounds contained in mucus; an increase or decrease in one or more mucus compounds; and/or an increase or decrease in the concentration or thickness of mucus.
52. The method of claim 51, wherein abnormal mucus composition comprises an increase in the ratio of MUC5AC:MUC5B; and/or wherein abnormal mucus composition comprises an increase in MUC5AC contained in mucus; and/or wherein abnormal mucus composition comprises an increase in thickness of mucus.
53. The method of claim 45, wherein the abnormal epithelium physiology is abnormal epithelium remodelling.
54. The method of any of claims 45-53, wherein the abnormal epithelium physiology is of the respiratory tract, preferably abnormal mucociliary physiology of the respiratory tract.
55. The method of claim 54, wherein the respiratory tract is the lower respiratory tract, preferably the bronchi.
56. The method for the prevention or treatment of an EGFR-mediated disease, by administering a therapeutically effective amount of an IL-33 antagonist.
57. The method of claim 56, wherein the EGFR-mediated disease is a RAGE-EGFR mediated disease.
58. The method of claim 56 or 57, wherein the EGFR mediated disease is characterised by aberrant EGFR activity.
59. The method of any of claims 56-58 wherein the EGFR mediated disease is characterised by abnormal epithelium physiology.
60. A method for the prevention or treatment of a disease, by administering a therapeutically effective amount of an IL-33 antagonist to improve epithelium physiology.
61. A method for the prevention or treatment of a disease, by administering a therapeutically effective amount of an IL-33 antagonist to inhibit an EGFR-mediated effect.
62. The method of claim 61, wherein the EGFR-mediated effect is EGFR signalling.
63. The method of claim 61 or 62, wherein the EGFR-mediated effect is a RAGE-EGFR-mediated effect.
64. The method of any of claims 61-63, wherein the EGFR-mediated effect is RAGE-EGFR-mediated signalling.
65. The method of any of claims 60-64, wherein the disease is a respiratory disease.
66. The method of claim 65, wherein the respiratory disease is characterised by abnormal epithelium physiology and/or aberrant EGFR activity.
67. The method of claim 65 or 66, wherein the respiratory disease is a lower respiratory disease, preferably a respiratory disease of the bronchi.
68. The method of any of claims 65-67, wherein the respiratory disease is selected from: COPD, bronchitis, emphysema, bronchiectasis, such as CF-bronchiectasis or -CF-bronchiectasis, asthma or asthma and COPD overlap (ACO).
69. The method of any of claims 65-68, wherein the respiratory disease is COPD, preferably bronchitic COPD.
70. The method of any of claims 65-69, wherein the respiratory disease is asthma, preferably bronchitic asthma.
71. The method of any of claims 45-70, wherein the method improves mucus clearance.
72. The method of any of claims 45-71, wherein the method inhibits or reduces abnormal mucus production.
73. The method of claim 72, wherein the abnormal mucus production is an increase in MUC5AC production.
74. The method of any of claims 45-73, wherein the method inhibits an abnormal mucus composition.
75. The method of claim 74, wherein the method inhibits or reduces the ratio of MUC5AC:MUC5B; and/or wherein the method inhibits or reduces MUC5AC in mucus; and/or wherein the method reduces the thickness of mucus.
76. The method of any of claims 45-75, wherein the method inhibits abnormal epithelium remodeling.
77. The method of any of claims 45-76, wherein the method inhibits or reduces abnormal goblet cell differentiation or proliferation.
78. The method of claim 77, wherein the method inhibits or reduces abnormal MUC5AC.sup.+ goblet cell differentiation or proliferation.
79. The method of any of claims 45-78, wherein the method reduces the thickness of the respiratory epithelium.
80. The method of claim 79, wherein the method reduces the amount of MUC5AC.sup.+ goblet cells in the total tissue area of the respiratory epithelium.
81. The method of any of claims 45-80, wherein the IL-33 antagonist inhibits the activity of oxidised IL-33.
82. The method of any of claims 45-81, wherein the IL-33 antagonist prevents binding of oxidised IL-33 to RAGE, thereby inhibiting RAGE-EGFR signalling.
83. The method of any of claims 45-82, wherein the IL-33 antagonist down-regulates or inhibits RAGE-EGFR dependent signalling and/or RAGE-EGFR mediated effects.
84. The method of any of claims 45-83, wherein the IL-33 antagonist is a binding molecule or fragment thereof which binds to IL-33, preferably reduced IL-33 or oxidised IL-33, preferably reduced IL-33.
85. The method of any of claims 45-84, wherein the IL-33 antagonist is an antibody or antigen-binding fragment thereof, preferably an anti-IL-33 antibody or antigen-binding fragment thereof, preferably an anti-reduced-IL-33 antibody or antigen-binding fragment thereof.
86. The method of any of claims 45-85, wherein the IL-33 antagonist is a binding molecule which comprises complementarity determining regions (CDRs) of a variable heavy domain (VH) and a variable light domain (VL) pair selected from Table 1.
87. The method of any of claims 45-86, wherein the IL-33 antagonist is a binding molecule which comprises a variable heavy domain (VH) and variable light domain (VL) pair selected from Table 1.
88. The method of any of claims 45-87, wherein the IL-33 antagonist is a binding molecule which comprises a VHCDR1 having the sequence of SEQ ID NO: 37, a VHCDR2 having the sequence of SEQ ID NO: 38, a VHCDR3 having the sequence of SEQ ID NO: 39, a VLCDR1 having the sequence of SEQ ID NO: 40, a VLCDR2 having the sequence of SEQ ID NO: 41, and a VLCDR3 having the sequence of SEQ ID NO: 42.
89. The method of any of claims 45-88, or the IL-33 antagonist for use according to any of claims 1-44, wherein the IL-33 antagonist is an anti-IL33 antibody or antigen binding fragment thereof comprising a VH domain of the sequence of SEQ ID NO:1 and a VL domain of the sequence of SEQ ID NO:19.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0297] Embodiments will be described, by way of example, with reference to the following drawings, in which:
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EXAMPLES
Example 1—Oxidised IL-33 Drives Formation of a Signaling Complex Between RAGE and EGFR
[0338] In Cohen, E. S. et al. Nat. Commun. 6:8327 (2015), the applicant described the discovery of an oxidized, disulphide bonded form of IL-33 (DSB IL-33) and showed that this form does not bind ST2 and cannot activate ST2-dependent signalling. Subsequently (see WO2016156440A1), the applicant showed that oxIL-33 binds the Receptor for Advanced Glycation End products (RAGE) and signals in a RAGE-dependent manner to activate STAT5 and affect epithelial migration.
[0339] To further explore the function of oxIL-33, epithelial cells were stimulated with IL-33 in reduced or oxidised forms and signaling pathways were investigated. Here the inventors show that oxIL-33 is a novel ligand for a complex of the receptor for advanced glycation end products (RAGE) and the epidermal growth factor receptor (EGFR), leading to profound effects on epithelial function.
[0340] 1. Cloning and Expression of Human Mature and Cysteine-Mutated Variants of IL33
[0341] cDNA molecules encoding the mature component of human IL-33 (112-270); accession number (UniProt) 095760 (also referred to as IL33-01 or IL-33), and a variant with the 4 cysteine residues mutated to serine (also referred to as IL33-16 or IL-33[C->S]) were synthesized by primer extension PCR and cloned into pJexpress 411 (DNA 2.0). The wild type (WT) and mutant IL-33 coding sequences were modified to contain a 10×His, Avitag, and Factor-Xa protease cleavage site (MHHHHHHHHHHAAGLNDIFEAQKIEWHEAAIEGR SEQ ID NO:43) at the N-terminus of the proteins. N-terminal tagged His10/Avitag IL33-01 (WT, SEQ ID NO:44) and N-terminal tagged His10/Avitag IL33-16 (WT, SEQ ID NO:45) were generated by transforming E. coli BL21(DE3) cells. Transformed cells were cultured in autoinduction media (Overnight Express™ Autoinduction System 1, Merck Millipore, 71300-4) at 37° C. for 18 hours before cells were harvested by centrifugation and stored at −20° C. Cells were resuspended in 2×DPBS containing complete EDTA-free protease inhibitor cocktail tablets (Roche, 11697498001) and 50 U/ml Benzonase nuclease (Merck Millipore, 70746-3) and lysed by sonication. The cell lysate was clarified by centrifugation at 50,000×g for 30 min at 4° C. IL-33 proteins were purified from the supernatant by immobilized metal affinity chromatography, loading on a HisTrap excel column (GE Healthcare, 17371205) equilibrated in 2×DPBS, 1 mM DTT at 5 ml/min. The column was washed with 2×DPBS, 1 mM DTT, 20 mM Imidazole, pH 7.4 to remove impurities and then 2×DPBS, 0.1% Triton X-114 to deplete the immobilised protein of endotoxin. Following further washing with 2×DPBS, 1 mM DTT, 20 mM Imidazole, pH 7.4, the sample was eluted with 2×DPBS, 1 mM DTT, 400 mM Imidazole, pH 7.4. IL-33 was further purified by size exclusion chromatography using a HiLoad Superdex 75 26/600 pg column (GE Healthcare, 28989334) in 2×DPBS at 2.5 ml/min. Peak fractions were analysed by SDS PAGE. Fractions containing pure IL-33 were pooled and the concentration determined by absorbance at 280 nm. Final samples were analysed by SDS-PAGE.
[0342] To generate untagged IL-33, N-terminal tagged His10/Avitag IL33 was incubated with 10 units of Factor Xa (GE healthcare, 27084901) per mg of protein in 2×DPBS buffer at RT for 1 hour. Untagged IL-33 was purified using SEC chromatography in 2×DPBS on a HiLoad 16/600 Superdex 75 pg column (GE healthcare, 28989333) with a flow rate of 1 ml/min.
[0343] 2. Generation and Purification of Oxidised IL-33 (oxIL-33)
[0344] Reduced IL33-01 was oxidised by dilution to a final concentration of 0.5 mg/ml in 60% IMDM medium (with no phenol red), 40% DPBS and incubation at 37° C. for 18 hours. Aggregates generated during the oxidation process were removed from the sample by loading it on a HiTrap Capto Q ImpRes anion exchange column (GE Healthcare, 17547055). Prior to loading, the sample was modified by the addition of 1 M Tris, pH 9.0 until the pH reached 8.3 and the addition of 5 M NaCl to a final concentration of 125 mM—under these loading conditions, aggregates bound to the column and monomeric oxIL-33 flowed through without binding and was collected. Tags were cleaved from the oxIL-33 by incubation with Factor Xa (NEB, P8010L) at a final concentration of 1 μg Factor Xa per 50 μg of oxIL-33 for 120 min at 22° C. To deplete the sample of any remaining reduced IL-33, soluble human ST2S extracellular domain fused to human IgG1 Fc-His6 was incubated with the sample for 30 min at 22° C. and bound the reduced IL-33. The sample was concentrated in a centrifugal concentrator with a 3,000 Da cut-off and loaded on a HiLoad Superdex 75 26/600 pg column (GE Healthcare, 28989334) at a flow rate of 2 ml/min, which separated the monomeric oxIL-33 from the other sample components. Fractions containing pure oxIL-33 were pooled and concentrated and the final concentration of the sample was determined via UV absorbance spectroscopy at 280 nm. Final product quality was assessed by SDS-PAGE, HP-SEC and RP-HPLC.
[0345] 3. Cloning, Expression and Purification of Human ST2 ECD
[0346] A cDNA encoding the naturally occurring ST2S soluble isoform of ST2 (UniProt accession Q01638-2) without the endogenous signal peptide (amino acid residues 19-328) was amplified by PCR with primers encoding extensions compatible with Gibson assembly and a CD33 signal peptide fused to the N-terminus of the ST2S coding sequence. A coding sequence for human IgG1 Fc with a C-terminal His6-tag was similarly amplified. The ST2S cDNA and IgG1 Fc-His6 cDNA were assembled using Gibson assembly with pDEST12.2 OriP, a mammalian, CMV-promoter driven expression vector bearing the OriP origin of replication from EBV, allowing episomal maintenance in cell lines expressing the EBNA-1 protein. For protein expression, the plasmid was transiently transformed into a suspension culture of CHO cells overexpressing EBNA-1 using polyethyleneimine as the transfection reagent. Conditioned medium containing the secreted ST2S-Fc-His6 fusion protein was collected 7 days post-transfection and loaded on a HiTrap MabSelect SuRe (Protein A, GE Healthcare, 11-0034-95) affinity chromatography column at 2 ml/min. The column was washed with 2×DPBS and the protein eluted with 25 mM Sodium acetate, pH 3.6. Fractions containing ST2S-Fc-His6 were pooled and loaded on a HiLoad Superdex 200 26/600 pg column (GE Healthcare, 28989336) equilibrated in 2×DPBS at 2 ml/min. Fractions containing pure ST2S-Fc-His6 protein were pooled and the concentration determined by absorbance at 280 nm. Final samples were analysed by SDS-PAGE.
[0347] 4. Cloning, Expression and Purification of Human Asialoglycoprotein Receptor (ASGPR) ECD
[0348] A cDNA encoding the extracellular domain (ECD) of the Asialoglycoprotein receptor (UniProt accession P07306) without the cytoplasmic and transmembrane domains (amino acid residues 62-291) was chemically synthesized at Geneart with a CD33 signal peptide followed by an His10_Avi Tag sequence fused to the N-terminus of the ECD domain. The construct was cloned directly into pDEST12.2 OriP, a mammalian, CMV-promoter driven expression vector bearing the OriP origin of replication from EBV, allowing episomal maintenance in cell lines expressing the EBNA-1 protein. For protein expression, the plasmid was transiently transformed into a suspension culture of HEK Freestyle 293F cells using 293 Fectin as the transfection reagent. Conditioned medium containing the secreted HisAVi_hASGPR ECD fusion protein was collected 7 days post-transfection by immobilized metal affinity chromatography, loading on a HisTrap excel column (GE Healthcare, 17371205) equilibrated in 2×DPBS, at 4 ml/min. The column was washed with 2×DPBS, 40 mM Imidazole, pH 7.4 to remove impurities and the sample was eluted with 2×DPBS, 400 mM Imidazole, pH 7.4. Human ASGPR ECD was further purified by size exclusion chromatography using a HiLoad Superdex 75 16/600 pg column (GE Healthcare, 28-9893-33) in 2×DPBS at 1 ml/min. Peak fractions were analysed by SDS PAGE. Fractions containing pure monomeric ASGPR were pooled and the concentration determined by absorbance at 280 nm. Final samples were analysed by SDS-PAGE.
[0349] 5. The Oxidised Form of IL-33 Activates MAP Kinase Pathways
[0350] Normal Human Bronchial Epithelial (NHBE) cells (CC-2540) were obtained from Lonza and were maintained in complete BEGM media (Lonza) according to the manufacturer's protocol. NHBEs were harvested with accutase (PAA, #L11-007) and seeded at 1×10.sup.6/2 ml in a 6-well dish (Corning Costar, 3516) in culture media [BEGM (Lonza CC-3171) and supplement kit (Lonza CC-4175)]. Cells were incubated at 37° C., 5% CO.sub.2 for 18-24 hours. After this time, media was aspirated, and the cells were washed twice with 1 ml PBS before the addition of starve media (BEGM (Lonza CC-3171) supplemented with 1% Penicillin/Streptomycin). The plates were then incubated at 37° C., 5% CO.sub.2 for a further 18-24 hours before stimulation.
[0351] MAP kinase phosphorylation antibody array kits (ab211061) were purchased from Abcam and experiments were carried out as per the manufacturer's instructions. NHBEs in a 6 well dish that had been starved for 18-24 h were left untreated or treated with 30 ng/ml of either reduced IL-33, IL-33-16 or oxidised IL-33 before being returned to an incubator 37° C., 5% CO.sub.2 for 10 mins (see Table 2 for activators used in this assay). The plates were removed from the incubator and the cells washed with ice-cold PBS before the addition of 100 μl/per well of 1× lysis buffer supplied with the kits. Protein extracts were transferred to 1.5 ml tubes before being clarified at 14,000 rpm at 4° C. Protein concentration was determined using the BCA technique (Thermo, 23225) and 250 μg of total protein was used per array membrane. All subsequent steps were carried out following the manufacturer's instructions. Membranes were visualised on a LiCor C-digit and quantified using Image Lite studio.
TABLE-US-00002 TABLE 2 Final conc Agonist Identifier Reconstitute in (μg/ml) Untagged oxidised IL33-01 RD15 PBS 100 Untagged IL33-01 Jul. 24, 2015 PBS 100 Untagged IL33-16 Nov. 12, 2015 PBS 100 EGF 236-EG-200 PBS 100
[0352] In contrast to the wild type (IL-33) and C->S (IL-33[C->S]) reduced forms of IL-33 (IL33-01 and IL33-16, respectively), oxidised IL33-01 (oxIL-33) activated multiple key signalling molecules (
[0353] 6. The Oxidised Form of IL-33 Activates Epidermal Growth Factor Receptor (EGFR)
[0354] To try and identify receptor tyrosine kinases (RTK) that were activated by oxIL-33, screening was performed using a 71 RTK array. RTK phosphorylation antibody array kits (ab193662) were purchased from Abcam and experiments were carried out as per the manufacturer's instructions. NHBEs were cultured and seeded at 1×10.sup.6/2 ml in a 6-well plate (Corning Costar, 3516) in culture media [BEGM (Lonza CC-3171) and supplement kit (Lonza CC-4175)]. Cells were incubated at 37° C., 5% CO.sub.2 for 18-24 hours. After this time, media was aspirated, and the cells were washed twice with 1 ml PBS before the addition of starve media (BEGM (Lonza CC-3171) without supplement kit). The plates were then incubated at 37° C., 5% CO.sub.2 for a further 18-24 hours before stimulation. Following the same steps previously described for the MAP kinase array, cells were activated (Table 2 activators), lysed and 250 μg of total protein was used per array membrane. All subsequent steps were carried out following the manufacturer's instructions. Membranes were visualised on a LiCor C-digit and quantified using Image Lite studio. There was no response detected to either reduced wild type (IL-33) or C->S (IL-33[C->S]) IL-33 (IL33-01 and IL33-16, respectively). However, oxIL-33 (oxidised IL-33-01) triggered a positive signal on the RTK array corresponding to epidermal growth factor receptor (EGFR) (
[0355] The ability of oxIL-33 (oxidised IL-33-01) to stimulate EGFR signalling was confirmed by additional methods. Upon activation, EGFR is phosphorylated at Tyr1068 and this phospho-EGFR can be detected using a homogeneous FRET (fluorescence resonance energy transfer) HTRF® (Homogeneous Time-Resolved Fluorescence, Cisbio International) assay (Cisbio kit #64EG1PEH). Briefly, NHBEs were plated at 5×10.sup.5/100 μl in a 96-well plate (Corning Costar, 3598) in culture media [BEGM (Lonza CC-3171) and supplement kit (Lonza CC-4175)]. The plates were incubated at 37° C., 5% CO.sub.2 for 18-24 hours. After this time, media was aspirated, and the cells were washed twice with 0.2 ml PBS before the addition of starve media (BEGM (Lonza CC-3171) without supplement kit). The plates were then incubated at 37° C., 5% CO.sub.2 for a further 18-24 hours before stimulating with increasing concentrations of IL-33-01, IL-33-16 and oxIL-33 (oxidised IL-33-01) and EGFR ligands (Tables 2 & 3) before being returned to an incubator 37° C., 5% CO.sub.2 for 10 mins. The media was aspirated and replaced with 50 μl of lysis buffer per well (Cisbio, 64EG1PEH). The assay was then carried out as per the manufacturer's instructions (Cisbio, 64EG1PEH). Time resolved fluorescence was read at 620 nm and 665 nm emission wavelengths using an EnVision plate reader (Perkin Elmer). Data were analysed by calculating the 665/620 nm ratio and EC50 values determined using GraphPad Prism software by curve fitting using a four-parameter logistic equation.
TABLE-US-00003 TABLE 3 Final conc Agonist Supplier Identifier Reconstituted in (μg/ml) TGFα R&D 239-A-100 10 mM 100 systems acetic acid HB-EGF R&D 259-HE-050/CF PBS 100 systems Amphiregulin R&D 262-AR-100/CF PBS 100 (AREG) systems Betacellulin/ R&D 261-CE-010/CF PBS 100 BTC systems Epiregulin R&D 1195-EP-025/CF PBS 100 systems Epigen R&D 6629-EP-025/CF PBS 100 systems HMGB1 R&D 1690-HMB-050 PBS 200 systems S100A8/A9 R&D 8226-S8-050 PBS 500 systems S100A12 R&D 1052-ER-050 PBS 200 systems S100B R&D 1820-SB-050 PBS 200 systems
[0356] Similarly, EGFR phosphorylation was assessed in the epithelial cell line A549 utilizing HTRF assay as previously mentioned in this section. Briefly, A549s were obtained from ATCC and cultured in RPMI GlutaMax medium supplemented with 1% Penicillin/Streptomycin and 10% FBS. Cells were harvested with accutase (PAA, #L11-007) and seeded into 96 well plates at 5×10.sup.5/100 μl and incubated at 37° C., 5% CO.sub.2 for 18-24 hours. The wells were then washed twice with 100 μl of PBS before addition of 100 μl of starve media (RPMI GlutaMax medium supplemented with 1% Penicillin/Streptomycin) and incubated at 37° C., 5% CO.sub.2 for 18-24 hours. Cells were stimulated with increasing concentrations of IL-33-01, IL-33-16 and oxIL-33-01 (synonym of oxidised IL-33-01), EGFR ligands and RAGE ligands (Tables 2 & 3) before being returned to an incubator 37° C., 5% CO.sub.2 for 10 mins. The media was aspirated and replaced with 50 μl of lysis buffer per well (Cisbio, 64EG1PEH). The assay was then carried out as per the manufacturer's instructions (Cisbio, 64EG1PEH). Time resolved fluorescence was read at 620 nm and 665 nm emission wavelengths using an EnVision plate reader (Perkin Elmer). Data were analysed by calculating the 665/620 nm ratio and EC50 values determined using GraphPad Prism software by curve fitting using a four-parameter logistic equation.
[0357] In both NHBE and A549 cells, oxIL-33 promoted phosphorylation of the EGFR similarly to a bona fide agonist, EGF (
[0358] 7. Western Blotting of Signaling Components
[0359] Western blot experiments were performed to further investigate which elements of the EGFR signalling complex are activated in response to oxIL-33 (oxidised IL33-01). NHBEs were cultured and plated in 6 well dishes as described above in section 5. Following serum starvation, cells were stimulated with oxIL-33 (30 ng/ml) for between 5 to 240 minutes. The media was then aspirated and the cells were washed with ice-cold PBS before the addition of 150 μl of lysis buffer [1×LDS sample buffer (Thermo, NP0008), 10 mM MgCl2 (VWR, 7786-30-3), 2.5% β-mercaptoethanol (Sigma, M6250) and 0.4 mg/ml benzonase (Millipore, 70746)]. Cells were left on ice for 10 mins before lysate was transferred to 1.5 ml tubes and heated to 90° C. for 5 mins. Solutions were transferred to new 1.5 ml tubes and 10 μl of sample along with 5 μl of protein ladder (BioRad, 1610374) was run on a 4-12% SDS-PAGE gel (Thermo, NW04127BOX) in IVIES running buffer (B0002). Gels were transferred onto PVDF membranes (BioRad, 1704156) using a Transblot Turbo (BioRad). PVDF membranes were blocked in PBS-tween solution containing 5% skimmed milk powder (Marvel) for 10 minutes. Membranes were then incubated with primary antibodies in PBS-tween containing 5% BSA over night at 4° C. The membranes were then washed five times with PBS-tween and then incubated with secondary HRP tagged antibodies in PBS-tween containing 5% skimmed milk powder for 1 hour at room temperature. The membranes were then washed five times with PBS-tween before the addition of ECL (BioRad, 1705062) and visualisation of a Licor C-digit.
[0360] The results show that oxIL-33-01 activated several EGFR signaling components (
[0361] 8. Ox-IL-33 Induces STAT-5 Phosphorylation, which is Blocked by EGFR-Neutralizing Ab
[0362] It was next sought to establish whether oxIL33-mediated STAT5 activation could be inhibited by preventing binding to EGFR. Briefly, A549 cells were cultured in RPMI GlutaMax medium supplemented with 1% Penicillin/Streptomycin and 10% FBS. Cells were harvested with accutase and seeded into 96 well plates at 5×10.sup.5/100 μl and incubated at 37° C., 5% CO.sub.2 for 18-24 hours. The wells were then washed twice with 100 μl of PBS before addition of 100 μl of starve media (RPMI GlutaMax medium supplemented with 1% Penicillin/Streptomycin) and incubated at 37° C., 5% CO.sub.2 for 18-24 hours. Anti-EGFR antibody (Clone LA1 (05-101, Millipore) or isotype control (MAB002, R&D Systems) was added in a dose dependent manner to the wells and the plate was returned to the incubator for 30 mins. The plates were then stimulated with oxidised IL-33 (30 ng/ml) for 30 mins before lysis using the phosho-STAT5 ELISA kit lysis buffer (85-86112-11, ThermoFischer Scientific) and developed following manufacturer's instructions before reading absorbance at 450 nM. As shown in
Example 2—Oxidised IL-33 Induces Complex Formation Between EGFR and RAGE
[0363] 9. OxIL-33 induces complex formation between EGFR and RAGE In order to understand how RAGE and EGFR are involved in promoting signaling of oxIL-33, immunoprecipitation experiments were performed to explore the signaling complex. Firstly, anti-EGFR antibodies were covalently coupled to Dynabeads. Two 100 pg vials of anti-EGFR antibodies (R&D systems, AF231) were incubated with 40 mg of Dynabeads (Thermo, 14311D) and covalently coupled as per the manufacturer's instructions. Following successful coupling the beads were resuspended in PBS at 30 mg/ml and kept at 4° C.
[0364] NHBEs were obtained from Lonza (CC-2540) and frozen vials seeded directly into 15 cm dishes (Thermo, 157150) at 1×10.sup.6 cells per dish. NHBEs were maintained in complete BEGM media (Lonza) according to the manufacturer's protocol for one month with a media change every three days until the cells reached confluency. The plates were incubated at 37° C., 5% CO.sub.2 for the duration of this time. The day before stimulation, the plates were washed twice with 20 ml PBS before the addition of 15 ml starve media (BEGM (Lonza CC-3171) without supplement kit). The plates were then incubated at 37° C., 5% CO.sub.2 for a further 18-24 hours before stimulation with media alone (unstimulated control), 30 ng/ml reduced IL-33-01, 30 ng/mL oxidised IL-33-01 or 30 ng/mL EGF and returned to 37° C., 5% CO.sub.2 for 10 mins. Media was aspirated, and the plates were washed twice with ice-cold PBS before the addition of 1 ml lysis buffer (Abcam, ab152163) containing phosphatase and protease inhibitors (Thermo, 78440) per 15 cm dish. The cells were scraped into the lysis buffer before being transferred into 2 ml Protein LoBind tubes (Eppendorf, Z666513) and clarified by spinning at 14,000 rpm at 4° C. Protein concentration was determined using a BCA kit (Thermo, 23225) and all protein extracts were normalised to 3 mg/ml with lysis buffer. 6 mg of total protein extract was incubated in a clean 2 ml LoBind tube with 100 μl of anti-EGFR Dynabeads (described above). The tubes were then placed on an end-over-end mixer at 4° C. for 5 h. Using a magnet (BioRad, 1614916) the Dynabeads were immobilised and the protein extract was aspirated and replaced with 2 ml wash buffer 1 (50 mM Tris-HCl pH 7.5 (Thermo, 15567027), 0.5% TritonX 100 (Sigma, X100), 0.3 M NaCl. This was repeated four more times. The beads were then washed a further ten times in the same manner with wash buffer 2 (50 mM Tris-HCl pH 7.5). After the final washing step, 50 μl of 1% Rapigest (w/v) (Waters, 186001861), in 50 mM Tris-HCl pH8.0, was added to the beads and heated at 60° C. for 10 min. The supernatant was then transferred to a new LoBind 2 ml tube. A further 100 μl of 50 mM Tris-HCl pH8.0 was added to the resin and mixed before it was combined with the first elution. TCEP (Sigma, 646547) was then added to a final concentration of 5 mM and the sample was heated at 60° C. for 10 min. The eluates were then alkylated by addition of iodoacetamide (Sigma, 16125) to 10 mM in the dark at room temperature for 20 min. The alkylation was quenched by the addition of DTT (Sigma, D5545) to 10 mM. Tris-HCl buffer 50 mM pH8.0 was then added to give a final sample volume of 500 μl. 0.5 μg of trypsin (Promega, V5111) per tube was added and samples were digested at 30° C. overnight at on a shaking platform at 400 rpm. The samples were then acidified with trifluoroacetic acid (Sigma, 302031) to a final concentration of 2.0% (v/v) and incubated at 37° C. for 1 h. Samples were then centrifuged at 14,000 rpm for 30 min and the supernatant was transferred to a new 2 ml LoBind tube. Samples were then processed through C18 columns (Thermo, 87784) as per the manufacturer's instructions. Samples were then dried using a speed-vac before being stored at −20° C. Samples were then analysed by peptide mass fingerprinting mass spectrometry (PMF-LC-MS). Scaffold software was used to analyse the results.
[0365] EGFR was detected similarly across all 4 conditions suggesting that the immunoprecipitation had worked well across all the samples. RAGE and IL-33 were detected in samples that had been treated with oxIL-33, in contrast to those treated with IL33-01 (IL-33) or EGF, suggesting that oxIL-33 and RAGE were associated with EGFR during signaling. Consistent with prior observations of EGFR activation in these cells with oxIL-33 and EGF, proteins previously reported to be involved in EGFR signaling and endocytosis were detected after stimulation with these ligands, but not reduced IL33-01 (Table 4).
[0366] Table 4 shows LCMS analysis of NHBE stimulated with reduced IL-33-01 (IL-33), oxIL-33 (oxidised IL-33-01) or EGF. IL-33 and RAGE are detected in complex with EGFR following stimulation with oxIL-33, but not after stimulation with reduced IL33-01 (IL-33) or EGF. Parentheses indicate the number of unique peptides identified for each protein.
TABLE-US-00004 TABLE 4 Unstimulated IL-33 oxIL-33 EGF EGFR (63) EGFR (62) EGFR (60) EGFR (57) — — IL-33 (11) — — — RAGE (11) — — — AP-2α1 (20) AP-2α1 (14) — — AP-2α2 (16) AP-2α2 (10) — — AP-2β (15) AP-2β (16) — — AP-2μ (20) AP-2μ (20) — — AP-2σ (10) AP-2σ (11) — — CBL-B (5) CBL-B (4)
[0367] To confirm these observations, Immunoprecipitation and Western blotting was also performed on cell lysates prepared according to the above protocol. Following NHBE protein extract concentration determination, 3 mg of total protein was incubated in a 1.5 ml tube with 6 μg of anti-EGFR antibody (R&D systems, AF231) and placed on an end-over-end mixer at 4° C. for 2.5 h. 1.5 mg of protein A/G magnetic beads (Thermo, 88802) were then added to each tube and the tubes were then returned to 4° C. for another 1 h with mixing. The beads were then collected with a magnet (BioRad, 1614916) and washed three times with 500 μl of (50 mM Tris (pH 7.5), 1% TritonX and 0.25 M NaCl) and once with 500 μl of 10 mM Tris (pH 7.5). The proteins were then released from the magnetic beads using 35 μl of LDS sample buffer (Thermo, NP0008) with reducing agent (Thermo, NP0004) and heating at 95° C. for 5 minutes. Solutions were transferred to new 1.5 ml tubes and 10 μl of sample along with 5 μl of protein ladder (BioRad, 1610374) was run on a 4-12% SDS-PAGE gel (Thermo, NW04127BOX) in IVIES running buffer (B0002). Gels were transferred onto PVDF membranes (BioRad, 1704156) using a Transblot Turbo (BioRad). PVDF membranes were blocked in PBS-tween solution containing 5% skimmed milk powder (Marvel) for 10 minutes. Membranes were then incubated with primary antibodies (anti-EGFR (Cell Signaling Technology, 2232), anti-RAGE (Cell Signaling Technology, 6996) or anti-IL-33 (R&D systems, AF3625) in PBS-tween containing 5% BSA over night at 4° C. The membranes were then washed five times with PBS-tween and then incubated with anti-rabbit secondary HRP tagged antibodies (Cell Signalling Technology, 7074) or anti-goat secondary HRP tagged antibodies (R&D systems, HAF109) in PBS-tween containing 5% skimmed milk powder for 1 hour at room temperature. The membranes were then washed five times with PBS-tween before the addition of ECL (BioRad, 1705062) and visualisation of a Licor C-digit. Western blotting confirmed that RAGE coprecipitated with EGFR in the presence of oxIL-33 whereas no RAGE was detected with EGF stimulation (
[0368] 10. RAGE is Required for oxIL-33 to Form a Complex with EGFR
[0369] The experiments described above have shown that oxIL-33 is a ligand for a complex of the EGF Receptor (EGFR), which results in downstream signaling. The experiments in this section are designed to determine whether oxIL-33 is a direct binding ligand for either RAGE or EGFR. To understand more about the formation of the signaling complex and assess whether oxIL-33 directly interact with EGFR, an ELISA format was used to explore binding of oxIL-33 to RAGE, ST2-Fc and EGFR.
[0370] Proteins and Modifications: Proteins containing the Avitag sequence motif (GLNDIFEAQKIEWHE SEQ ID NO:46) were biotinylated using the biotin ligase (BirA) enzyme (Avidty, Bulk BirA) following the manufacturer's protocol. All modified proteins without Avitag used herein were biotinylated via free amines using EZ link Sulfo-NHS-LC-Biotin (Thermo/Pierce, 21335) following manufacturer protocols. Table 5 is the list of biotinylated proteins used.
TABLE-US-00005 TABLE 5 Reagent Biotinylated EGF (Thermo) Avitag-Human ASGPR Avitag_IL-33-01 (reduced IL-33) Avitag_IL-33-01 (oxidised IL-33) Avitag_IL-33-16 HMGB1
[0371] Streptavidin plates (Thermo Scientific, AB-1226) were coated with 100 μl/well of biotinylated antigen (10 μg/ml in PBS) at room temperature for 1 hour. Plates were washed 3× with 200 μl PBS-T (PBS+1% (v/v) Tween-20) and blocked with 300 μl/well blocking buffer (PBS with 1% BSA (Sigma, A9576)) for 1 hour. Plates were washed 3× with PBS-T. RAGE-Fc (R&D Systems #1145-RG) or ST2-Fc (R&D Systems #523-ST) were diluted to 10 μg/mL in PBS in blocking buffer, added to the relevant wells and incubated at room temperature for 1 hour. Alternatively, 100 μl of EGFR-Fc (R&D Systems #344-ER-050) at 10 μg/mL in PBS was added in the presence or absence of untagged RAGE (Sino Biological, 11629-HCCH) at 10 μg/mL in PBS for 1 hour. Plates were washed with 200 μl PBS-T three times. Then RAGE-Fc, ST2-Fc and EGFR-Fc were detected with anti-human IgG HRP (Sigma A0170, 5.1 mg/mL) diluted 1:10000 in blocking buffer, 100 μl/well for 1 hour at room temperature. Plates were washed 3× with PBS-T and developed with TMB, 100 μl/well (Sigma, T0440). The reaction was quenched with 50 μl/well 0.1M H.sub.2SO.sub.4. Absorbance was read at 450 nm on the Cytation Gen5 or similar equipment. The results show that oxIL-33 displayed a clear interaction with RAGE (
[0372] The need of RAGE in EGFR signaling triggered by oxIL-33 was further confirmed making use of RAGE-deficient cell lines. A RAGE knockout A549 cell line was generated as follows:
[0373] A mammalian plasmid was generated containing expression vectors for red fluorescent protein (RFP), guide RNA targeted to Exon 3 of AGER (TGAGGGGATTTTCCGGTGC SEQ ID NO:47) and Cas9 endonuclease. A549 conditioned media was generated by growing A549 cells in F12K nut mix (Gibco, supplemented with 10% FBS and 1% Penicillin/Streptomycin) in T-175 flasks for two days. Spent media was taken off the A549s, filtered, and diluted five-fold in fresh Gibco F12K nut mix (supplemented with 20% FBS and 1% Penicillin/Streptomycin). A549s were seeded into three T-75 flasks at 2×10.sup.5 cells/ml in 15 ml total and placed in a 37° C., 5% CO.sub.2 incubator overnight. Transfection mix was prepared using 1.6 ml of F12K nut mix (supplemented with 1% Penicillin/Streptomycin) with 8 μg of the AGER guide RNA plasmid and 22.5 μg PEI (Polysciences, 23966-2). The mix was then vortexed for 10 seconds and left at room temperature for 15 mins. 0.75 ml of the transfection mix was then added to each T-75 flask. The flasks were returned to the incubator for two days. The A549 cells were then detached using Accutase and transferred into PBS containing 1% FBS and single cell sorted on an Aria cell sorter (BD) based on expression of RFP into a 96-well dish. The cells were fed every 3-5 days with conditioned media. Once cells became over 50% confluent, they were transferred to 24-well plates and grown up. This process of upscaling continued until each successful clone was split into T15 flasks. Cells were then split into 12 well plates and grown until over 50% confluent before analysis genomic PCR for successful knockouts. Cells were lysed in 100 μl DNA lysis buffer (Viagen Bitoech, 301-C, supplemented with 0.3 pg/ml proteinase K) per well. These samples were incubated at 55° C. for 4 hours followed by 15 min at 85° C. PCR of RAGE was performed with forward and reverse primers having the following sequences: forward—gttgcagcctcccaacttc (SEQ ID NO:48), reverse—aatgaggccagtggaagtca (SEQ ID NO:49). The reaction and cycling was set up as follows in a 50 μl reaction volume [25 μl Q5 polymerase mix, 2.5 μl forward primer (10 pM stock), 2.5 μl reverse primer (10 pM stock), 2 μl of template DNA lysate, 18 μl nuclease-free water]. The PCR reaction was run with initial denaturation at 98° C. for 30 seconds, followed by 35 cycles of 98° C. for 5 seconds, 57° C. for 10 seconds and 72° C. for 20 seconds before a final step at 72° C. for 2 minutes. 4 μl of the PCR product was mixed with 6 μl of nuclease-free water and 2 μl of 6×DNA loading buffer (Thermo Scientific, R0611). Samples were run on a 1% agarose gel (1:10000 SYBR safe) at 90V for 1 hour before visualisation on Versadoc Imager. The remainder of the PCR products were then cleaned up with the QIAquick PCR purification kit (Qiagen, 28104), following the manufacturer's protocol. DNA-50 concentration was measured using a nanodrop. Several clones (selected from results) were sent for in-house sequencing. Results showed successful insertion of stop codon in clones RAGE09 and RAGE10.
[0374] In order to ascertain the essentiality of RAGE to oxIL-33-mediated EGFR signaling, immunoprecipitation and Western blotting were then performed on A549 and the RAGE-deficient A549 cells. Briefly, cell lines were activated at various time points (0-15 minutes) with oxIL-33-01. Subsequent immunoprecipitation of EGFR or RAGE was followed by western blotting with anti-RAGE, anti-EGFR and anti-IL-33 following the relevant experimental protocols detailed in section 9. The results show the essential role of RAGE in the formation of a complex with oxIL-33 and EGFR (
[0375] 11. Oxidised IL-33 Induces STAT5 Phosphorylation which is Blocked by RAGE, but not ST2 Neutralizing Antibody
[0376] To confirm the importance of RAGE over ST2 in oxIL-33 signaling, blocking antibodies were tested. Briefly, A549s were cultured in RPMI GlutaMax medium supplemented with 1% Penicillin/Streptomycin and 10% FBS. Cells were harvested with accutase and seeded into 96 well plates at 5×10.sup.5/100 μl and incubated at 37° C., 5% CO.sub.2 for 18-24 hours. The wells were then washed twice with 100 μl of PBS before addition of 100 μl of starve media (RPMI GlutaMax medium supplemented with 1% Penicillin/Streptomycin) and incubated at 37° C., 5% CO.sub.2 for 18-24 hours. Anti-RAGE (M4F4; WO 2008137552); Anti-ST2 (AF532; RnD Systems) or isotype control (MAB002, R&D Systems) was added in a dose dependent manner to the wells and the plate was returned to the incubator for 30 mins. The plates were then stimulated with oxidised IL-33 (30 ng/ml) for 30 mins before lysis using the phosho-STAT5 ELISA kit lysis buffer (85-86112-11, ThermoFisher Scientific) and developed following manufacturer's instructions before reading absorbance at 450 nM. As shown in
Example 3—OxIL-33 Triggers the Internalization of EGFR in Epithelial Cells
[0377] It was next investigated whether oxIL-33 induces changes in the dynamics of EGFR as compared to EGF.
[0378] 12. Confocal Experiments of EGF Internalization
[0379] This experiment aims to investigate the dynamics of EGFR in epithelial cells after stimulation with EGF, reduced or oxidised forms of IL-33 utilizing confocal imaging. EGFR-GFP A549 epithelial cell line (Sigma, CLL1141-1VL) were plated at concentration of 20000 cells/ml (RPMI medium+10% FCS+Pen/Strep), 1 ml per 24 well glass bottom plate (Greiner, 662892). The EGF receptor linked to Green Fluorescence Protein (GFP) allows EGFR membrane dynamics and internalisation to be tracked. Cells were washed once with PBS and incubated in RPMI media (without FCS). After 24 hours of starvation, cells were washed with RPMI and incubated with 0.5 ml of RPMI media with CellMask (Invitrogen C10046) deep red at 1:5000 dilution. Cells were stained briefly with CellMask before treatment for membrane marking and live imaged immediately at treatment on confocal at 1 frame/min, to record the dynamics of EGFR-GFP. Cells were stained at 37° C. for 5 minutes, washed once with PBS and stimulated with oxIL-33 (oxidised IL-33-01) or IL-33-16 at a concentration of 200 ng/ml in 0.5 ml serum free RPMI/well. Confocal images were taken immediately, 40× oil objective, 1 min/frame, 5 stacks spacing 2 μm for 25 minutes (about 30 minutes after adding the proteins). Disrupted (dotted) pattern of the GFP signal indicates clustering of the receptor on the membrane and internalisation. Pixel intensity histograms of the membrane area (masked by CellMask) and the intracellular area (masked by inverted CellMask, not shown) were generated at different time points from live imaging, showing depletion of EGFR in non-clustered area (left shift of the histogram bell shape peaks), and increased numbers of saturated pixels (intensity 255) caused by clustering. oxIL-33 induced clustering and internalization of EGF receptor, although EGF stimulation resulted in most evident EGFR clustering. In contrast, reduced form of IL-33 (IL-33-16) did not display major changes in EGFR cell distribution (
Example 4—OxIL-33 Induces the Secretion of IL-8 by Epithelial Cells, Similar to EGF
[0380] 13. Selective Secretion of IL-8 by oxIL-33
[0381] Human bronchial epithelial cells from healthy subjects (NHBE; Lonza CC-2540) and chronic obstructive pulmonary disease (COPD) (DHBE; Lonza 00195275) were maintained in complete BEGM media (Lonza) according to the manufacturers protocol for one month with a media change every three days until the cells reached confluency. Cells were harvested with accutase and seeded at 5×10.sup.5/100 μl in a 96-well plate (Corning 3596) in culture media. The plates were incubated at 37° C., 5% CO.sub.2 for 18-24 hours. After this time, media was aspirated, and the cells were washed twice with 100 μl PBS before the addition of starve media (BEGM (Lonza CC-3171) without supplement kit supplemented with 1% Penicillin/Streptomycin). The plates were then incubated at 37° C., 5% CO.sub.2 for a further 18-24 hours before stimulation with media alone (unstimulated control), 30 ng/ml reduced IL-33-01, 30 ng/mL IL-33-16, 30 ng/mL oxidised IL-33-01 or 30 ng/mL EGF and returned to 37° C., 5% CO.sub.2. 24 hours after stimulation, supernatant were collected and evaluated for chemokine production using a multiplex assay (Mesoscale Discovery K15047D-2). As shown in
Example 5—OxIL-33 Impairs Scratch Wound Repair Response in Submerged Monolayer Epithelial Cultures
[0382] 14. oxIL-33 Impairs Scratch Wound Closure in A549 and NHBE Cells, in Contrast to EGF
[0383] A549s were obtained from ATCC and cultured in RPMI GlutaMax medium supplemented with 1% Penicillin/Streptomycin and 10% FBS. Cells were harvested with accutase (PAA, #L11-007) and seeded into 96 well plates at 5×10.sup.5/100 μl and incubated at 37° C., 5% CO.sub.2 for 18-24 hours. The wells were then washed twice with 100 μl of PBS before addition of 100 μl of starve media (RPMI GlutaMax medium supplemented with 1% Penicillin/Streptomycin) and incubated at 37° C., 5% CO.sub.2 for 18-24 hours. Using a WoundMaker™ (Essen Bioscience), cells were scratched and then wells were washed 2× with 200 μl of PBS before addition of RPMI GlutaMax medium supplemented with 0.1% FBS (v/v) and 1% (v/v) Penicillin/Streptomycin containing the indicated stimulations; media alone (unstimulated control), 30 ng/ml reduced IL-33-01, 30 ng/mL oxidised IL-33-01 or 30 ng/mL EGF and returned to 37° C., 5% CO.sub.2. Plates were placed into an IncucyteZoom for wound healing imaging and analysis over a 48 hour period. Relative Wound Density was calculated through the wound healing algorithm within the Incucyte Zoom software.
[0384] NHBEs (CC-2540) were obtained from Lonza and were maintained in complete BEGM media [BEGM (Lonza CC-3171) and supplement kit (Lonza CC-4175)] according to the manufacturer's protocol. Cells were harvested with accutase and seeded at 5×10.sup.5/100 μl in a 96-well ImageLock plate (Sartorius, 4379) in culture media. The plates were incubated at 37° C., 5% CO.sub.2 for 18-24 hours. After this time, media was aspirated, and the cells were washed twice with 100 μl PBS before the addition of starve media (BEGM (Lonza CC-3171) without supplement kit supplemented with 1% Penicillin/Streptomycin). The plates were then incubated at 37° C., 5% CO.sub.2 for a further 18-24 hours before scratch wounding. Using a WoundMaker™ (Essen Bioscience), cells were scratched and then wells were washed 2× with 200 μl of PBS before addition of BEBM media (Lonza) supplemented with 0.1% FBS (v/v) and 1% (v/v) Penicillin/Streptomycin containing the indicated stimulations; media alone (unstimulated control), 30 ng/ml reduced IL-33-01, 30 ng/mL oxidised IL-33-01 or 30 ng/mL EGF and returned to 37° C., 5% CO.sub.2. Plates were placed into an IncucyteZoom for wound healing imaging and analysis over a 48 hour period. Relative Wound Density was calculated through the wound healing algorithm within the Incucyte Zoom software. As shown in
[0385] 15. The Impairment of Scratch Wound Closure by Oxidised IL-33 can be Prevented by Antibodies Neutralising RAGE or EGFR but not ST2
[0386] To understand whether these functional effects of oxIL-33 were mediated through RAGE/EGFR, the scratch assay was performed in NHBE cells as described in section 14, but in the presence of antibodies that neutralised different receptor components. NHBE cells were treated with media alone (unstimulated control), reduced IL-33, or oxidised IL-33, in the presence of 10 μg/mL anti-ST2 (AF532, R&D Systems), anti-RAGE (M4F4, WO 2008137552) or anti-EGFR (Clone LA1, 05-101 Millipore). OxIL-33, but not reduced IL-33, inhibits scratch closure. This effect of oxIL-33 is reversed by anti-RAGE and anti-EGFR but not anti-ST2, again demonstrating that RAGE and EGFR are essential receptors involved in the oxidised IL-33 signalling pathway (
Example 6—Anti-IL-33 Improves the Phenotype of COPD Cells in Submerged Cultures
[0387] 16. OxIL-33 can Drive a COPD-Like Response in Healthy NHBEs in a Scratch Wound Closure Assay
[0388] Next, the effect of oxidised IL-33 in healthy, smokers and COPD bronchial epithelial cells was investigated. NHBEs (CC-2540), NHBEs from smokers (CC-2540) and DHBEs (COPD, 00195275) were obtained from Lonza and were maintained in complete BEGM media (Lonza) according to the manufacturers protocol. A scratch assay was performed as described in Section 14. Cells were treated with media alone (unstimulated control), or 30 ng/mL oxidised IL-33. Bronchial epithelial cells from smokers or COPD showed an impaired ability for scratch closure compared with cells from healthy subjects that was similar to the impairment observed after treatment of healthy cells with oxIL-33 (
[0389] 17. Blockade of Endogenous IL-33 Through the RAGE/EGFR Pathway can Improve the Impaired Scratch Wound Repair Phenotype of COPD Basal Cells.
[0390] Since epithelial cells are known to produce IL-33, it was possible that autocrine IL-33 secretion could account for the impaired scratch repair phenotype observed in the COPD cells. To investigate, a scratch closure assay was performed in bronchial epithelial cells from COPD in the presence of IL-33 neutralisation. NHBEs (Lonza CC-2540) and DHBEs (Lonza, COPD 00195275) were maintained in complete BEGM media (Lonza) according to the manufacturers protocol. Cells were harvested with accutase and seeded at 5×10.sup.5/100 μl in a 96-well ImageLock plate (Sartorius, 4379) in culture media. The plates were incubated at 37° C., 5% CO.sub.2 for 18-24 hours. After this time, media was aspirated, and the cells were washed twice with 100 μl PBS before the addition of starve media (BEGM (Lonza CC-3171) without supplement kit supplemented with 1% Penicillin/Streptomycin). The plates were then incubated at 37° C., 5% CO.sub.2 for a further 18-24 hours before scratch wounding. Using a WoundMaker™ (Essen Bioscience), cells were scratched and then wells were washed 2× with 200 μl of PBS before addition of BEBM media (Lonza) supplemented with 0.1% FBS (v/v) and 1% (v/v) Penicillin/Streptomycin containing 10 μg/mL of anti-IL-33 (33_640087-7B, described in WO2016/156440), anti-ST2 (AF532, R&D Systems), anti-RAGE (M4F4, WO 2008137552) or NIP228 (IgG1 isotype control), and returned to 37° C., 5% CO.sub.2. Plates were placed into an IncucyteZoom for wound healing imaging and analysis over a 48 hour period. Relative Wound Density was calculated through the wound healing algorithm within the Incucyte Zoom software. As observed previously, COPD cells were impaired in their scratch closure response compared with cells derived from healthy subjects. Anti-IL-33 and anti-RAGE, but not anti-ST2, were able to improve the scratch closure response of the COPD cells to a level similar to healthy cells (
Example 7—Anti-IL-33 Reduces Goblet Cells in 3D Epithelial Cultures
[0391] 18. Air-Liquid Interface (ALI) Culture of Airway Basal Cells
[0392] Next the inventors sought to determine the relevance of IL-33 signalling in Air-liquid interface cell cultures (“ALI cultures”). ALI culturing is a method by which basal cells are grown with their basal surfaces in contact with media and the top (apical) cellular layer exposed to the air. ALI culturing enables the development of a three-dimensional cellular structure in vitro with the mucociliary phenotype of a pseudostratified epithelium, similar to the tracheal epithelium. ALI cultures can therefore be used to study fundamental aspects of the respiratory epithelium, such as cell-to-cell signalling, disease modelling, and respiratory regeneration.
[0393] Cryovials of frozen lung basal cells from healthy controls or COPD patients were received from the University of North Carolina and the University of Pittsburgh. Cells were thawed and plated on a T-75 flask coated with Purecol Type I Bovine Collagen (Advanced BioMatrix, San Diego, Calif.) diluted 1:70 in 1×PBS (Gibco, Waltham, Mass.) and grown in Epix Media (276-201, Propagenix, Rockville, Md.). After reaching confluency, these cells were split once to an appropriate number of T-75 flasks before being harvested for ALI culture. Transwells for ALI culture containing 12 mm, 0.4 μM polyester membrane inserts (Costar, Corning, N.Y.) were prepared by coating the inserts with 1:70 Purecol solution and incubating at 37° C. for between 1-16 hours. The Purecol solution was removed and the Transwells were placed under a UV light for 30 minutes and then washed with PBS. The basal cells in T-75 flasks were detached using 4 ml of trypsin solution (ThermoFisher, 15400054). The cell suspension was added to a 50 ml tube containing 5 ml of FBS and then counted on a ViCell counter (Beckman Coulter, Brea, Calif.) and spun down at 1,000 RPM for 5 minutes. The cells were then resuspended in Pneumacult ALI media (Stemcell Tech, Vancouver, BC) at a density of 3.57×10.sup.5/ml and 700 μl was dispensed onto each Transwell. 1 mL of ALI media was added into the space below the insert. Cells were left submerged in ALI media until confluent and tight junctions are formed (typically 7 days), at which point the media was removed from the apical side and cells were differentiated for 2 weeks, with media change on the basal side every other day. Fully differentiated cultures were treated with no antibody, 1 mg/ml anti-IL-33 (33_640087-7B) or 1 mg/ml NIP228 (IgG1 isotype control) for 7 days by inclusion of treatments in the media supplied to the basal side of the culture. A media change was performed every other day (containing relevant treatments).
[0394] 19. IHC Triplex Staining (Basal, Goblet and Ciliated) and Quantitation
[0395] ALI cultures from COPD donors were generated and treated as described in section 18. ALI epithelial cultures were fixed in 10% neutral buffered formalin for 24-hours and embedded in paraffin. Paraffin sections (4 um) were mounted on positively charged slides and stained on the Ventana Discovery Ultra with a sequential 3 plex chromogenic assay. Antigen retrieval was done with cell conditioner 1 (CC1) (cat #5424569001, Roche) and endogenous peroxidase was blocked with Discovery Inhibitor (cat #7017944001, Roche) for 12 min. Anti-p63 (clone 4A4) (cat #790-4509, Roche, Basel, Switzerland) was applied for 24 min at 36° C. and visualized with mouse anti-HQ (12 min) (cat #7017782001, Roche) and anti-HQ HRP (12 min) (cat #7017936001, Roche), and incubated in the Teal substrate (cat #8254338001, Roche) for 12 min. The slides were treated with an antibody denature step (100° C. for 24 min) with cell conditioner 2 (CC2) (cat #5424542001, Roche) and then anti-tubulin (cat # ab24610, Abcam, Cambridge, UK) diluted 0.01 μg/ml with Dako Antibody Diluent (cat # S3022) for 16 min and detected with mouse OmniMap-HRP (8 min) (cat #5269652001, Roche) and visualized with Discovery Purple substrate (cat #7053983001, Roche) for 16 min. The slides were subjected to an additional antibody denaturation with CC2 and then a cocktail of rabbit anti-Mucin 5AC 1.1 μg/ml and rabbit anti-Mucin 5B 7 μg/ml (cat # ab198294 and cat # ab87376, Abcam respectively), were applied for 20 min and visualized with anti-rabbit NP (4 min) (cat #7425317001, Roche), anti-NP-AP (8 min) (cat #7425325001, Roche) and then Discovery Yellow (cat #7698445001, Roche) for 20 min. The stained slides were rinsed with Dawn detergent, counterstained with hematoxylin (cat #5277965001, Roche), rinsed, dehydrated with graded series of ethanol and xylene and mounted with permanent mounting media. Quantification using HALO software showed a decrease in goblet cells in ALI culture derived from healthy donors that had been treated with anti-IL-33 (
Example 8—Anti-IL-33 Regulates Mucins in 3D Epithelial Cultures from COPD and Improves Mucus Movement
[0396] 20. IHC Duplex IF Staining (Mucin5B+Mucin5AC)
[0397] ALI cultures from COPD donors were generated and treated as described in section 18. ALI epithelial cultures were fixed in 10% neutral buffered formalin for 24-hours and embedded in paraffin. Paraffin sections (4 um) were mounted on positively charged slides and stained on the Ventana Discovery Ultra with a sequential 2 plex immunofluorescent assay. Antigen retrieval was done with cell conditioner 1 (CC1) and endogenous peroxidase was blocked with Discovery Inhibitor for 12 min and the blocked 8 min with S Block (RUO) Roche Diagnostics (cat #760-4212) and incubated in anti-Mucin5B used at 7 μg/ml diluted in Dako Ab Diluent, S3022, for 24 min at 36C, and detected with anti-rabbit-HQ (Roche Diagnostics Cat #760-4815) for 4 min and anti-HQ-HRP (Roche Diagnostics cat #760-4820) for 8 min. The samples were then incubated with Discovery FITC, a tyramide conjugate, (Roche Diagnostics cat #760-232) for 8 min. Dual Sequence was selected in the Discovery Ultra program and the samples were treated with an antibody denature step, (100° C. for 24 min) with cell conditioner 2 (CC2) and then neutralized with Discovery Inhibitor (40C, 24 min) before application of anti-Mucin5AC, 1.1 μg/ml, for 20 min at 36° C. The Mucin5AC was detected with anti-rabbit-HQ (Roche Diagnostics Cat #760-4815) for 4 min and anti-HQ-HRP (Roche Diagnostics cat #760-4820) for 8 min, and visualized with Discovery Red610, a tyramide conjugate, for 8 min. After completion of this step, the stained slides were removed from the Discovery Ultra Autostainer and were rinsed with Dawn detergent and then de-ionized water. The samples were the incubated in 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI Nucleic Acid Stain), ThermoFisher cat # D1306, diluted in de-ionized water at 1 μg/ml for 2 min. Samples were rinsed with de-ionized water and coverslipped with ProLong Gold Antifade mounting media (ThermoFisher, cat # P36930) and stored in light tight slide box. Stained slides were imaged with Zeiss LSM 880 confocal microscope (Carl Zeiss Microscopy, LLC, White Plains, N.Y.).
[0398] 21. Anti-IL-33 Reverses the Impaired Mucociliary Clearance Observed in COPD ALI Cultures
[0399] ALI cultures from COPD donors were generated and treated as described in section 18. 30 μl of 0.2 μM FluoSpheres (ThermoFisher, F8811) diluted 1:33 in PBS was then added to the apical surface and using a Zeiss LSM800 microscope a short video of the FluoSphere movement was captured and shows that mucociliary movement increases after treatment with anti-IL-33 (33_640087-7B) but not control antibody.
Example 9—Single Cell RNA Analysis of ALI Cultures Shows Goblet Cell Changes after Treatment with Anti-IL-33
[0400] ALI cultures from COPD donors were generated and treated as described in section 18. To obtain a single-cell suspension, the filter insert was incubated with 0.25% trypsin for 5 min at 37° C. degrees. Epithelial cells were gently detached from the filter by washing with PBS pipetting up and down and then transferred to a 15 ml Falcon tube. Cells were centrifuged at 1000 RPM for 5 min at 4° C. After removing the supernatant, the cells were resuspended in 0.4% BSA in PBS and the cell concentration was adjusted to 1000 cells/μl for sequencing. Cell suspension was loaded according to the standard protocol in Chromium single cell 3′ kit to capture between 5000 and 10.000 cells/channel. Version 2 chemistry was used. Single cell 3′ libraries for Illumina sequencing were obtained followed the manufacturer's protocol (Chromium™ Single Cell 3′ Reagent Kit, v2 Chemistry). Libraries were assessed for quality (TapeStation 4200, Agilent) and then sequenced on NextSeq 500 or NovaSeq 6000 instruments (Illumina). Initial data processing was performed using the Cell Ranger version 2.0 pipeline (10× Genomics). Post-processing was performed using the Seurat package for exclusion of low cell quality and normalization. Each sample was analyzed as independent data to capture within-sample heterogeneity (cell subtyping). Clustering and visualization were realized with t-Distributed Stochastic Neighbor Embedding (tSNE). Identification of cell clusters in COPD was guided by marker genes. For other samples, initial clusters were inspected manually then Seurat's Label Transfer algorithm was applied for subtype identification. MUC5AC and MUC5B gene expression analysis was performed for each cluster between cells from COPD ALI cultures with and without anti-IL-33 treatment as mentioned in section 18. Heatmaps and tSNE plots were generated using Seurat.
Example 10—Anti-IL-33 Reduces Goblet Cells in COPD 3D Epithelial Cultures
[0401] 22. Air Liquid Interface (ALI) Culture of Airway Basal Cells
[0402] In order to quantify and interrogate the effects of oxIL-33 in a physiologically relevant Air liquid interface (ALI) culture system, a flow cytometry assay aiming to discriminate between goblet cell types (MUC5ac vs MUC5b) and the rest of epithelial populations (Mucin negative) was developed.
[0403] Cryovials of frozen lung basal cells from healthy (CC-2540) controls or COPD (195275) patients were received from Lonza. One vial per donor was thawed and plated on 4×T-175 flasks in Epix Media (276-201, Propagenix, Rockville, Md.). After reaching confluency, these cells were frozen down at 1e6 cells per vial at P2. Cells at P2 were initiated into 2×T-75 flasks in Epix Media and grown until 80% confluent. Transwells for ALI culture containing 12 mm or 6.5 mm 0.4 μM polyester membrane inserts (Costar, Corning, N.Y.) were prepared by coating the inserts with 1× Collagen I solution (Celladhere™ Collagen I—Stemcell #07001, prepared in dH2O) and incubating at 37° C. for between 1-16 hours. The Collagen I solution was removed and the Transwells were washed with PBS. The basal cells in T-75 flasks were washed with PBS and detached using 6 ml of trypsin solution (Lonza trypsin subculture pack—#CC-5034). The trypsin was neutralised with 6 ml of trypsin neutralising solution (Lonza trypsin subculture pack—#CC-5034) and the cell suspension was added to a 15 ml tube, counted and tube spun down at 1,200 RPM for 5 minutes. The cells were then resuspended in Pneumacult ALI media (Stemcell Tech, Vancouver, BC) at a density of 8×10.sup.5/ml and 0.5 ml was dispensed onto each 12 mm Transwell and 0.25 ml onto each 6.5 mm Transwell. 1 mL of ALI media was added into the space below the 12 ml insert and 0.5 ml below the 6.5 mm insert. Cells were left submerged in ALI media until confluent and tight junctions are formed (typically 7 days), at which point the media was removed from the apical side and cells were differentiated for 3 weeks, with media change on the basal side every Monday, Wednesday and Friday. Fully differentiated normal cultures were left untreated or treated with reduced or oxidised untagged IL33-01 (30 ng/ml), untagged IL33-16 (30 ng/ml), IL-13 (10 ng/ml), EGF (30 ng/ml) or HMGB1 (30 ng/ml) for 7 days by inclusion of treatments in the media supplied to the basal side of the culture (7 day treatments). Fully differentiated COPD cultures were left untreated or treated with 1 μg/ml anti-IL-33 (33_640087-7B), 1 μg/ml NIP228 (IgG1 isotype control), 10 μg/ml mNIP228, 10 μg/ml anti-ST2, 1 μg/ml anti-RAGE or 1 μg/ml anti-EGFR for 7 days by inclusion of treatments in the media supplied to the basal side of the culture. A media change was performed every Monday, Wednesday and Friday (containing relevant treatments).
TABLE-US-00006 TABLE 6 Antibody Identifier hIgG1 NIP228_SP14-266 anti-IL-33 (33_640087-7B) SP15-124 mIgG1 mNIP228_SP14-108 Anti-ST2 Ab1440361 Anti-RAGE M4F4 Anti-EGFR LA1 (Merk, 05-101)
[0404] 23. FACS Analysis of Goblet Cells in ALI Cultures
[0405] Following a 7-day treatments (Table 6), 4-week old normal control or COPD ALI cultures on 6.5 mm inserts were analysed by flow cytometry. 200 μl 37° C. PBS was added to the apical region (Transwell surface) of each Transwell and placed in an incubator for 30 min. The apical wash was stored at −80 for mucin analysis. 150 μl trypsin (Lonza trypsin subculture pack—#CC-5034) was added onto both the apical and basolateral (beneath Transwell) compartments. Transwells were returned to the incubator for 30 mins. The ALIs were dissociated by gently pipetting the trypsin up and down. 150 μl trypsin neutralising solution (Lonza trypsin subculture pack—#CC-5034) was added to each apical chamber and mixed. The cell suspension was moved to U-shaped 90-well plate, cells were counted and centrifuged at 1200 RPM at 4° C. for 5 min. The trypsin/TNS was removed and 200 μl of live dead stain (eBioscience™ Fixable Viability Dye eFluor™ 780 Thermo 65-0865-14, dilute 1:2000 in PBS) was added to each well. The cells were re-suspended and incubated for 10 min on ice in the dark. The plate was centrifuged at 1200 RPM at 4° C. for 5 min, the live dead stain was removed and 200 μl PBS was added to each well. The plate was centrifuged at 1200 RPM at 4° C. for 5 min and the PBS was removed and replaced with 200 μl fixation/permeabilization solution (Thermo 00-5123 and 00-5223). The plate incubated for 40 min on ice in the dark. The plate was centrifuged at 1200 RPM at 4° C. for 5 min and the solution was removed. Cells were re-suspend in 300 μl of 1× permeabilization solution (Thermo 00-8333). 5e4 cells from each well were added to a new 96-well U bottom plate centrifuged at 1200 RPM at 4° C. for 5 min and the cells re-suspended in 50 μl of 1× permeabilization solution. 50 μl of antibody stain cocktail (anti-Muc5AC at 1:400 and anti-Muc5B at 1:800) or isotype stain cocktail at the same dilutions. The plate was incubated for 30 mins on ice in the dark. The plate was centrifuged at 1200 RPM at 4° C. for 5 min and the solution was removed. The cells were washed with PBS, centrifuged and then re-suspended in 150 μl of PBS. Data was then acquired on a BD FACSymphony™ and analysed using FlowJo software.
TABLE-US-00007 TABLE 7 Name Identifier Supplier Fluorophore Cell marker Anti-Muc5AC ab3649 Abcam Coupled to AF488 Goblet (Expedeon, 332-0005) Anti-Muc5B ab105460 Abcam Coupled to AF647 Goblet (Expedeon, 336-0005) AF488 Isotype 400109 BioLegend FITC Isotype AF647 Isotype 400130 BioLegend AF647 Isotype
[0406] 24. qPCR/Bulk RNA Seq Analysis of ALI Cultures.
[0407] Following a 7-day treatment (Table 6), 4-week old normal control or COPD ALI cultures on 6.5 mm inserts were lysed for RNA analysis. First 200 μl 37° C. PBS was added to each ALI apical surface and the plates were returned to an incubator for 30 min. The apical wash was stored at −80 for mucin analysis. The MagMAX™-96 Total RNA Isolation Kit (Thermo, AM1830) was used to lyse the ALI cultures and extract RNA. RNA was then used to synthesise cDNA using the High-Capacity RNA-to-cDNA™ Kit (Thermo, 4388950). Whereby 9 μl of each RNA sample was incubated with 10 μl of 2×RT Buffer Mix and 1 μl of 20×RT Enzyme Mix in PCR Tubes (Thermo, AM12230) and placed on a thermo cycler and incubated at for 37° C. for 60 minutes. The reaction was stopped by heating to 95° C. for 5 minutes and holding at 4° C. 60 μl of nuclease free water (Thermo, 750024) was added to each tube containing 20 μl of cDNA. For RT-qPCR, 4 μl of cDNA was added to a MicroAmp™ EnduraPlate™ Optical 384-Well Clear Reaction Plates with Barcode (Thermo. 4483273) with 5 μl of TaqMan Fast Advanced Master Mix (Thermo, 4444557) and 0.5 μl of Muc5AC FAM probe (Thermo, Hs01365616_m1) and 0.5 μl of GAPDH VIC probe (Thermo, Hs02786624_g1). Plates were sealed and briefly centrifuged before analysis using a QuantStudio™ 7 Flex Real-Time PCR System (Thermo). Delta-delta-ct was then calculated by normalising the data to an untreated normal control.
[0408] oxIL-33 but not reduced IL-33 leads to increased goblet cell number, in particular the MUC5AC+ goblet cells subset (
[0409] 25. IHC Triplex Staining (Basal, Goblet and Ciliated) and Quantitation
[0410] Next, quantitative image analysis from ALI immunohistochemistry was evaluated. ALI cultures from COPD donors were generated and treated as described in section 22; Air Liquid Interface (ALI) culture of airway basal cells. ALI epithelial cultures were fixed in 10% neutral buffered formalin for 24-hours and embedded in paraffin. Paraffin sections (4 um) were mounted on positively charged slides and stained on the Ventana Discovery Ultra with a sequential 3 plex chromogenic assay. Antigen retrieval was done with cell conditioner 1 (Ultra CC1) (cat #5424569001, Roche) and endogenous peroxidase was blocked with Discovery Inhibitor (cat #7017944001, Roche) for 12 min. Anti-p63 (clone 4A4) (cat #790-4509, Roche, Basel, Switzerland) was applied for 24 min and visualized with anti-Mouse HQ (12 min) (cat #7017782001, Roche) and anti-HQ HRP (12 min) (cat #7017936001, Roche), and incubated with the Discovery Purple kit (cat #07053983001, Roche) for 12 min. The slides were treated with an antibody denature step (92° C. for 24 min) with cell conditioner 2 (Ultra CC2) (cat #5424542001, Roche) and then anti-tubulin (cat # ab24610, Abcam, Cambridge, UK) diluted with Dako Antibody Diluent (cat # S3022) for 16 min (concentration on slide 0.003 μg/ml) and detected with mouse OmniMap-HRP (8 min) (cat #5269652001, Roche) and visualized with Discovery Teal HRP kit (cat #82544338001, Roche). The slides were subjected to an additional antibody denaturation with CC2 and then a cocktail of rabbit anti-Mucin 5AC 1.1 μg/ml (dispenser concentration) and rabbit anti-Mucin 5B 7 μg/ml (dispenser concentration) (cat # ab198294 and cat # ab87376, Abcam respectively), were applied for 20 min and visualized with anti-rabbit NP (4 min) (cat #7425317001, Roche), anti-NP-AP (8 min) (cat #7425325001, Roche) and then Discovery Yellow kit (cat #7698445001, Roche) for 20 min. The stained slides were counterstained with Hematoxylin II (8 min) (cat #5277965001, Roche) and Bluing reagent (4 min) (cat #5266769001, Roche), rinsed with dish washing detergent, dehydrated with graded series of ethanol and xylene and mounted with permanent mounting media.
[0411] IHC images were analysed in HALO v3.1 (Indica Labs), where they were first annotated manually to exclude out of focus and tissue damage areas. A random forest classifier was trained to recognise the epithelium and separate it from transmembrane and glass slide background. For cilia area quantification, another random forest classifier was trained for coarse detection of tubulin staining, followed by fine detection using algorithm Area Quantification v2.1.7. For Mucin area quantification Area Quantification v2.1.7 was directly used to detect the staining. For basal (p63+) cell counting, algorithm CytoNuclear 2.0.9 was used to segment cells based on nuclear staining, basal cells were further detected by counting p63 positive nuclei. All quantification methods were validated against human recognition and had more than 90% accuracy.
[0412] In line with previous findings, oxIL-33 profoundly affected the number of goblet cells (MUC5ac+b) (
[0413] Together, these studies showed a role for oxIL-33 in promoting differentiation of goblet cells within the lung epithelia. This suggest that epithelia chronically exposed to ox-IL33 evolve towards a goblet hyperplasic phenotype that negatively impacts lung function.
[0414] 26. Reversal of COPD Goblet Phenotype with Blocking Agents
[0415] An important hallmark of COPD is the excess of mucus, due to increases in goblet cells and in mucus secretion (Gohy et al 2019 Sci Rep 9:17963). To investigate whether oxidised IL-33 could play a direct role in the goblet COPD phenotype, ALI cultures from COPD donors were established with readouts as described in sections 22-25.
[0416] COPD ALI were cultured in the presence of anti-IL-33 (33-640087_7B), anti-RAGE or anti-EGFR neutralizing antibodies. All three treatments resulted in reduced MUC5AC+goblet cell numbers (
[0417] Lastly, MUC5AC and MUC5B released in the apical mucus from both healthy and COPD ALI cultures were measured using ELISAs. For quantification of mucin released from ALI cultures apical supernatants were analysed for levels of MUC5AC by immunoassay (Novus NBP2-76703) according to manufacturer's protocol. Samples were diluted 1:2000 in sample diluent and concentrations extrapolated from recombinant MUC5AC protein standard curves. As shown in
[0418] Overall this Example highlights a role for oxidised IL-33 in the dysregulation of epithelial cell differentiation in the lung. The results imply that, when uncontrolled, oxidised IL-33 may be responsible for the goblet cell hyperplasia and excessive mucus production seen in some phenotypes of COPD. Therefore, treatment with oxIL-33 signaling axis antagonists, such as anti-IL-33, anti-RAGE or anti-EGFR binding molecules, may be of great therapeutic benefit to COPD patients, by restoring normal epithelium physiology, for example, by decreasing goblet cell numbers and reducing of excessive mucus production.
TABLE-US-00008 Additional Sequences Further to the sequences listed in Table 1, we provide the following additional CDR sequences SEQ ID NO 37: SYAMS SEQ ID NO 38: GISAIDQSTYYADSVKG SEQ ID NO 39: QKFMQLWGGGLRYPFGY SEQ ID NO 40: SGEGMGDKYAA SEQ ID NO 41: RDTKRPS SEQ ID NO 42: GVIQDNTGV N terminal His10/Avitag/Factor Xa protease cleavage site SEQ ID NO 43: MHHHHHHHHHHAAGLNDIFEAQKIEWHEAAIEGR IL-33-01 SEQ ID NO 44: SITGISPITEYLASLSTYNDQSITFALEDESYEIY VEDLKKDEKKDKVLLSYYESQHPSNESGDGVDGKM LMVTLSPTKDFWLHANNKEHSVELHKCEKPLPDQA FFVLHNMHSNCVSFECKTDPGVFIGVKDNHLALIK VDSSENLCTENILFKLSET IL-33-16 SEQ ID NO 45: SITGISPITEYLASLSTYNDQSITFALEDESYEIY VEDLKKDEKKDKVLLSYYESQHPSNESGDGVDGKM LMVTLSPTKDFWLHANNKEHSVELHKSEKPLPDQA FFVLHNMHSNSVSFESKTDPGVFIGVKDNHL ALIKVDSSENLSTENILFKLSET Avitag sequence motif SEQ ID NO 46: GLNDIFEAQKIEWHE gRNA vector targeting RAGE exon 3 SEQ ID NO 47: TGAGGGGATTTTCCGGTGC RAGE forward primer SEQ ID NO 48: gttgcagcctcccaacttc RAGE reverse primer SEQ ID NO 49: aatgaggccagtggaagtca Human ST2S (signal peptide underlined) SEQ ID NO 50: MGFWILAILTILMYSTAAKFSKQSWGLENEALIVR CPRQGKPSYTVDWYYSQTNKSIPTQERNRVFASGQ LLKFLPAAVADSGIYTCIVRSPTFNRTGYANVTIY KKQSDCNVPDYLMYSTVSGSEKNSKIYCPTIDLYN WTAPLEWFKNCQALQGSRYRAHKSFLVIDNVMTED AGDYTCKFIHNENGANYSVTATRSFTVKDEQGFSL FPVIGAPAQNEIKEVEIGKNANLTCSACFGKGTQF LAAVLWQLNGTKITDFGEPRIQQEEGQNQSFSNGL ACLDMVLRIADVKEEDLLLQYDCLALNLHGLRRHT VRLSRKNPSKECF Human ST2S-huIgGl Fc-His6 (signal peptide underlined) SEQ ID NO 51: MPLLLLLPLLWAGALAKFSKQSWGLENEALIVRCP RQGKPSYTVDWYYSQTNKSIPTQERNRVFASGQLL KFLPAAVADSGIYTCIVRSPTFNRTGYANVTIYKK QSDCNVPDYLMYSTVSGSEKNSKIYCPTIDLYNWT APLEWFKNCQALQGSRYRAHKSFLVIDNVMTEDAG DYTCKFIHNENGANYSVTATRSFTVKDEQGFSLFP VIGAPAQNEIKEVEIGKNANLTCSACFGKGTQFLA AVLWQLNGTKITDFGEPRIQQEEGQNQSFSNGLAC LDMVLRIADVKEEDLLLQYDCLALNLHGLRRHTVR LSRKNPSKECFEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHH HHHH His10/Avitag human ASGPR ECD (signal peptide underlined and tags double underlined) SEQ ID NO 52: MPLLLLLPLLWAGALAHHHHHHHHHHggs GLNDIFEAQKIEWHEGGSQNSQLQEELRGLRETFS NFTASTEAQVKGLSTQGGNVGRKMKSLESQLEKQQ KDLSEDHSSLLLHVKQFVSDLRSLSCQMAALQGNG SERTCCPVNWVEHERSCYWFSRSGKAWADADNYCR LEDAHLVVVTSWEEQKFVQHHIGPVNTWMGLHDQN GPWKWVDGTDYETGFKNWRPEQPDDWYGHGLGGGE DCAHFTDDGRWNDDVCQRPYRWVCETELDKASQEP PLL