IL-18 binding protein (IL-18BP) in inflammatory diseases

Abstract

The present invention provides means and methods for treating Interleukin 18 (IL-18)-associated diseases and disorders. In particular, the present invention discloses antibodies specific for free IL-18 and IL-18 Binding Protein (IL-18BP) for use in such treatments and for the diagnosis of the indications.

Claims

1. A method for treating an IL-18 associated disease or disorder in a subject, wherein the IL-18 associated disease or disorder is selected from chronic obstructive pulmonary disease (COPD) or Adult-onset Still's disease (AOSD), the method comprising administering to said subject a therapeutically effective amount of an IL-18 inhibitor, wherein the IL-18 inhibitor is an IL-18 binding protein (IL-18BP).

2. The method of claim 1, wherein the subject has abnormal levels of free IL-18 and/or an abnormal ratio of free IL-18/IL-18 binding protein (IL-18BP) in the body fluids compared to the levels in the body fluids of a healthy control subject.

3. The method of claim 1, wherein said IL-18 associated disease or disorder is chronic obstructive pulmonary disease (COPD).

4. The method of claim 1, wherein said IL-18 associated disease or disorder is Adult-onset Still's disease (AOSD).

5. The method of claim 1, wherein administration of the IL-18 inhibitor leads to inhibition of increased expression of IFNγ, IL-13 or IL-17A in treated subjects compared to untreated subjects suffering from said disease or disorder.

6. The method of claim 2, wherein said abnormal level of free IL-18 in the body fluids exceeds the level in body fluids of a healthy control subject by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100%.

7. The method of claim 6, wherein said abnormal levels of free IL-18 are elevated levels of free IL-18, wherein the elevated levels of free IL-18 are ≥5 pg/mL; and wherein the body fluid is serum.

8. The method of claim 1, wherein the IL-18BP is a human IL-18BP (hIL-18BP).

9. The method of claim 8, wherein the hIL-18BP is a recombinant human IL-18BP.

10. The method of claim 1, wherein the IL-18BP is an isoform of IL-18BP.

11. The method of claim 1, wherein the subject is diagnosed with having abnormal levels of free IL-18 and/or an abnormal ratio of free IL-18/IL-18 binding protein (IL-18BP) in the body fluids compared to the levels in the body fluids of a healthy control subject, wherein the level of free IL-18 in the body fluids of the subjects has been determined by an immunoassay.

12. The method of claim 11, wherein the immunoassay comprises an IL-18BP as a capturing molecule.

13. The method of claim 12, wherein the IL-18BP is a human IL-18BP (hIL-18BP).

14. The method of claim 13, wherein the hIL-18BP is a recombinant hIL-18BP.

15. The method of claim 12, wherein the IL-18BP is an isoform of IL-18BP.

16. The method of claim 11, wherein the abnormal levels of free IL-18 and/or abnormal ratio of free IL-18/IL-18BP in the body fluids compared to the levels in the body fluids of a healthy control subject have been determined by a method comprising the steps of: a) bringing a sample or a specific body part or body area suspected to contain free IL-18 into contact with an IL-18 binding molecule, which specifically binds to free IL-18, but not to IL-18 bound in a complex; b) allowing the IL-18 binding molecule to bind to free IL-18; c) detecting the binding of IL-18 to the IL-18 binding molecule and determining the amount of free IL-18 and/or the ratio of free IL-18/IL-18BP in the sample.

17. The method of claim 11, wherein the level of free IL-18 in the body fluids of the subjects has not been determined by calculation based on the Law of Mass Action.

18. The method of claim 11, wherein said abnormal level of free IL-18 in the body fluids exceeds the level in body fluids of a healthy control subject by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100%.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1: Comparison of total and free IL-18 in individual sepsis patients. Adapted from Novick et al 2001. The level of free IL-18 (closed circles) in sera of sepsis patients upon admission was calculated based on the concentration of total IL-18 (open circles) and IL-18BPa, taking into account a 1:1 complex of IL-18 and IL-18BPa and a calculated KD of 400 pM. Each vertical line links total and free IL-18 in an individual serum sample. The above ELISA assays are performed with the pair of antibodies developed by Taniguchi et al 1997 1, namely antibodies 125-2H as primary/capture antibody and 159-12B as secondary/developing antibody.

(2) FIG. 2: Detection of total IL-18 with antibodies 125-2H and 159-12B. The data indicates that both antibodies quantify total IL-18.

(3) FIG. 3: Titration of 400 pg/ml IL-18 as a function of IL-18BP level

(4) FIG. 4: Mouse IL-18 induction in the lung airway space at day 5 after 1) air exposure, 2) tobacco smoke (TS), 3) p[I:C] alone, 4) p[I:C] combined to tobacco smoke at day 4 (induction of exacerbation). Dotted line indicates lower limit of detection. Statistical analyses were performed using either the unpaired t-test.

(5) FIG. 5: Inhibition of total cell infiltration in the mouse lung airway space at day 5 after 1) air exposure, 2) tobacco smoke (TS), 3) p[I:C] alone, 4) p[I:C] combined to tobacco smoke at day 4 (induction of exacerbation), 5-7) p[I:C] combined to tobacco smoke at day 4 under IL-18BP treatment at either 1, 3 or 10 mg/kg, 8) dexamethasone treatment at 10 mg/kg. Statistical analyses were performed using either the unpaired t-test.

(6) FIG. 6: Inhibition of total cell infiltration in the mouse lung airway space at day 5 after 1) air exposure, 2) tobacco smoke (TS), 3) p[I:C] alone, 4) p[I:C] combined to tobacco smoke at day 4 (induction of exacerbation), 5) p[I:C] combined to tobacco smoke at day 4 under IL-18BP treatment at 10 mg/kg, 6) dexamethasone treatment at 10 mg/kg. Statistical analyses were performed using ANOVA test (post-test Sidak's).

(7) FIG. 7: Inhibition of neutrophil infiltration by IL-18BP. Neutrophil infiltration in the mouse lung airway space was monitored at day 5 after 1) air exposure, 2) tobacco smoke (TS), 3) p[I:C] alone, 4) p[I:C] combined to tobacco smoke at day 4 (induction of exacerbation), 5-7) p[I:C] combined to tobacco smoke at day 4 under IL-18BP treatment at either 1, 3 or 10 mg/kg, 8) dexamethasone treatment at 10 mg/kg. Statistical analyses were performed using ANOVA test (post-test Sidak's).

(8) FIG. 8: Inhibition of neutrophil infiltration by IL-18BP. Neutrophil infiltration in the mouse lung airway space was monitored at day 5 after 1) air exposure, 2) tobacco smoke (TS), 3) p[I:C] alone, 4) p[I:C] combined to tobacco smoke at day 4 (induction of exacerbation), 5) p[I:C] combined to tobacco smoke at day 4 under IL-18BP treatment at 10 mg/kg, 6) dexamethasone treatment at 10 mg/kg. Statistical analyses were performed using ANOVA test (post-test Sidak's).

(9) FIG. 9: Inhibition of G-CSF pathway by IL-18BP. The presence of G-CSF (pg/ml) was monitored in the mouse lung airway space by ELISA at day 5 after 1) air exposure, 2) tobacco smoke (TS), 3) p[I:C] alone, 4) p[I:C] combined to tobacco smoke at day 4 (induction of exacerbation), 5-7) p[I:C] combined to tobacco smoke at day 4 under IL-18BP treatment at either 1, 3 or 10 mg/kg, 8) dexamethasone treatment at 10 mg/kg. Dotted line indicates lower limit of detection. Statistical analyses were performed using Students t-test.

(10) FIG. 10: Mitigation of weight loss by IL-18BP. Mouse weight loss was monitored at day 5 after 1) air exposure, 2) tobacco smoke (TS), 3) p[I:C] alone, 4) p[I:C] combined to tobacco smoke at day 4 (induction of exacerbation), 5) p[I:C] combined to tobacco smoke at day 4 under IL-18BP treatment at 10 mg/kg, 7) dexamethasone treatment at 10 mg/kg. Statistical analyses were performed using ANOVA test (post-test Sidak's).

(11) FIGS. 11A-11M: shows show the amino acid sequences of the variable heavy chain (vh) and the variable light chain (vk) of antibodies produced by different clones. The complementary determining regions CDR 1, CDR 2 and CDR 3 are identified by underlining the respective sequences as determined by the IMGT numbering system (Lefranc, M.-P. et al., Nucleic Acids Research, 27, 209-212 (1999)). From left to right, the first underlined sequence in each of the VII and VK sequences shown represents CDR1, the second underlined sequence represents CDR 2 and the third underlined sequence represents CDR 3. The variable domain is highlighted in BOLD.

SEQUENCES

(12) SEQ ID NO 1: IL-18 Epitope 1: Tyr-Phe-Gly-Lys-Leu-Glu-Ser-Lys-Leu-Ser-Val-Ile-Arg-Asn

(13) SEQ ID NO 2: IL-18 Epitope 2: Phe-Ile-Ile-Ser-Met-Tyr-Lys-Asp-Ser-Gln-Pro-Arg-Gly-Met-Ala-Val-Thre-Ile-Ser-Val-Lys

(14) SEQ ID NO 3: IL-18 Epitope 3: Glu-Met-Asn-Pro-Pro-Asp-Asn-Ile-Lys-Asp-Thr-Lys-Ser-Asp-Ile-Ile-Phe

(15) SEQ ID NO 4: IL-18 Epitope 4: Tyr-Phe-Gly-Lys-Leu-Glu-Ser

(16) SEQ ID NO 5: IL-18 Epitope 5: Tyr-Lys-Asp-Ser-Gln-Pro-Arg-Gly-Met-Ala

(17) SEQ ID NO 6: IL-18 Epitope 6: Asp-Asn-Ile-Lys-Asp-Thr-Lys

(18) SEQ ID NO 7: IL-18 Binding Protein (IL-18BP)

(19) SEQ ID NO: 8: 13-amino acid Linker Sequence: Glu-Phe-Gly-Ala-Gly-Leu-Val-Leu-Gly-Gly-Gln-Phe-Met

(20) SEQ ID NO: 9: Antibody 107C6 VH sequence

(21) SEQ ID NO:10: Antibody 107C6 VK sequence

(22) SEQ ID NO: 11: Antibody 108F8 VH sequence

(23) SEQ ID NO: 12: Antibody 108F8 VK sequence

(24) SEQ ID NO: 13: Antibody 109A6 VH sequence

(25) SEQ ID NO: 14: Antibody 109A6 VK sequence

(26) SEQ ID NO: 15: Antibody 111A6 VH sequence

(27) SEQ ID NO: 16: Antibody 111A6 VK sequence 1

(28) SEQ ID NO: 17: Antibody 111A6 VK sequence 2

(29) SEQ ID NO: 18: Antibody 131B4 VH sequence

(30) SEQ ID NO: 19: Antibody 131B4 VK sequence

(31) SEQ ID NO: 20: Antibody 131E8 VH sequence 1

(32) SEQ ID NO: 21: Antibody 131E8 VH sequence 2

(33) SEQ ID NO: 22: Antibody 131E8 VK sequence

(34) SEQ ID NO: 23: Antibody 132H4 VH sequence

(35) SEQ ID NO: 24: Antibody 132H4 VK sequence

(36) SEQ ID NO: 25: Antibody 133A6 VH sequence

(37) SEQ ID NO: 26: Antibody 133A6 VK sequence

(38) SEQ ID NO: 27: Antibody 107C6 VH sequence CDR1: Gly Tyr Thr Phe Thr Asn Tyr Gly

(39) SEQ ID NO: 28: Antibody 107C6 VH sequence CDR2; Ile Asn Thr Tyr Ser Gly Val Pro

(40) SEQ ID NO: 29: Antibody 107C6 VH sequence CDR3: Ala Arg Glu Gly Tyr Ser Thr Thr Arg Ser Met Asp Tyr

(41) SEQ ID NO: 30: Antibody 107C6 VK sequence CDR1: Gln Ser Leu Leu Asp Ser Arg Thr Arg Lys Asn Tyr

(42) SEQ ID NO: 31: Antibody 107C6 VK sequence CDR2: Trp Ala Ser

(43) SEQ ID NO: 32: Antibody 107C6 VK sequence CDR3: Lys Gln Ser Tyr Asn Leu Arg Thr

(44) SEQ ID NO: 33: Antibody 108F8 VH sequence CDR1: Gly Tyr Thr Phe Thr Asn Tyr Gly

(45) SEQ ID NO: 34: Antibody 108F8 VH sequence CDR2: Ile Asn Thr Tyr Ser Gly Val Pro

(46) SEQ ID NO: 35: Antibody 108F8 VH sequence CDR3: Ala Arg Glu Gly Tyr Ser Thr Thr Arg Ser Met Asp Tyr

(47) SEQ ID NO: 36: Antibody 108F8 VK sequence CDR1: Gln Ser Leu Leu Asp Ser Arg Thr Arg Lys Asn Tyr

(48) SEQ ID NO: 37: Antibody 108F8 VK sequence CDR2: Trp Ala Ser

(49) SEQ D NO: 38: Antibody 108F8 VK sequence CDR3: Lys Gln Ser Tyr Asn Leu Arg Thr

(50) SEQ ID NO: 39: Antibody 109A6 VH sequence CDR1: Gly Phe Lys Ile Lys Asp Thr Tyr

(51) SEQ ID NO: 40: Antibody 109A6 VH sequence CDR2: Ile Asp Pro Ala Asn Gly Asn Thr

(52) SEQ ID NO: 41: Antibody 109A6 VH sequence CDR3: Ala Gly Tyr Val Trp Phe Ala Tyr

(53) SEQ ID NO: 42: Antibody 109A6 VK sequence CDR1: Gln Arg Leu Val His Ser Asn Gly Asn Thr Tyr

(54) SEQ ID NO: 43: Antibody 109A6 VK sequence CDR2: Thr Val Ser

(55) SEQ D NO: 44: Antibody 109A6 VK sequence CDR3: Ser Gln Ser Thr Leu Val Pro Trp Thr

(56) SEQ ID NO: 45: Antibody 111A6 VH sequence CDR1: Gly Phe Lys Ile Lys Asp Thr Tyr

(57) SEQ ID NO: 46 Antibody 111A6 VH sequence CDR2: Ile Asp Pro Ala Asn Gly Asn Thr

(58) SEQ ID NO: 47: Antibody 111A6 VH sequence CDR3: Ala Gly Tyr Val Trp Phe Ala Tyr

(59) SEQ ID NO: 48: Antibody 111A6 VK sequence 1 CDR1: Ser Ser Val Ser Ser Ser Tyr

(60) SEQ ID NO: 49: Antibody 111A6 VK sequence 1 CDR2: Ser Thr Ser

(61) SEQ ID NO 50: Antibody 111A6 VK sequence 1 CDR3: Gln Gln Tyr Ser Gly Tyr Pro Leu Thr

(62) SEQ ID NO: 51: Antibody 111A6 VK sequence 2 CDR1: Gln Arg Leu Val His Ser Asn Gly Asn Thr Tyr

(63) SEQ ID NO: 52: Antibody 111A6 VK sequence 2 CDR2: Thr Val Ser

(64) SEQ ID NO: 53: Antibody 111A6 VK sequence 2 CDR2: Ser Gln Ser Thr Leu Val Pro Trp Thr

(65) SEQ ID NO: 54: Antibody 131B4 VH sequence CDR1: Gly Phe Lys Ile Lys Asp Thr Tyr

(66) SEQ ID NO: 55: Antibody 131B4 VH sequence CDR2: Ile Asp Pro Ala Asn Gly Asn Thr

(67) SEQ ID NO: 56: Antibody 131B4 VH sequence CDR3: Ala Gly Tyr Val Trp Phe Ala Tyr

(68) SEQ ID NO: 57: Antibody 131B4 VK sequence CDR1: Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr

(69) SEQ ID NO: 58: Antibody 131B4 VK sequence CDR2: Lys Val Ser

(70) SEQ ID NO: 59: Antibody 131B4 VK sequence CDR3: Ser Gln Ser Ser Leu Val Pro Trp Thr

(71) SEQ ID NO: 60: Antibody 131E8 VH sequence 1 CDR1: Gly Phe Ser Leu Pro Asn Tyr Gly

(72) SEQ ID NO: 61: Antibody 131E8 VH sequence 1 CDR2: Ile Trp Ser Gly Gly Ser Thr

(73) SEQ ID NO: 62: Antibody 131E8 VH sequence 1 CDR3: Ala Arg Asn Phe Tyr Ser Lys Tyr Asp Tyr Ala Met Asp Tyr

(74) SEQ ID NO: 63: Antibody 131E8 VH sequence 2 CDR1: Gly Tyr Thr Phe Thr Ser Tyr Trp

(75) SEQ ID NO: 64: Antibody 131E8 VH sequence 2 CDR2: Ile Asn Pro Asn Ser Gly Ser Thr

(76) SEQ ID NO: 65: Antibody 131E8 VH sequence 2 CDR3: Ala Arg Leu Gly Asp Tyr

(77) SEQ ID NO: 66: Antibody 131E8 VK sequence CDR1: Ser Ser Val Ser Tyr

(78) SEQ ID NO: 67: Antibody 131E8 VK sequence CDR2: Asp Thr Ser

(79) SEQ ID NO: 68: Antibody 131E8 VK sequence CDR3: Phe Gln Gly Ser Gly Tyr Pro Leu Thr

(80) SEQ ID NO: 69: Antibody 132H4 VH sequence CDR1: Gly Phe Thr Phe Ser Asn Tyr Ala

(81) SEQ ID NO: 70: Antibody 132H4 VH sequence CDR2: Ile Ser Ser Gly Gly Ala Asn Ile

(82) SEQ ID NO: 71: Antibody 132H4 VH sequence CDR3: Ala Arg Gly Asp Tyr Phe Asn His Phe Trp Phe Ala Tyr

(83) SEQ ID NO: 72: Antibody 132H4 VK sequence CDR1: Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

(84) SEQ ID NO: 73: Antibody 132H4 VK sequence CDR2: Lys Val Ser

(85) SEQ ID NO: 74: Antibody 132H4 VK sequence CDR3: Phe Gln Gly Ser His Val Pro Trp Thr

(86) SEQ ID NO: 75: Antibody 133A6 VH sequence CDR1: Gly Phe Thr Phe Ser Asn Tyr Ala

(87) SEQ ID NO: 76: Antibody 133A6 VH sequence CDR2: Ile Ser Ser Gly Gly Gly Asn Ile

(88) SEQ ID NO: 77: Antibody 133A6 VH sequence CDR3: Ala Arg Gly Asp Tyr Ser Asn Tyr Phe Trp Phe Ala Tyr

(89) SEQ ID NO: 78: Antibody 133A6 VK sequence CDR1: Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

(90) SEQ ID NO: 79: Antibody 133A6 VK sequence CDR2: Lys Val Ser

(91) SEQ ID NO: 80: Antibody 133A6 VK sequence CDR3: Phe Gln Gly Ser His Val Pro Trp Thr

EXAMPLES

A. Detection of Free IL-18 Versus Complex IL-18/IL-18BP

1. Common Detection of IL-18 in Patients

(92) Human IL-18 quantification in patients is performed with ELISA assays detecting total IL-18 (both free form and IL-18BP complex). The ELISA comprises commercially available antibodies (see Table 8 below). Most common ELISA assays are performed with the pair of anti-IL-18 antibodies developed by Taniguchi et al 1997 and sold by different suppliers, namely monoclonal mouse antibody 125-2H as primary/capture antibody and monoclonal rat 159-12B as secondary/developing antibody.

(93) TABLE-US-00001 TABLE 8 Scientific publications reporting IL-18 quantifications in human patients Antibodies and References Assay, disease commercial source Wong CK IL-18 and IL-12 levels in 1. Human IL-18 ELISA kit et al 2000 plasma, Systemic Lupus from MBL, #7620 Erythematosus 2. Human IL-12 ELISA kit from R&D Systems, #DP400 Park MC IL-18 level in serum, Human IL-18 ELISA kit et al 2004 Systemic Lupus from R&D Systems same Erythematosus as MBL kit #7620 Novick D IL-18 and IL-18BP in 1. Two human IL-18 et al 2001 serum, Sepsis antibodies from R&D systems (mouse monoclonal biotinylated as capture # N/A and rabbit polyclonal ruthenylated as detection #N/A) 2. Two IL-18BP antibodies developed by Interpharm and Serono that are not commercially available, clone MAb No. 582.10 as capture antibody (see above, paragraph 2.2. IL-18BP detection in human serum and urine) and rabbit polyclonal antibody for detection Novick D IL-18 and IL18BP levels Same as Novick et al et al 2010 in serum, Systemic Lupus 2001, see previous row Erythematosus Chen DY IL-18 levels in serum, Human IL-18 ELISA kit et al 2004 Adult Still's Disease from Bender MedSystems (now eBioscience) comprising 2 human IL-18 antibodies called BMS267/2MST: 1. Monoclonal capture antibody # N/A 2. Monoclonal detection antibody labeled with biotin # N/A and reaction revealed with streptavidin-HRP

2. Estimations of Free IL-18 Levels

(94) To date, there are no reports of measured levels of free IL-18. Estimations of free IL-18 are made by extrapolation using the calculation described by Novick et al 2004 (see below). The data compares levels of IL-18 and IL-18BP in human. In these studies, researchers used the pair of commercial monoclonal anti-IL-18 antibodies 125-2H and 159-12B, where antibody 125-2H is used for capture and is known to bind the IL-18/IL-18BP complex (Argiradi et al 2009). To calculate free IL-18 in patient sera, they applied the Law of Mass Action assuming that the binding of IL-18 antibodies is reversible. The calculation is performed as follow:
K.sub.D=0.4 nM=([IL-18]×[IL-18BP])/[IL-18-IL18BP]
or [IL-18] in nM=(0.4×[IL-18-IL18BP])/[IL-18BP] Where: IL-18-IL-18BP is a complex Dissociation constant as calculated by Kim et al 2000, K.sub.D=0.4 nM Stoichiometry 1:1 in the complex IL-18-IL-18BP Concentration of IL-18 is determined by electro-chemiluminescence Concentration of IL-18BP is determined by ELISA

(95) It is important to note that the authors find large variations of free IL-18 versus the total IL-18 between patients that do not reflect the ratio of IL-18 versus IL-18BP. Interestingly, this IL18/IL-18BP ratio is not reported in the cited publications. Furthermore, anti-IL18 antibodies are not able to distinguish between free IL-18 and the complex form IL-18/IL-18BP. Finally, as described by Novick et al 2001, the anti-IL-18BP antibodies do not detect IL-18BP free form but total IL-18BP since they were reported not to block the interaction between IL-18BP and IL-18, respectively monoclonal antibodies 582.10 and 657.27. Consequently, the calculation of free IL-18 using the concentration of IL-18BP lacks accuracy. Even though encouraging, the data variation indicates that free IL-18 detection could be improved with a more appropriate assay combining antibodies specifically targeting the region of IL-18 that binds to IL-18BP.

3. Confirmation that Commonly Used Commercially Antibodies do not Detect Free IL-18

(96) Eleven commercially available anti-IL-18 monoclonal antibodies were tested for their ability to prevent any IL-18 interaction with IL-18BP. The below data demonstrates that this is not the case and that none of the antibodies tested bind to the site of interaction between IL-18 and IL-18BP. Consequently, the detection of free IL-18 in human samples requires specific design and approaches targeting for example the IL-18 binding site/epitope to IL-18BP.

(97) The commonly used 125-2H and 159-12B antibodies were tested for both as capture and developing antibodies (see FIG. 2). The data indicates that both antibodies do not recognize the IL-18 epitope for IL-18BP and consequently provide only a quantification of total IL-18 (both forms free and complex to IL-18BP).

(98) In parallel to antibodies 125-2H and 159-12G, nine other commercial monoclonal antibodies were tested for their potential to detect free IL-18 in the same conditions as above. As described above, such antibody will be valuable to detect free IL-18 in biological samples. The list of tested commercial antibodies is given in the table 9 below.

(99) TABLE-US-00002 TABLE 9 Tested monoclonal anti-IL-18 antibodies Company Antibody name MBL International D043-3, clone 25-2G D-045-6 159-12B biotin Santa Cruz sc-13602 (1.51E3E1) Biotechnologies sc-133127 (E-8) Abnova MAB 1308, clone mxsghk-18 MAB8223, clone SB116c1 MAB8224, cone SB116b1 MAB9935, clone 2 Millipore 04-1503 Anti-Interleukin 18 (clone CPTC-IL18-1) Lifespan LS-C137620 (clone 50008-2)

(100) The collected data indicates that none of the commercially available antibodies was able to distinguish the free IL-18 from its complex with IL-18BP.

4. ELISA Set Up to Detect Free IL-18

(101) 4.1. Capture of Free IL-18 with IL-18BP

(102) Microplate wells are coated with an appropriate volume phosphate buffer saline solution containing recombinant human IL-18BP. Plates are incubated for a period of time at 4° C. and then stabilized with a blocking buffer containing bovine serum albumin or other appropriate blocking agents. Once the reaction is finished, microplates are sealed and stored at 4° C. until used for detection of free IL-18. Microplates can also be dried in a stabilizing solution allowing storage at room temperature and then be reconstituted by hydration when needed for assay.

(103) As an example, for a final reaction volume of 100 μl, dispense first 80 μl of biotin/antibody conjugate. Samples or biological fluids containing free IL-18 are tested with the IL-18BP coated microplates. After that, 20 μl sample volume containing biological fluid or standard is dispensed per microplate well. Non-diluted or diluted biological fluid can be but is not restricted to serum, urine, tear, saliva, bile, sweat, exhalation or expiration, sputum, bronchoalveolar fluid, sebum, cellular, gland, mucosa or tissue secretion, biopsy, homogenized tissue. The free IL-18 standard concentrations range between 4.2 pg/ml to 3000 pg/ml. Standard and concentrations were prepared from commercially available recombinant human IL-18. The plates are sealed and then incubated under gentle shaking for free IL-18 capture. A suitable period of time is allowed for the reaction ranging from minutes to hours at room temperature, 37° C. or other temperatures that do not affect the stability of the samples and reagents. The microplate wells are the washed extensively with the appropriate buffer and then, 100 μl buffer developing mixture is added to each well. The developing mixture contains a streptavidin-conjugated enzyme such as peroxidase or alkaline phosphatase. The microplate wells are sealed and the reaction is allowed for a period of time at A suitable period of time ranging from minutes to hours at room temperature, 37° C. or other temperatures that do not affect the stability of the samples and reagents. The resulting reactions are then monitored with a microplate reader at an appropriate nanometer wavelength for absorbance or fluorescence of the produced reagent.

(104) 4.2. Capture of Free IL-18 with Anti-IL-18 Antibody

(105) Microplate wells are coated with an appropriate volume phosphate buffer saline solution containing Antibody X. Plates are incubated for a period of time at 4° C. and then stabilized with a blocking buffer containing bovine serum albumin or other appropriate blocking agents. Once the reaction is finished, microplates are sealed and stored at 4° C. until used for detection of free IL-18. Microplates can also be dried in a stabilizing solution allowing storage at room temperature and then be reconstituted by hydration when needed for assay.

(106) As an example, for a final reaction volume of 100 μl, dispense first 80 μl of biotin/antibody conjugate. Samples or biological fluids containing free IL-18 are tested with the IL-18BP coated microplates. After that, 20 μl sample volume containing biological fluid or standard is dispensed per microplate well. Non-diluted or diluted biological fluid can be but is not restricted to serum, urine, tear, saliva, bile, sweat, exhalation or expiration, sputum, bronchoalveolar fluid, sebum, cellular, gland, mucosa or tissue secretion, biopsy, homogenized tissue. The free IL-18 standard concentrations range between 4.2 pg/ml to 3000 pg/ml. Standard and concentrations were prepared from commercially available recombinant human IL-18. The plates are sealed and then incubated under gentle shaking for free IL-18 capture. A suitable period of time is allowed for the reaction ranging from minutes to hours at room temperature, 37° C. or other temperatures that do not affect the stability of the samples and reagents. The microplate wells are the washed extensively with the appropriate buffer and then, 100 μl buffer developing mixture is added to each well. The developing mixture contains a streptavidin-conjugated enzyme such as peroxidase or alkaline phosphatase. The microplate wells are sealed and the reaction is allowed for a period of time at A suitable period of time ranging from minutes to hours at room temperature, 37° C. or other temperatures that do not affect the stability of the samples and reagents. The resulting reactions are then monitored with a microplate reader at an appropriate nanometer wavelength for absorbance or fluorescence.

(107) 4.3. Titration of Free IL-18 as a Function of IL-18BP Level

(108) A constant quantity of recombinant IL-18 was titrated as a function of different and well defined quantities of IL-18BP in order to understand when free IL-18 is not any more detectable. A PBS solution of 400 pg/mL IL-18 supplemented by 5% BSA was spiked with defined quantities of IL-18BP ranging from 0 to 10′000 pg/mL. The molar ratios were calculated according to the respective molecular weight of IL-18 and IL-18BP. The free IL-18 detection was performed with ELISA using IL-18BP for IL-18 as described above. The collected data presented in FIG. 1 indicates that 400 pg/mL IL-18 detection are near background detection level when IL-18BP concentration is equal or higher to 6000 pg/mL representing a molar ratio IL-18BP/IL-18 of ˜15 fold higher IL-18BP. In contrast, when molar ratio is lower than 15, free IL-18 is easily detectable.

(109) 4.4. Revised Calculation of Dissociation Constant (K.sub.D) Between Human IL-18 and IL-18BP

(110) 4.4.1 K.sub.D Calculation by Titration

(111) A K.sub.D of 400 pM is reported in the literature based on BIAcore measurements (Kim et al 2000.sup.8). However, due to the above results, the K.sub.D was revisited with the above ELISA set up. Titration of 10 pM IL-18 was performed with increasing concentrations of IL-18BP (60 pM-3 nM) in either a) healthy volunteer sera depleted in endogenous IL-18BP or b) PBS supplemented by 5% BSA. The free IL-18 ELISA in addition to commercially available assays for total IL-18 and total IL-18BP allows the determination of K.sub.D in solution which should reflect better the affinity of IL-18 to its binding protein in body fluids than data from solid-phase BIAcore method. Example of results are exposed in Table 10.

(112) TABLE-US-00003 TABLE 10 Titration of IL-18 in serum or 5% BSA solution containing 1.87 nM IL-18BP IL-18 Titration IL-18 IL-18 spiked spiked Final into into 5% Standard curve IL18 serum BSA nM nM pg/mL OD450 spiked OD450 OD450 IL- IL- IL-18 nm ng/mL nm nm 18 18BP 2000 2.894 24 0.474 1.151 1.3953 1.87 666.7 2.292 20 0.342 0.897 1.1628 1.87 222.2 0.875 16 0.286 0.735 0.9302 1.87 74.1 0.303 12 0.200 0.511 0.6977 1.87 24.7 0.114 8 0.157 0.348 0.4651 1.87 8.2 0.061 4 0.091 0.188 0.2326 1.87 2.7 0.042 2 0.065 0.155 0.1163 1.87 0 0.039 0 0.040 0.037 0 1.87

(113) K.sub.D was calculated based on the following formula:
K.sub.D=[free IL-18]×[free IL-18BP]/[IL-18/IL-18BP complex]
[free IL-18BP]=[total IL-18BP]−[free IL-18]
[IL-18/IL-18BP complex]=[total IL-18]−[free IL-18]

(114) Result: K.sub.D=50 pM (Serum diluent); 35 pM (5% BSA diluent)

(115) The titration result indicates a K.sub.D of respectively 50 pM in serum diluent and 35 pM in PBS supplemented by 5% BSA. In contrast to the previous estimations of the K.sub.D between human IL-18BP and IL-18, the newly calculated K.sub.D indicates that previous estimations of free IL-18 based on the K.sub.D of 400 pM reported by Kim et al 2000 are not accurate.

(116) 4.4.2 K.sub.D Estimation by BIAcore

(117) Following the above K.sub.D results obtain by titration, we tested the binding affinity of IL-18BP to IL-18 with a simpler BIAcore setup consisting of binding IL-18BP to the BIAcore chip and then testing its affinity to IL-18. The method setup is the contrary of Kim et al 2000.sup.8, who bound IL-18 to the BIAcore chip with a monoclonal antibody and then tested the affinity of the complex antibody-IL-18 to IL-18BP. Importantly, the new BIAcore setup collected data that are aligned completely to the above titration findings, i.e. a K.sub.D ranging between 20 and 30 pM. The data is presented in Table 11 below.

(118) TABLE-US-00004 TABLE 11 New BIAcore estimation of human IL-18BP affinity to human IL-18 K.sub.a (10.sup.+5/Ms) K.sub.d (10.sup.−6 1/s) K.sub.D (10.sup.−11 M) 5.3 ± 1.2 13.3 ± 2.7 25.9 ± 4.8

(119) 4.5. Titration of Spiked IL-18 in Serum or 5% BSA Solution Containing IL-18BP

(120) Human serum contains significant levels of endogenous as well as complexed IL-18 to IL-18BP, respectively at ng/mL and pg/mL levels. Both are detectable with commercially available antibodies. However, no commercially available assays are available to detect free IL-18. In order to verify the above ELISA setup for the detection of free IL-18, we spiked recombinant human IL-18 in human serum to find levels of detection. For this, nanograms of IL-18 were spiked in either serum containing endogenous 35 ng/mL IL-18BP or PBS solution supplemented by 5% BSA and 35 ng/mL IL-18BP. Resulting free IL-18 was monitored with the ELISA procedure described above. Results are presented in Table 12 below.

(121) TABLE-US-00005 TABLE 12 Spiked IL-18 detection in serum or 5% BSA containing 35 ng/ml IL-18BP IL-18 Titration Standard curve Final IL-18 spiked IL-18 spiked pg/mL OD450 IL18 spiked into serum into 5% BSA IL-18 nm ng/mL OD450 nm OD450 nm 2000 3.171 100 3.5 3.5 666.7 1.388 80 3.5 3.5 222.2 0.477 70 2.37 3.5 74.1 0.183 60 0.99 3.37 24.7 0.085 50 0.68 2.05 8.2 0.050 40 0.46 1.17 2.7 0.043 30 0.298 0.75 0 0.043 20 0.185 0.44 10 0.11 0.16 5 0.06 0.09 2 0.05 0.07 0 0.04 0.04

5. Detection of Free IL-18 in Patients

(122) 5.1. Detection of Free IL-18 in Serum and Synovial Fluid from Patients Suffering from Different Inflammatory Diseases

(123) Samples coming from patients suffering of different inflammatory diseases were tested with the ELISA described above. For this, we selected different disease and stress conditions reported with higher levels of IL-18 such as rheumatoid arthritis, psoriasis, systemic lupus erythematosus and intensive care unit. To our knowledge, no free IL-18 has been identified in those patients, only by calculation with the Law of Mass Action and the K.sub.D of 400 pM reported by Kim et al 2000. According to the above data, it was expected that possible levels of free IL-18 will be difficult to detect due to the total IL-18 levels ranging in serum below or close to 1000 pg/mL as reported in scientific publications. Furthermore, we tested samples from healthy age-matched controls to verify the performance of our ELISA setup. As expected and contrary to the reported Law of Mass Action estimations, the levels of free IL-18 were not detectable neither in serum nor synovial fluid whereas total IL-18 and IL-18BP were (see Table 13).

(124) TABLE-US-00006 TABLE 13 Detection of free IL-18 in patients from intensive care unit, with psoriasis, lupus and rheumatoid arthritis Calculated Free free IL-18 Patient Total IL18 pg/ml Patient condition/ Biological IL18 pg/ K.sub.D = 4 × IL18BP # disease fluid pg/ml ml 10.sup.−10 M ng/ml 1 Healthy Serum 209.7 — 40.4 29.7 2 Healthy Serum 125.5 — 24.7 28.9 3 Healthy Serum 189.7 — 28.2 40.6 4 Healthy Serum 284.7 — 65.9 23.6 5 Healthy Serum 227.2 — 55.5 22.0 6 Healthy Serum 319.7 — 56.4 33.2 7 Healthy Serum 145.5 — 26.4 32.0 8 Healthy Serum 206.3 — 59.4 17.6 9 Healthy Serum 323.0 — 56.5 33.5 10 Healthy Serum 208.0 — 49.6 22.7 11 Intensive Serum 1158.8 — 52 151.3 care** 12 Intensive Serum 3769.0 — 170.9 151.9 care** 13 Intensive Serum 623.8 — 27.9 151.3 care** 14 Intensive Serum 1978.8 — 104.7 128.0 care** 15 Intensive care Serum 611.3 — 66.9 57.9 16 Intensive care Serum 434.7 — 34.3 82.7 17 Psoriasis Serum 713.8 — 70.6 64.9 arthritis serum 17 Psoriasis Synovial 533.0 — 78.1 41.5 arthritis fluid synovial fluid 18 Lupus serum Serum 510.5 — 123.4 22.5 18 Lupus Synovial 820.5 — 158.9 30.0 synovial fluid fluid 19 Lupus serum Serum 503.8 — 66.4 46.9 19 Lupus Synovial 236.3 — 24.9 60.1 synovial fluid fluid 20 Rheumatoid Plasma 416.3 — 39.9 66.8 arthritis 21 Rheumatoid Serum 281.3 — 67.5 22.6 arthritis 22 Rheumatoid Serum 490.5 — 42.7 74.4 arthritis 23 Rheumatoid Serum 337.2 — 52.2 38.8 arthritis 24 Rheumatoid Serum 342.2 — 53.5 38.3 arthritis 25 Rheumatoid Serum 677.2 — 90.6 46.2 arthritis 26 Rheumatoid Serum 238.8 — 41 34.2 arthritis 27 Rheumatoid Serum 183.8 — 41 24.7 arthritis 28 Rheumatoid Serum 385.5 — 41.6 58.6 arthritis 29 Rheumatoid Serum 345.5 — 42.5 50.6 arthritis —: not detectable, levels comparable to the background signal **: High IL-18BP levels not within standard curve

(125) 5.2. Detection of Free IL-18 in Serum from Patients Suffering from Adult Onset Still's Disease

(126) Following the results and in contrast to the above indications having reasonably low levels of total IL-18, we tested Adult onset Still's Disease patient samples which is known for its elevated levels of total IL-18 in serum (Kawashima et al 2001 and Chen et al 2004). As described by Kawashima et al 2001 and elsewhere, elevated total IL-18 serum levels correlate with Adult onset Still's Disease activity such as a) pyrexia, arthralgia, arthritis, cartilage damage, b) higher levels of Ferritin and c) liver enzymes (LDH). Thanks to the above ELISA set up, we report for the first time free IL-18 levels in Adult onset Still's Disease patients (see Table 14). As for the other tested indications, calculated free IL-18 levels do not correspond to the detected free IL-18 levels. The collected data indicates at least 70% of patients were positive to free IL-18.

(127) TABLE-US-00007 TABLE 14 Detection of free IL-18 in ASD patient serum and synovial fluid Calculated Free IL-18 Sample Bio- Total Free pg/ml IL- Patient collection logical IL-18 IL-18 K.sub.D = 4 × 18BP number date fluid pg/ml pg/ml 10.sup.−10 M ng/ml 1 Serum 6699 9.6 1366.5 32.6 1 Synovial 439 15.8 439 — fluid 2 Serum 713 22.5 564.3 2.0 3 Serum 106026 3.2* 59030 50.4 4 Serum 225456 24.9 157207 68.1 5 Serum 175589 23.6 139614 36.1 6 Serum 35045 2.5* 8908 45.6 7 Serum 17714 22.4 634.8 206.0 7 Synovial 133325 21.3 11162 193.6 fluid 8 Serum 25020 21.1 1277.4 153.7 9 Serum 3625 24.9 394.7 60.8 10 17 Feb. 2006 Serum 11401 7.7 6062 11.3 10 11 Jun. 2007 Serum 79942 31.6 62035 19.1 10 6 Apr. 2009 Serum 37372 18.9 22252 19.2 10 6 Aug. 2010 Serum 185157 12.1 10566 282.9 10 6 Jun. 2012 Serum 131561 11.2 4091 341.2 11 3 Jan. 2006 Serum 150669 34.3 114012 37.2 11 4 Apr. 2007 Serum 106026 26.2 63543 45.2 11 20 Oct. 2008 Serum 225456 23.6 70633 163.0 11 21 Apr. 2010 Serum 175589 23.3 116583 59.8 12 2 Jun. 2009 Serum 3625 8.0 1633 10.5 13 10 Mar. 2010 Serum 439 4.8** 151.2 13.7 14 17 Jul. 2009 Serum 133325 19.3 21118 144.4 15 24 Jul. 2006 Serum 35045 14.3 14628 29.3 16 25 Apr. 2007 Serum 17714 8.0 4075 36.6 16 10 Jun. 2010 Serum 25020 6.4 2592 82.4 *: Level comparable to the background signal **: Level comparable to the lower limit of detection —: not detectable, level comparable to the background signal

6. Conclusions

(128) The data in both publications and the above experimental setup demonstrate that commercial monoclonal antibodies detect total IL-18 but not free IL-18. Furthermore, the most commonly used antibodies to quantify IL-18, namely 125-2H and 159-12B, are confirmed as well in detecting total IL-18.

(129) The estimation of free IL-18 using the Law of Mass Action is an interesting approach. Nevertheless, the large error bars obtained do not support its use in clinical monitoring. Furthermore, the anti-IL-18BP antibodies detect total IL-18BP and not the free form. Consequently, the calculation of free IL-18 using the concentration of IL-18BP lacks accuracy.

(130) The proposed approach to quantify free IL-18 by targeting IL-18 binding site to IL-18BP seems more appropriate and is demonstrated for the first time to be more accurate than extrapolated quantifications with the Law of Mass Action. In addition, the affinity of IL-18BP is higher than reported by Kim et al 2000 with a K.sub.D ranging near 50 pM in serum and 20-30 pM with a new BIAcore setup.

(131) Finally, patients suffering of Adult onset Still's Disease were diagnosed as positive to free IL-18 for the first time with the ELISA approach and a set up is presented in the present invention. The data support earlier findings on total IL-18 for Adult onset Still's Disease as reported by Kawashima et al 2001 and Chen et al 2004 reporting high levels of total IL-18. For the first time, the new ELISA approach presented in this application demonstrates presence of free, not complexed and biologically active pro-inflammatory IL-18 in Adult onset Still's Disease patients.

B. IL-18BP Efficacy in COPD Exacerbation Mouse Model

(132) The aim of the study was to determine the effect of IL-18BP, administered at three dose levels, by the sub-cutaneous route, on Polyinosinic:polycytidylic acid-induced exacerbation of tobacco-smoke induced pulmonary inflammation, in C57BL/6J mice. High level of dexamethasone, dosed orally, was included in the study as a reference agent.

1. General Methodology: Four-Day Exacerbation/Tobacco Smoke Mouse Model

(133) Mice received either vehicle (PBS) or IL-18BP. IL-18BP was given subcutaneously to 3 groups of animals respectively at 1, 3 or 10 mg/kg 2 h prior to the initial tobacco smoke exposure from Day 1 to Day 4. Mice received orally either vehicle or dexamethasone (10 mg/kg) 1 h prior to each twice daily exposure. Mice received by intranasal administration either the vehicle or Polyinosinic:polycytidylic acid (2 mg/kg) 2 h prior to the initial air or tobacco smoke exposure on Day 4 to induce lung inflammation exacerbation. Tobacco smoke exposure was performed during the morning and afternoon as follow: Day 1 for 15 min, Day 2 for 25 min, Day 3 for 30 min and Day 4 for 30 min.

(134) Animal groups and their respective treatment regimes are summarized in Table 1.

(135) TABLE-US-00008 TABLE 15 Treatment regimes for tobacco smoke mouse model Treatment Treatment Dose Exposure s.c./oral Code n mg/kg Challenge Frequency Air Veh/Veh A 10 -/- Veh Sub- TS Veh/Veh B 10 -/- Veh cutaneous Air Veh/Veh C 10 -/- p[I:C] 2 h prior to 2 mg/kg initial TS TS Veh/Veh D 10 -/- p[I:C] on each 2 mg/kg day TS IL-18BP/ E 10 1/- p[I:C] Oral 1 h Veh 2 mg/kg prior to TS IL-18BP/ F 10 3/- p[I:C] each TS Veh 2 mg/kg exposure TS IL-18BP/ G 10 10/- p[I:C] on each Veh 2 mg/kg day TS Veh/ Dex H 10 -/10 p[I:C] p[I:C] 2 mg/kg intranasal 2 h prior to TS exposure on day 4 TS: Tobacco smoke; Veh: Vehicule; Dex: Dexamethazone, p[I:C]: Polyinosinic:polycytidylic acid

(136) Following the above treatments, animals were terminally anaesthetised on Day 5. After that, a blood sample was taken via the sub-clavian artery (plasma) and the animals were bronchoalveloar lavaged with 3×0.4 ml of PBS for further cellular and cytokine/mediator analysis. Bronchoalveolar lavage supernatants were stored at −80° C. for cytokine/mediator analysis. Cells recovered from the BALF were counted using the Sysmex cell counter. Finally, the collected data was statistically analyzed by Students t-test and ANOVA (Sidak's was used in the case of data passed normality test or Kruskal Wallis test if data did not pass normality test).

2. Confirmation of IL-18 Pathway Activation in the Four-Day Exacerbation/Tobacco Smoke Mouse Model

(137) Mouse IL-18 was tested in the BAL using a commercial ELISA in order to confirm the mouse model for IL-18 pathway activation. The collected data indicates a clear induction of IL-18 in the lung airway space (see FIG. 5). IL-18 is not detectable in the control (air only). Interestingly, IL-18 is expressed under smoke exposure but is not significantly induced under poly[I:C] alone (under the lower limit of detection). In contrast and as expected, the combination of smoke and poly[I:C] raises considerably IL-18 to much higher levels in the BAL than smoke or poly[I:C] alone.

3. Exacerbated Inflammation Mitigation by IL-18BP in Exacerbation/Tobacco Smoke Mouse Model

(138) 3.1. Inhibition of Total Cell Infiltration and Exacerbated Inflammation in the Lung Airway Space by IL-18BP

(139) Mice treated by IL-18BP had a significant mitigation of total cell infiltration in the lung following induction of exacerbated inflammation. Doses of either 3 and 10 mg/kg indicated statistically valuable efficacy compared to the positive control dexamethasone (see FIG. 5). It is important to note that dexamethasone had no sign of efficacy at 3 mg/kg doses in the mouse model (data not shown), indicating that the high dexamethasone dose of 10 mg/kg is potentially inducing apoptosis in certain cell types such as macrophages, eosinophils and lymphocytes (data not shown). Similar observation was made with Roflumilast [3-(cyclopropylmethoxy)-N-(3,5-dichloropyridin-4-yl)-4-(difluoromethoxy) benzamide] in the mouse model where no hint of cell infiltration inhibition was observed with 2.5 mg/kg dose (data not shown). FIG. 6 shows clear and statistically relevant efficacy of IL-18BP at 10 mg/kg in exacerbated inflammation inhibition in the current mouse model.

(140) 3.2. Inhibition of Neutrophil Infiltration in the Lung Airway Space by IL-18BP

(141) Neutrophil infiltration was inhibited by IL-18BP in tobacco smoke-exacerbated lungs. Doses of either 3 and 10 mg/kg IL-18BP indicated statistically valuable efficacy compared to the positive control dexamethasone (see FIG. 7). In the current mouse model conditions, IL-18BP 10 mg/kg dose seems to have the best statistical efficacy (see FIG. 8).

(142) 3.3. Inhibition of Granulocyte Colony-Stimulating Factor (G-CSF) Pathway in the Lung Airway Space by IL-18BP

(143) G-CSF is well acknowledged as key cytokine stimulating the survival, proliferation, differentiation, and function of neutrophil precursors and mature neutrophils. Consequently, mitigation of G-CSF pathway-induced by smoke-p[I:C] is an significant factor demonstrating an effect of IL-18BP on neutrophil recruitment in the mouse lung airway space. The presence of G-CSF in the BALF was monitored with a commercially available ELISA kit. FIG. 9 demonstrates that administration of IL-18BP mitigates G-CSF release in the lung airways, thereby confirming the inhibition of neutrophil infiltration. The three tested IL-18BP doses have a statistically relevant effect in the mouse model.

(144) 3.4. IL-18BP Safety: Effect on Weight Loss in Exacerbation/Tobacco Smoke Mouse Model

(145) IL-18BP administration appeared to be well tolerated by exacerbation/tobacco smoke mouse model. As an example, weight loss was mitigated by IL-18BP even though both Students t-test and ANOVA statistical analyses were not significant (see FIG. 10). A large majority of mice receiving 3 and 10 mg/kg IL-18BP lost respectively 6-7% weight in contrast to the control exposed to tobacco smoke and p[I:C] that lost about 9%. Hence, weight loss alleviation data indicates that IL-18BP is not providing additional stress to the animal model. It is interesting to note that mice receiving only p[I:C] did not lose weight compared to mice receiving the combination of p[I:C] and tobacco smoke [see FIG. 8, treatment 3) and 4)].

C. Generation of Anti-IL-18 Monoclonal Antibodies

1. Mouse Immunization and Monoclonal Antibody Screening

(146) Mice were vaccinated aginst human interleukin-18 using a technology allowing immunization with properly folded proteins. Prior to immunization, genetically modified mice were selected for major histocompatibility complexes supposedly sensitive to IL-18 surface area epitopes binding IL-18BP. Following immunization, B cells were isolated from spleen and hybridized following standard hybridoma technology. Hybridoma were sorted onto microplates and then tested for expression of monoclonal anti-IL-18 antibodies targeting IL-18 epitopes included in IL-18BP binding site. The screening was performed in 3 sequential and selective steps: First step. Positive antibody screening attempt was performed with IL-18 attached to Luminex beads confirming cell expressing monoclonal anti-IL-18 antibodies. Second step. Potential antibodies targeting IL-18 on IL-18BP binding site were rescreened in competition with IL-18BP. For this, monoclonal antibodies were bound to Luminex beads carrying IL-18. The complex was then exposed to biotinylated IL-18BP in order to identify interference to previously identified anti-IL-18 antibodies (see Table 1, Column #2). The second screening carried more than 300 positive antibody candidates (see Table 1, Column #3). The number of positive candidates was surprisingly high suggesting an excellent mouse immunization to the targeted epitope area. However, inhibitions were not sufficient due to diminished but still persistent fluorescence signals, thus indicating binding of IL-18BP to the complexed antibody IL-18. Nevertheless and importantly, such standard screening method reported elsewhere does not take into account a potential steric hindrance of the large antibody molecule (about 160 kDa) against the much smaller IL-18BP (about 18 kDa, peptide only). Third step. A third screening program was undertaken with Luminex beads linked to IL-18BP and then complexed to interleukin-18, assuring the presentation of properly folded recombinant IL-18 to positive antibody candidates. The resulting screening was considerably more selective because most of the above antibodies still bound the Luminex-IL-18 beads thereby indicating that their previous inhibitory effect to IL-18BP was due to steric hindrance. Finally, a total of 12 antibodies were finally considered as targeting IL-18 on the IL-18BP protein due to their very low fluorescence signal after binding IL-18 in the presence of IL-18BP, namely clone #107C6, 108F8, 109A6, 111A6, 129C3, 131B4, 131E8, 131H1, 132C12, 132H4, 133A6 and 134B2 (see Table 16, Column #4, selected clones representing inhibition means of more than 500 fold compared to Column #2). The positive antibodies versus a set of negatives are presented in Table 16 below.

(147) The collected data from third screening step (Table 16, Column #4) promoted further mRNA sequencing and clone dilution work to enrich positive monoclonal cells out of #107C6, 108F8, 109A6, 111A6, 129C3, 131B4, 131E8, 131H1, 132C12, 132H4, 133A6 and 134B2. All of these monoclonal antibodies were confirmed to bind to IL-18 on the IL-18BP binding site.

(148) TABLE-US-00009 TABLE 16 Screening of monoclonal antibodies targeting IL-18 on the IL-18BP binding site Column #1 Column #2 Column #3 Monoclonal IL-18BP binding on Monoclonal antibody antibodies IL-18 previously binding on IL-18 binding on complexed to previously complexed Clone IL-18 monoclonal antibody to IL-18BP name Fluorescence intensity Examples of negative antibodies not following selection criteria 101D2 26 963 1 226 1 544   104H10 26 508 1 199 2 499   105A2 21 528 1 886 1 840   106H1 27 178 1 011 1 324   108F3 23 496 1 964 2 383   108G6 25 652 1 137 2 507   115E6 25 752 1 604 2 649   119E9 25 420 1 307 2 931   Positive antibodies following selection criteria 107C6 26 250 1 389 33 108F8 25 126 1 292 45 109A6 25 848 .sup. 913 33 111A6 25 855 1 398 42 131B4 24 838 1 656 41 131E8 25 411 1 389 36 131H1 24 806 1 026 24 132C12 24 541 1 515 48 132H4 23 839 1 488 28 133A6 23 273 1 631 25 134B2 24 278 1 261 48 129C3 25 412 .sup. 760 44

REFERENCES

(149) Argiriadi M A, Xiang T, Wu C, Ghayur T and Borhani D W. Unusual water-mediated antigenic recognition of the proinflammatory cytokine interleukin-18. J Biol Chem 2009; 284(36)24478-24489. Azoulay E, Eddahibi S, Marcos E, Levame M, Harf A, Schlemmer B, Adnot S and Delclaux C. Granulocyte colony-stimulating factor enhances alpha-naphthylthiourea-induced pulmonary hypertension. J Appl Physiol 2003; 94:2027-2033. Baron R M, Choi A J S, Owen C A and Choi A M K. Genetically manipulated mouse models of lung disease: potential and pitfalls. Am J Physiol Lung Cell Mol Physiol 2012; L485-L497. Chen D Y, Lan J L, Lin F J and Hsieh T Y. Proinflammatory cytokine profiles in sera and pathological tissues of patients with active untreated adult onset Still's disease. J Rheumatol 2004; 31:2189-2198. Chen D Y, Lan J L, Lin F J, Hsieh T Y and Wen M C. Predominance of Th1 cytokine in peripheral blood and pathological tissues of patients with active untreated adult onset Still's disease. Ann Rheum Dis 2004; 63(10): 1300-1306. Cunningham R E. Tissue disaggregation. Methods Mol Biol 1994; 34:225-228. Daley E, Emson C, Guignabert C, de Waal Malefyt R, Louten J, Kurup V P, Hogaboam C, Taraseviciene-Stewart L, Voelkel N F, Rabinovitch M, et al. Pulmonary arterial remodeling induced by a Th2 immune response. J Exp Med 2008; 205:361-372. Elias J A, Kang M J, Crothers K et al. Mechanistic heterogeneity in chronic obstructive pulmonary disease: insights from transgenic mice. Proc Am Thorac Soc 2006; 494-498. Eltom S, Stevenson C S and Rastrick J. P2X7 Receptor and Caspase-1 Activation Are Central to Airway Inflammation Observed after Exposure to Tobacco Smoke PLoS ONE 2011; 6(9):e24097. Hackett B P, Shimizu N and Gitlin J D. Clara cell secretory protein gene expression in bronchiolar epithelium. Am J Physiol 1992; 262:L399-L404. Halbower A C, Mason R J, Abman S H and Tuder R M. Agarose infiltration improves morphology of cryostat sections of lung. Lab Invest 1994; 71: 149-153. Hautamaki R D, Kobayashi D K, Senior R M and Shapiro S D. Requirement for macrophage elastase for cigarette smoke-induced emphysema in mice. Science 1997; 277: 2002-2004. Hoshino T, Kawase Y, Okamoto M et al. IL-18-transgenic mice: in vivo evidence of a broad role for IL-18 in modulating immune function. J Immunol 2001; 7014-7018. Hoshino T, Kato S, Oka N et al. Pulmonary inflammation and emphysema: role of the cytokines IL-18 and IL-13. Am J Respir Crit Care Med 2007; 49-62. Helmut Fenner. Targeting IL-18 in Chronic Obstructive Pulmonary Disease: Background and rationale. Mar. 22, 2013 CONFIDENTIAL Page 19. Hou S, Li B, Wang L, Qian W, Zhang D, Hong X, Wang H, Guo Y (July 2008). “Humanization of an anti-CD34 monoclonal antibody by complementarity-determining region grafting based on computer-assisted molecular modeling.”. J Biochem 144 (1): 115-20 Imaoka H, Hoshino T, Takei S, Kinoshita T et al. Interleukin-18 production and pulmonary function in COPD. Eur Respir J 2008; 287-97. Jaatinen T, Laine J. Isolation of mononuclear cells from human cord blood by Ficoll-Paque density gradient. Curr Protoc Stem Cell Biol 2007; Chapter 2:Unit 2A.1. Kang M J, Lee C G, Lee J Y, Dela Cruz C S, Chen Z J, Enelow R, Elias J A. Cigarette smoke selectively enhances viral PAMP- and virus-induced pulmonary innate immune and remodeling responses in mice. J Clin Invest 2008; 118:2771-2784. Kang M J, Homer R J, Gallo A et al. IL-18 is induced and IL-18 receptor alpha plays a critical role in the pathogenesis of cigarette smoke-induced pulmonary emphysema and inflammation. J Immunol 2007; 1948-1959. Kang M J, Choi J M, Kim B H et al. IL-18 induces emphysema and airway and vascular remodeling via IFNγ, IL-17A, and IL-13. Am J Respir Crit Care Med 2012; 1205-1217. Kashmiri S V, De Pascalis R, Gonzales N R, Schlom J. (May 2005). “SDR grafting—a new approach to antibody humanization.”. Methods 36 (1): 25-34 Kawashima, M. et al. Levels of interleukin-18 and its binding inhibitors in the blood circulation of patients with adult-onset Still's disease. Arthritis Rheum 2001; 44(3):550-560. Kim, S. H. et al. Structural requirements of six naturally occurring isoforms of the IL-18 binding protein to inhibit IL-18. Proc Natl Acad Sci USA 2000; 97(3):1190-1195. Kratzer A, Salys J, Nold-Petry El al. Role of IL-18 in second hand smoke-induced emphysema. Am J Respir Cell Mol Biol 2013; 48(6):725-32. Lee C G, Link H, Baluk P, Homer R J, Chapoval S, Bhandari V, Kang M J, Cohn L, Kim Y K, McDonald D M, et al. Vascular endothelial growth factor (VEGF) induces remodeling and enhances TH2-mediated sensitization and inflammation in the lung. Nat Med 2004; 10:1095-1103. Lee C G, Hartl D, Lee G R, Koller B, Matsuura H, Da Silva C A, Sohn M H, Cohn L, Homer R J, Kozhich A A, et al. Role of breast regression protein 39 (BRP-39)/chitinase 3-like-1 in Th2 and IL-13-induced tissue responses and apoptosis. J Exp Med 2009; 206:1149-1166. Liu J et al. Requirement for tumor necrosis factor-receptor 2 in alveolar chemokine expression depends upon the form of the ligand. Am J Respir Cell Mol Biol 2005; 33:463-469. Londhe V A, Maisonet T M, Lopez B, Jeng J M, Li C, Minoo P. A subset of epithelial cells with CCSP promoter activity participates in alveolar development. Am J Respir Cell Mol Biol 2011; 44:804-812. Loza M J, Watt R, Baribaud F, Barnathan E S and Rennard S I. Systemic inflammatory profile and response to anti-tumor necrosis factor therapy in chronic obstructive pulmonary disease. Respir Res 2012; 13:12. Ma B, Kang M J, Lee C G, Chapoval S, Liu W, Chen Q, Coyle A J, Lora J M, Picarella D, Homer R J and Elias J A. Role of CCR5 in IFN-γ-induced and cigarette smoke-induced emphysema. J Clin Invest 2005; 115:3460-3472. Nakajima T and Owen C. Interleukin-18: The Master Regulator Driving Destructive and Remodeling Processes in the Lungs of Patients with Chronic Obstructive Pulmonary Disease? Am J Respir Crit Care Med 2012; 1137-1138. Novick D et al. A novel IL-18BP ELISA shows elevated serum IL-18BP in sepsis and extensive decrease of free IL-18. Cytokine 2001; 14, 334-342. Novick D et al. High circulating levels of free interleukin-18 in patients with active SLE in the presence of elevated levels of interleukin-18 binding protein. J Autoimmun 2010; 34, 121-126. Park M C, Park Y B and Lee S K. Elevated interleukin-18 levels correlated with disease activity in systemic lupus erythematosus. Clin Rheumatol 2004; 23, 225-229. Petersen A M W, Penkowa M and Iversen M. Elevated Levels of IL-18 in Plasma and Skeletal Muscle in Chronic Obstructive Pulmonary Disease. Lung 2007; 161-171. Queen C, Schneider W P, Selick H E, Payne P W, Landolfi N F, Duncan J F, Avdalovic N M, Levitt M, Junghans R P, Waldmann T A. (December 1989). “A humanized antibody that binds to the interleukin 2 receptor.”. Proc Natl Acad Sci USA. 86 (24): 10029-33 Rastrick J M D, Stevenson C S, Eltom S, Grace M, Davies M, et al. Cigarette Smoke Induced Airway Inflammation Is Independent of NF-κB Signalling. PLoS ONE 2013; 8(1):e54128. Ray P, Tang W, Wang P, Homer R, Kuhn C III, Flavell R A and Elias J A. Regulated overexpression of interleukin 11 in the lung: use to dissociate development-dependent and -independent phenotypes. J Clin Invest 1997; 100:2501-2511. Reed L J and Muench H. A simple method of estimating 50% endpoints. Am J Hyg 1938; 27:493-497. Riechmann L, Clark M, Waldmann H, Winter G (1988). “Reshaping human antibodies for therapy”. Nature 332 (6162): 332-323 Rovina N, Dima E and Gerassimou C. Interleukin-18 in induced sputum: Association with lung function in chronic obstructive pulmonary disease. Respiratory Medicine 2009; 1056-1062. Shapiro D S. Transgenic and gene-targeted mice as models for chronic obstructive pulmonary disease. Eur J Respir 2007; 375-378. Taniguchi, M. et al. Characterization of anti-human interleukin-18 (IL-18)/interferon-gamma-inducing factor (IGIF) monoclonal antibodies and their application in the measurement of human IL-18 by ELISA. J Immunol Methods 1997; 206, 107-113. Wang Z, Zheng, Zhu T Z, Homer R J, Riese R J, Chapman H A, Shapiro S D, and Elias J A. Interferon γ induction of pulmonary emphysema in the adult murine lung. J Exp Med 2000; 192: 1587-1600. Wright J L, Cosio M and Churg A. Animal models of chronic obstructive disease. Am J Physiol Lung Cell Mol Physiol 2008; L1-L15. Wong C K, Li E K, Ho C Y and Lam C W. Elevation of plasma interleukin-18 concentration is correlated with disease activity in systemic lupus erythematosus. Rheumatology (Oxford) 2000; 39:1078-1081. Zhang J, Dong Z, Zhou R, Luo D, Wei H and Tian Z. Isolation of lymphocytes and their innate immune characterizations from liver, intestine, lung and uterus. Cell Mol Immunol 2005; 2:271-280. Zheng T, Zhu Z, Wang Z, Homer R J, Ma B, Riese R, Chapman H, Shapiro S D and Elias J A. Inducible targeting of IL-13 to the adult lung causes matrix metalloproteinase- and cathepsin-dependent emphysema. J Clin Invest 2000; 106:1081-1093. Zheng T et al. Role of cathepsin S-dependent epithelial cell apoptosis in IFN-gamma-induced alveolar remodeling and pulmonary emphysema. J Immunol 2005; 174:8106-8115.

Further Embodiments of the Invention

(150) 1. An IL-18 inhibitor for use in the treatment of an IL-18 associated disease or disorder in a subject diagnosed of having abnormal levels of free IL-18 and/or an abnormal ratio of free IL-18/IL-18BP in the body fluids compared to the levels in body fluids of a healthy control subject. 2. The IL-18 inhibitor for use according to embodiment 1, wherein said abnormal level of free IL-18 in the body fluids exceeds the level in body fluids of a healthy control subject by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100%. 3. The IL-18 inhibitor for use in any one of the preceding embodiments, wherein the subject to be treated belongs to a group of subjects which have been determined to have elevated levels of free IL-18 and/or an abnormal ratio of free IL-18/IL-18BP (IL-18BP) in the body fluids, particularly serum, sputum, broncho-alveolar lavage fluid (BALF), synovial fluid and/or circulation compared to the levels in the body fluids of a healthy subject. 4. The IL-18 inhibitor for use in embodiments 2 or 3, wherein said elevated levels of free IL-18 in serum are in the range of 5 to 10000 pg/mL, whereas the amount of free IL-18 in serum of healthy subject, particularly a healthy human is pg/mL. 5. The IL-18 inhibitor for use according to any one of the preceding embodiments, wherein said IL-18 associated disease or disorder is one selected from the group consisting of chronic obstructive pulmonary disease (COPD), transfusion-related lung injury, bronchopulmonary dysplasia (BPD), acute respiratory distress syndrome (ARDS), Adult Still's disease, juvenile Still's disease, interstitial lung disease (ILD), idiopathic pulmonary fibrosis, cystic fibrosis, pulmonary arterial hypertension, asthma, bronchiectasis, heart failure, amyotrophic lateral sclerosis (ALS), dry eye disease (DED), keratitis, corneal ulcer and abrasion, corneal neovascularization, pathological intraocular neovascularization, iritis, glaucoma, macular degeneration, Sjögren's syndrome, autoimmune uveitis, Behçet's disease, conjunctivitis, allergic conjunctivitis, dermatitis of eyelid, diabetes type 2, non-alcoholic fatty liver disease (NAFLD), steato hepatitis, solid organ and hematologic transplantation, ischemia reperfusion injury, familial Mediterranean fever, tumor necrosis factor receptor 1-associated periodic syndromes, cryopyrin-associated periodic fever syndromes, hyper-IgD syndromes, gout, Schnitzler syndrome, Wegener's granulomatosis also called granulomatosis with polyangitis (GPA), Hashimoto's thyroiditis, Crohn's disease, ulcerative colitis, immunoglobulin-4 (IgG4)-related diseases and stem cell therapies. 6. The IL-18 inhibitor for use according to any one of the preceding embodiments, wherein said IL-18 associated disease or disorder is induced by smoking or second-hand smoke exposure, in particular tobacco smoke exposure. 7. The IL-18 inhibitor for use according to any one of the preceding embodiments, wherein said IL-18 associated disease or disorder is induced by viral infection. 8. The IL-18 inhibitor for use according to any one of the preceding embodiments, wherein said IL-18 associated disease or disorder is an IL-18 induced systemic manifestation of inflammation and associated comorbidities selected from the group consisting of emphysema, tissue inflammation, tissue destruction, lung resection, disappearance of the vasculature, apoptosis of endothelial cells, mucos metaplasia, cardiac hypertrophy, decrease of VEGF in the lung tissue, pulmonary vessel loss, vessel muscularization, vascular remodeling, collagen deposition, aberrant elastin layers in the lung, fibrotic airway remodeling, airspace enlargement, chronic remodeling of the airways and pulmonary vessels and decreased pulmonary function. 9. The IL-18 inhibitor for use in any one of the preceding embodiments, wherein treatment comprises prevention, halting, alleviation or reversion of symptoms associated with said disease or disorder. 10. The IL-18 inhibitor for use in any one of the preceding embodiments, wherein IL-18 binding is restricted or inhibited, particularly binding of free IL-18 to IL-18R, but especially binding of free IL-18 to IL-18Rα. 11. The IL-18 inhibitor for use in any one of the preceding embodiments, wherein IL-18-dependent downstream signaling pathways are modified, particularly inhibited. 12. The IL-18 inhibitor for use in any one of the preceding embodiments, wherein increased expression of IFNγ, IL-13 or IL-17A is modified, particularly inhibited, compared to untreated subjects suffering from said disease or disorder. 13. The IL-18 inhibitor for use in any one of the preceding embodiments, wherein the IL-18 inhibitor compensates the IL-18/IL-18BP imbalance by trapping and neutralizing the excess of free IL-18 in tissue and circulation. 14. An IL-18 specific antibody including any functionally equivalent antibody or parts thereof, which antibody or part thereof binds to IL-18 at the binding site of IL-18BP or in the vicinity of the binding site of IL-18BP. 15. The IL-18 specific antibody including any functionally equivalent antibody or parts thereof according to embodiment 14, which antibody of part thereof binds free IL-18 protein, but not IL-18/IL-18BP complexes. 16. The IL-18 specific antibody including any functionally equivalent antibody or parts thereof according to embodiment 14 or embodiment 15, wherein said antibody or part thereof sterically hinders or prevents the binding of IL-18BP to IL-18. 17. The IL-18 specific antibody including any functionally equivalent antibody or parts thereof according to embodiments 14 to 16, wherein said antibody or part thereof specifically binds to a single epitope, a combination of two epitopes or a combination of 3 epitopes comprised in a sequence selected from a group of sequences depicted in SEQ ID NO.:1, SEQ ID NO: 2 and SEQ ID NO: 3. 18. The IL-18 specific antibody including any functionally equivalent antibody or parts thereof according to embodiment 17, wherein said epitope has a sequence which has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to a sequence selected from a group of sequences depicted in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. 19. The IL-18 specific antibody including any functionally equivalent antibody or parts thereof according to embodiment 18, wherein said epitope is selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. 20. The IL-18 specific antibody including any functionally equivalent antibody or parts thereof according to any one of the preceding embodiments, wherein said antibody or part thereof is a monoclonal antibody or a polyclonal antibody. 21. The IL-18 specific antibody including any functionally equivalent antibody or parts thereof according to any one of the preceding embodiments, wherein said antibody or part thereof is a chimeric, single chain, bispecific, simianized, human and humanized antibody. 22. The IL-18 specific antibody including any functionally equivalent antibody or parts thereof according to any one of the preceding embodiments, wherein said antibody or part thereof binds to human IL-18. 23. The IL-18 specific antibody including any functionally equivalent antibody or parts thereof according to any one of the preceding embodiments, wherein binding of IL-18 to IL-18 receptor, particularly binding to IL-18Rα is reduced by at least 5%, particularly by at least 10%, particularly by at least 15%, particularly by at least 20%, particularly by at least 25%, particularly by at least 30%, particularly by at least 40%, particularly by at least 45%, particularly by at least 50%, particularly by at least 55%, particularly by at least 60%, particularly by at least 65%, particularly by at least 70, particularly by at least 75, particularly by at least 80, particularly by at least 85%, particularly by at least 90%, particularly by at least 95%, particularly by 100%. 24. The IL-18 specific antibody including any functionally equivalent antibody or parts thereof according to any one of the preceding embodiments, wherein said antibody or part thereof neutralizes free IL-18 by restricting or preventing IL-18 binding to IL-18 receptor (IL-18R), especially free IL-18 binding to IL-18Rα. 25. The IL-18 specific antibody including any functionally equivalent antibody or parts thereof according to any one of the preceding embodiments, wherein said antibody or parts thereof a) specifically binds to a single epitope, a combination of two epitopes or a combination of 3 epitopes comprised in a sequence selected from a group of sequences depicted in SEQ ID NO:1, SEQ ID NO: 2 and SEQ ID NO: 3; and/or b) specifically binds to an epitope, which has a sequence identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% to the sequence depicted in SEQ ID NO: 4, SEQ ID NO:5 or SEQ ID NO: 6; and c) specifically binds to IL-18 at the binding site of IL-18BP or in the vicinity of the binding site of IL-18BP; and d) specifically binds to free IL-18 protein, but not IL-18/IL-18BP complexes; and e) sterically hinders the binding of IL-18BP to IL-18; and f) reduces binding of IL-18 to IL-18 receptor, particularly binding to IL-18Rα by at least 5%, particularly by at least 10%, particularly by at least 15%, particularly by at least 20%, particularly by at least 25%, particularly by at least 30%, particularly by at least 40%, particularly by at least 45%, particularly by at least 50%, particularly by at least 55%, particularly by at least 60%, particularly by at least 65%, particularly by at least 70, particularly by at least 75, particularly by at least 80, particularly by at least 85%, particularly by at least 90%, particularly by at least 95%, particularly by 100%. 26. The IL-18 inhibitor for use according to any one of embodiments 1 to 13, wherein the inhibitor is an antibody, particularly an antibody specific for free IL-18, particularly an antagonistic antibody, which prevents binding of free IL-18 to IL-18 receptor, especially free IL-18 binding to IL-18Rα. 27. The IL-18 inhibitor for use according to embodiment 26, wherein said antibody is the antibody of any one of embodiments 14-25. 28. The IL-18 inhibitor for use according to any one of embodiments 1-13, wherein said abnormal levels of free IL-18 in the body fluids has been determined by use of an antibody according to any one of embodiments 14-25. 29. The IL-18 inhibitor for use according to any one of the embodiments 1 to 13, wherein the inhibitor is IL-18BP, particularly human IL-18BP (hIL-18BP), particularly IL-18BP including any functionally equivalent or parts thereof, particularly an IL-18BP as shown in SEQ ID NO: 7. 30. The IL-18 inhibitor for use in according to embodiments 26-29, which is a full-length protein or a mutein, functional derivative, functional fragment, biologically active peptide, fraction, circularly permuted derivative, fused protein, isoform or a salt thereof. 31. IL-18BP for use in the treatment of chronic obstructive pulmonary disease (COPD), heart disease, dry eye disease and/or diabetes type II. 32. The IL-18BP for use according to embodiment 31 for the treatment of chronic obstructive pulmonary disease (COPD). 33. The IL-18BP for use according to embodiment 31 for the treatment of heart disease. 34. The IL-18BP for use according to embodiment 31 for the treatment of dry eye disease. 35. The IL-18BP for use according to embodiment 31 for the treatment of diabetes type II. 36. The IL-18BP for use according to embodiments 31 to 35, wherein said disease or disorder is induced by smoking or second-hand smoke exposure, in particular tobacco smoke exposure. 37. The IL-18BP for use according to any one of the preceding embodiments, wherein said disease or disorder is induced by viral infection. 38. The IL-18BP for use according to any one of the preceding embodiments, wherein said disease or disorder is an IL-18 induced systemic manifestation of inflammation and associated comorbidities selected from the group consisting of emphysema, tissue inflammation, tissue destruction, lung resection, disappearance of the vasculature, mucos metaplasia, cardiac hypertrophy, decrease of VEGF in the lung tissue, pulmonary vessel loss, vessel muscularization, collagen deposition, aberrant elastin layers in the lung, fibrotic airway remodeling, airspace enlargement, chronic remodeling of the airways and pulmonary vessels and decreased pulmonary function. 39. The IL-18BP for use according to any one of the preceding embodiments, wherein IL-18 binding is restricted or inhibited, particularly binding of free IL-18 to IL-18R, but especially free IL-18 binding to IL-18Rα. 40. The IL-18BP for use according to any one of the preceding embodiments, wherein IL-18-dependent downstream signaling pathways are modified, particularly inhibited. 41. The IL-18BP for use according to any one of the preceding embodiments, wherein increased expression of IFNγ, IL-13 or IL-17A is modified, particularly inhibited, compared to untreated subjects suffering from said disease or disorder. 42. The IL-18BP for use according to any one of the preceding embodiments, wherein the IL-18 inhibitor compensates the IL-18/IL-18BP imbalance by trapping the excess of free IL-18 in tissue and circulation. 43. The IL-18BP for use according to any one of the preceding embodiments, wherein treatment comprises prevention, halting, alleviation or reversion of symptoms associated with said disease or disorder. 44. A pharmaceutical composition for use in the treatment of the disease or disorder as defined in any one of embodiments 1-13 in a subject suffering from such a disease or disorder or having a predisposition to develop such a disease or disorder as defined in any one of embodiments 1-13, wherein said composition comprises the IL-18 inhibitor according to any one of embodiments 1-13 and 26-30, particularly in a prophylactically and/or therapeutically effective amount. 45. The pharmaceutical composition of embodiment 44, wherein said composition optionally further provides another inhibitor of a pro-inflammatory cytokine or functional fragment thereof, or a regulatory factor, which induces in-situ expression of said inhibitor of pro-inflammatory cytokine or functional fragment thereof, co-therapeutic agents such as anti-inflammatory, bronchodilatory, antihistamine, decongestant or anti-tussive drug substances. 46. The pharmaceutical composition of embodiment 44 or 45, comprising a pharmaceutically acceptable carrier and/or excipient. 47. A pharmaceutical composition for use in the treatment of the disease or disorder as defined in any one of embodiments 31 to 43 in a subject suffering from such a disease or disorder or having a predisposition to develop such a disease or disorder as defined in any one of embodiments 31 to 43, wherein said composition comprises the IL-18BP according to embodiments 31 to 43, particularly in a prophylactically and/or therapeutically effective amount. 48. The pharmaceutical composition of embodiment 47, wherein said composition optionally further provides another inhibitor of a pro-inflammatory cytokine or functional fragment thereof, or a regulatory factor, which induces in-situ expression of said inhibitor of pro-inflammatory cytokine or functional fragment thereof, co-therapeutic agents such as anti-inflammatory, bronchodilatory, antihistamine, decongestant or anti-tussive drug substances. 49. The pharmaceutical composition of embodiment 47 or 48, comprising a pharmaceutically acceptable carrier and/or excipient. 50. An expression vector comprising a coding sequence of the IL-18 inhibitor according to any one of embodiments 1-13 and 26-30, which upon administration to a subject suffering from a disease or disorder or having a predisposition to develop such a disease or disorder as defined in the preceding embodiments leads to in situ expression of IL-18 inhibitor for use in the treatment of the disease or disorder as defined in any one of embodiments 1-13. 51. An expression vector comprising an IL-18 antisense expressing vector, which upon administration to a subject suffering from a disease or disorder or having a predisposition to develop such a disease or disorder as defined in embodiments 1-13, leads to in situ inhibition of the expression of IL-18 for use in the treatment of the disease or disorder as defined in any one of embodiments 1-13. 52. The expression vector of embodiment 50 or 51 for use in the treatment of the disease or disorder as defined in any one of embodiments 1-13 and 26-43, wherein said expression vector is administered to a subject suffering from such a disease or disorder, or having a predisposition to develop such a disease or disorder, alone or in combination with the IL-18 inhibitor according to any one of embodiments 1-13 and 26-30, the IL-18BP according to embodiments 31-43 or the pharmaceutical composition according to any one of embodiments 44-49. 53. An expression vector comprising the coding sequence of IL-18BP according to embodiments 31-43, which upon administration to a subject suffering from a disease or disorder or having a predisposition to develop such a disease or disorder as defined in the preceding embodiments, leads to in situ expression of IL-18BP for use in the treatment of the disease or disorder as defined in any one of embodiments 31-43. 54. The expression vector of embodiment 53 for use in the treatment of the disease or disorder as defined in any one of embodiments 1-13 and 26-43, wherein said expression vector is administered to a subject suffering from such a disease or disorder, or having a predisposition to develop such a disease or disorder, alone or in combination with the IL-18 inhibitor according to any one of embodiments 1-13 and 26-30, the IL-18BP according to embodiments 31-43 or the pharmaceutical composition according to any one of embodiments 44-49. 55. The IL-18 inhibitor for use according to any one of embodiments 1-13 and 26-30, the IL-18BP for use according to any one of embodiments 31-43, the pharmaceutical composition for use according to any one of embodiments 44-49 or the expression vector for use according to any one of embodiments 50-54, comprising administering to a subject in need thereof a prophylactically and/or therapeutically effective amount of said IL-18 inhibitor, IL-18BP, pharmaceutical composition, or expression vector, particularly by systemic, intranasal, buccal, oral, transmucosal, intratracheal, intravenous, subcutaneous, intraurinary tract, intravaginal, sublingual, intrabronchial, intrapulmonary, transdermal or intramuscular administration, in particular broncho-pulmonary administration. 56. The IL-18 inhibitor, the IL-18BP, the pharmaceutical composition or the expression vector for use according to embodiment 55, wherein said subject is a mammal, particularly said subject is a human. 57. A method for treating the disease or disorder as defined in any one of embodiments 1-13 and 26-43 in a subject suffering from such a disease or disorder, or having a predisposition to develop such a disease or disorder, comprising administering to said subject a therapeutically or prophylactically effective amount of the IL-18 inhibitor according to any one of embodiments 1-13 and 26-30, the IL-18BP according to embodiments 31-43 or the pharmaceutical composition according to any one of embodiments 44-49 and/or the expression vector according to any one of embodiments 50-54, particularly by systemic, intranasal, buccal, oral, transmucosal, intratracheal, intravenous, subcutaneous, intraurinary tract, intravaginal, sublingual, intrabronchial, intrapulmonary, transdermal or intramuscular administration, in particular broncho-pulmonary administration. 58. A method for diagnosis of the diseases or disorder as defined in any one of embodiments 1-13 and 26-43, for diagnosing a predisposition to the disease or disorder as defined in any one of embodiments 1-13 and 26-43, for monitoring minimal residual disease in a subject, or for predicting responsiveness of a subject to a treatment with IL-18 inhibitor according to embodiments 1-13 and 26-30, the IL-18BP according to embodiments 31-43 or the pharmaceutical composition comprising IL-18 inhibitor according to embodiments 44-49, comprising the steps: a) obtaining a sample of body fluid, particularly serum from a subject; b) testing said sample for the presence of free IL-18 by using the IL-18 antibody of any one of embodiments 14-25 or the IL-18BP of any one of embodiments . . . as capturing molecule and/or testing the said sample for the presence of free IL-18BP by using a first monoclonal IL-18BP specific capturing antibody and an IL-18BP specific detection antibody, which binds to a different site of IL-18BP than the capturing antibody, particularly one of said antibodies binds to the IL-18 binding site of IL-18BP; c) determining the amount of free IL-18 and/or free IL-18BP bound to the capturing molecule in the sample; d) comparing the amount of free IL-18 and/or free IL-18BP in the sample of the subject suffering from such a disease to the amount in the sample of a healthy subject. 59. A method for diagnosis of the diseases or disorder as defined in any one of embodiments 1-13 and 26-43, for diagnosing a predisposition to the disease or disorder as defined in any one of embodiments 1-13 and 26-43, for monitoring minimal residual disease in a subject, or for predicting responsiveness of a subject to a treatment with IL-18 inhibitor according to embodiments 1-13 and 26-30, the IL-18BP according to embodiments 31-43 or the pharmaceutical composition comprising IL-18 inhibitor according to embodiments 44-49 and a pharmaceutically acceptable carrier and/or excipient according to any one of the preceding embodiments, comprising the steps: a) obtaining a sample of body fluid, particularly sputum and serum from a subject; b) testing said sample for the presence of free IL-18 by using the IL-18 antibody of embodiments 14-25 or the IL-18BP as capturing molecule and/or testing the said sample for the presence of free IL-18BP by using a first monoclonal IL-18BP specific capturing antibody and an IL-18BP specific detection antibody, which binds to a different site of IL-18BP than the capturing antibody, particularly one of said IL-18BP specific antibodies binds to the IL-18 binding site of IL-18BP; c) testing said sample for the presence of total IL-18 total and/or total IL-18BP by using a first monoclonal IL-18BP specific antibody which does not bind to the IL-18 binding site of IL-18BP and a second IL-18 specific antibody, which does not bind to the IL-18BP binding site of IL-18; d) determining the amount of free and total IL-18 and/or free and total IL-18BP bound to the capturing molecule in the sample; e) comparing the amount of free and/or total IL-18 and/or free and/or total IL-18BP in the sample of the subject suffering from such a disease to the amount in the sample of a healthy subject. 60. The method for diagnosis of any one of the preceding embodiments, wherein the amount of free IL-18 in isolated serum of a subject, particularly a human, suffering from said disease ranges from 5 to 10000 pg/mL, whereas the amount of free IL-18 in serum of healthy subject, particularly a healthy human is pg/mL. 61. A set of biomarkers for use in the diagnosis of the diseases or disorder as defined in any one of embodiments 1-13 and 26-43, for use in diagnosing a predisposition to the disease or disorder as defined in any one of embodiments 1-13 and 26-43 or for use in monitoring minimal residual disease in a subject, or for predicting responsiveness of a subject to a treatment with IL-18 inhibitor according to embodiments 1-13 and 26-30, the IL-18BP according to embodiments 31-43 or the pharmaceutical composition comprising IL-18 inhibitor according to embodiments 44-49. 62. A method for diagnosis of the diseases or disorder as defined in any one of embodiments 1-13 and 26-43, for diagnosing a predisposition to the disease or disorder as defined in any one of embodiments 1-13 and 26-43 or for monitoring minimal residual disease in a subject, or for predicting responsiveness of a subject to a treatment with IL-18 inhibitor according to embodiments 1-13 and 26-30, the IL-18BP according to embodiments 31-43 or the pharmaceutical composition comprising IL-18 inhibitor according to embodiments 44-49, comprising the steps: e) obtaining a biomarker profile of a subject to be tested by taking a sample of a body fluid from said subject; f) obtaining a biomarker profile of a healthy reference population; g) obtaining a biomarker profile from a population which suffers from said disease or disorder and h) comparing the biomarker profile obtained in step a) with the profile obtained in step b) and step c). 63. A pharmaceutical kit comprising IL-18 inhibitor according to any one of embodiments 1-13 and 26-30, IL-18BP according to embodiments 31-43 or a pharmaceutical composition comprising IL-18 inhibitor according to embodiments 44-49 and a pharmaceutically acceptable carrier and/or excipient according to the invention in separate unit dosage forms, said forms being suitable for administration in effective amounts. 64. A diagnostic kit for detecting free IL-18, comprising an IL-18-specific antibody according to any one of embodiments 14-25 as capturing antibody or the IL-18BP as alternative capturing molecule, and a second IL-18 specific detection antibody or an IL-18-specific antibody according to any one of embodiments 14-25 as detection antibody and a second IL-18 specific capturing antibody, wherein the detection antibody bind to different sites of IL-18 than the capturing molecule. 65. A diagnostic kit for detecting total IL-18 or total IL-18BP, comprising a first monoclonal IL-18BP specific antibody which does not bind to the IL-18 binding site of IL-18BP and a second IL-18 specific antibody, which does not bind to the IL-18BP binding site of IL-18. 66. A diagnostic kit for detecting free IL-18BP, comprising a first monoclonal IL-18BP specific capturing antibody and an IL-18BP specific detection antibody, which binds to a different site of IL-18BP than the capturing antibody. 67. A diagnostic kit, which comprises all diagnostic kits of embodiments 64 to 66.