Anti-IL-1R3 antibodies for use in inflammatory conditions

Abstract

To provide uses of an antibody or antigen-binding fragment thereof that specifically binds to IL-1R3 in the manufacture of a medicament for treating an IL1R3-mediated inflammatory condition and / or disorder in a subject.SOLUTION: An antibody or antigen-binding fragment thereof that specifically binds to IL-1R3 comprises: a heavy chain variable (VH) region that is at least 95% identical to a VH region that contains a specific amino acid sequence; and a light chain variable (VL) region that is at least 95% identical to a VL region that contains a specific amino acid sequence. The antibody or antigen-binding fragment thereof comprises: a) a complementarity determining region that comprises CDR-H1, CDR-H2, and CDR-H3, each of which contains a specific amino acid sequence; and b) a complementarity determining region that comprises CDR-L1, CDR-L2, and CDR-L3, each of which contains a specific amino acid sequence. Provided are uses of the antibody or antigen-binding fragment thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for treating medical conditions and / or conditions characterized by unregulated or abnormal expression of members of the IL1R3 signaling pathway, such as IL-1α, IL-1β, IL-33, IL-36, IL-RA, and / or IL1R3. More particularly, the present invention relates to methods for treating inflammatory conditions and / or disorders. The present invention relates to methods of using anti-IL-1R3 antibodies. [Background technology]

[0002] Interleukin-1 receptor accessory protein (IL1RAP) (also called IL1R3) is a co-receptor for type 1 interleukin 1 receptor (IL1R1) and mediates IL-1 signal transduction. Upon binding of IL-1, IL-1R1 associates with IL-1RAcP to form a functional signaling receptor complex that stimulates NFκB activity.

[0003] IL-33, its receptor ST2, and IL-1RAcP also interact with NFκB as an IL-1β / IL-1R1 / IL-1RAcP complex. IL-36 (IL-36α (IL-1F6), IL-36β (IL-1F8), and IL-36γ (IL-1F9)), their receptors IL-36R, and IL-1RAcP also form a complex (IL-33 / ST2 / IL-1RAcP) with similar activity in terms of NFκB activation as the IL-1β / IL-1R1 / IL-1RAcP complex. The complex (IL-36 / IL-36R / IL-1RAcP) has the activity of

[0004] The interleukin-1 (IL-1) pathway is an intracellular signaling pathway that plays a key role in mammalian inflammatory responses and is associated with a wide range of immunological, metabolic, physiological, and hematopoietic activities. The IL-1 family includes three structurally related cytokines: IL-1 alpha, IL-1 beta, and IL-1 receptor antagonist (IL-1ra). Of the three, IL-1 alpha and IL-1 beta are proinflammatory agonists, while IL-1 receptor antagonist (IL-1ra) functions to block IL-1 alpha and IL-1 beta activity. All known biological functions of IL-1 are mediated through type I IL-1R. IL-1 alpha, IL-1 beta, and IL-1ra bind with high affinity to type I IL-1R. In contrast, IL-1 beta binds with high affinity to type II IL-1R. IL-1alpha and IL-1ra bind with low affinity to type II IL-1R, which has a significantly truncated cytoplasmic domain and does not signal to the cell upon binding IL-1, but instead participates in regulating IL-1-mediated responses by acting as a decoy receptor.

[0005] IL-1 production is triggered by infections, microbial toxins, inflammatory agents, and allergic reactions. The overall primary function of IL-1 is to regulate the amplitude and duration of immune and inflammatory responses at the site of inflammation or allergic immune response. When excessive IL-1 is produced or IL-1 expression is not properly regulated, disease states can develop. Therefore, IL-1 is implicated in a variety of inflammatory and immunoregulatory diseases and conditions. It has been proposed that systemic or local excess of IL-1 contributes to the development of many medical disorders. In addition to this proposal, IL-1ra, which blocks IL-1alpha and IL-1beta activity, has been shown to have varying degrees of efficacy in treating several diseases thought to be mediated by IL-1 signaling.

[0006] It has been suggested that suppression of IL-1 may be beneficial for patients suffering from a variety of disorders characterized by abnormal or excessive IL-1 expression or activity. IL-1ra and ICE inhibitors have had limited success as therapeutic agents for diseases associated with IL-1 activity. Although progress has been made in devising effective treatments for the disease, improved drugs and therapies are needed.

[0007] Unfortunately, Kineret, Ilaris, or Arcalyst Existing IL-1 pathway inhibitors, such as IL-1Ra, have certain drawbacks that prevent their use. For example, Kineret (IL-1Ra) has a short half-life and therefore requires very frequent treatment intervals (daily). Antibodies targeting a single cytokine (such as Ilaris) can result in unwanted signaling / activation of other IL-1 family cytokines. Lee's cytokines are known to induce both pro-inflammatory and IL-1R3-independent anti-inflammatory signaling, both of which are interfered with when targeting the cytokines or their alpha chain receptors. In contrast, the antibodies of the present invention combine the advantages of reduced treatment intervals, simultaneous inhibition of different IL-1 family cytokine-mediated signaling, and specificity for blocking inflammatory signaling pathways. Summary of the Invention

[0008] Thus, the present invention provides a method for treating IL1R3 signaling pathways having the preferred characteristics described below. The present invention provides new and improved methods for treating conditions associated with unregulated expression of members of the tract. [Brief explanation of the drawings]

[0009] [Figure 1-1]Sequences (amino acids in single letter code) Complete sequences of variable regions (VR): Heavy chain: VH complete: SEQ ID NOs: 1 to 34 and SEQ ID NOs: 173 and 176 Light chain: VL complete: SEQ ID NOs: 35 to 68 and SEQ ID NOs: 174 and 177 Complementarity determining regions (CDR): Heavy chain: CDRH1: SEQ ID NOs: 69 to 85 and 178 CDRH2: SEQ ID NOs: 86 to 102 and 179 CDRH3: SEQ ID NOs: 103 to 119 and 180 Light chain: CDRL1: SEQ ID NOs: 120 to 136 and 181 CDRL2: SEQ ID NOs: 137 to 153 and 182 CDRL3: SEQ ID NOs: 154 to 170, 175 and 183 Constant regions (CR): Light chain: CR-L: SEQ ID NO: 171 Heavy chain: CR-H: SEQ ID NO: 172 In the following figure, AF676 is a commercially available polyclonal antibody preparation purchased from the following link: https: / / www.rndsystems.com / products / human-il-1-racp-il-1-r3-antibody_af676. [Figure 1-2] Sequences (amino acids in single letter code) Complete sequences of variable regions (VR): Heavy chain: VH complete: SEQ ID NOs: 1 to 34 and SEQ ID NOs: 173 and 176 Light chain: VL complete: SEQ ID NOs: 35 to 68 and SEQ ID NOs: 174 and 177 Complementarity determining regions (CDR): Heavy chain: CDRH1: SEQ ID NOs: 69 to 85 and 178 CDRH2: SEQ ID NOs: 86 to 102 and 179 CDRH3: SEQ ID NOs: 103 to 119 and 180 Light chain: CDRL1: SEQ ID NOs: 120 to 136 and 181 CDRL2: SEQ ID NOs: 137 to 153 and 182 CDRL3: SEQ ID NOs: 154 to 170, 175 and 183 Constant regions (CR): Light chain: CR-L: SEQ ID NO: 171 Heavy chain: CR-H: SEQ ID NO: 172 In the following figure, AF676 is a commercially available polyclonal antibody preparation purchased from the following link: https: / / www.rndsystems.com / products / human-il-1-racp-il-1-r3-antibody_af676. [Figure 1-3]Sequences (amino acids in single letter code) Complete sequences of variable regions (VR): Heavy chain: VH complete: SEQ ID NOs: 1 to 34 and SEQ ID NOs: 173 and 176 Light chain: VL complete: SEQ ID NOs: 35 to 68 and SEQ ID NOs: 174 and 177 Complementarity determining regions (CDR): Heavy chain: CDRH1: SEQ ID NOs: 69 to 85 and 178 CDRH2: SEQ ID NOs: 86 to 102 and 179 CDRH3: SEQ ID NOs: 103 to 119 and 180 Light chain: CDRL1: SEQ ID NOs: 120 to 136 and 181 CDRL2: SEQ ID NOs: 137 to 153 and 182 CDRL3: SEQ ID NOs: 154 to 170, 175 and 183 Constant regions (CR): Light chain: CR-L: SEQ ID NO: 171 Heavy chain: CR-H: SEQ ID NO: 172 In the following figure, AF676 is a commercially available polyclonal antibody preparation purchased from the following link: https: / / www.rndsystems.com / products / human-il-1-racp-il-1-r3-antibody_af676. [Figure 1-4] Sequences (amino acids in single letter code) Complete sequences of variable regions (VR): Heavy chain: VH complete: SEQ ID NOs: 1 to 34 and SEQ ID NOs: 173 and 176 Light chain: VL complete: SEQ ID NOs: 35 to 68 and SEQ ID NOs: 174 and 177 Complementarity determining regions (CDR): Heavy chain: CDRH1: SEQ ID NOs: 69 to 85 and 178 CDRH2: SEQ ID NOs: 86 to 102 and 179 CDRH3: SEQ ID NOs: 103 to 119 and 180 Light chain: CDRL1: SEQ ID NOs: 120 to 136 and 181 CDRL2: SEQ ID NOs: 137 to 153 and 182 CDRL3: SEQ ID NOs: 154 to 170, 175 and 183 Constant regions (CR): Light chain: CR-L: SEQ ID NO: 171 Heavy chain: CR-H: SEQ ID NO: 172 In the following figure, AF676 is a commercially available polyclonal antibody preparation purchased from the following link: https: / / www.rndsystems.com / products / human-il-1-racp-il-1-r3-antibody_af676. [Figure 1-5]Sequences (amino acids in single letter code) Complete sequences of variable regions (VR): Heavy chain: VH complete: SEQ ID NOs: 1 to 34 and SEQ ID NOs: 173 and 176 Light chain: VL complete: SEQ ID NOs: 35 to 68 and SEQ ID NOs: 174 and 177 Complementarity determining regions (CDR): Heavy chain: CDRH1: SEQ ID NOs: 69 to 85 and 178 CDRH2: SEQ ID NOs: 86 to 102 and 179 CDRH3: SEQ ID NOs: 103 to 119 and 180 Light chain: CDRL1: SEQ ID NOs: 120 to 136 and 181 CDRL2: SEQ ID NOs: 137 to 153 and 182 CDRL3: SEQ ID NOs: 154 to 170, 175 and 183 Constant regions (CR): Light chain: CR-L: SEQ ID NO: 171 Heavy chain: CR-H: SEQ ID NO: 172 In the following figure, AF676 is a commercially available polyclonal antibody preparation purchased from the following link: https: / / www.rndsystems.com / products / human-il-1-racp-il-1-r3-antibody_af676. [Figure 1-6] Sequences (amino acids in single letter code) Complete sequences of variable regions (VR): Heavy chain: VH complete: SEQ ID NOs: 1 to 34 and SEQ ID NOs: 173 and 176 Light chain: VL complete: SEQ ID NOs: 35 to 68 and SEQ ID NOs: 174 and 177 Complementarity determining regions (CDR): Heavy chain: CDRH1: SEQ ID NOs: 69 to 85 and 178 CDRH2: SEQ ID NOs: 86 to 102 and 179 CDRH3: SEQ ID NOs: 103 to 119 and 180 Light chain: CDRL1: SEQ ID NOs: 120 to 136 and 181 CDRL2: SEQ ID NOs: 137 to 153 and 182 CDRL3: SEQ ID NOs: 154 to 170, 175 and 183 Constant regions (CR): Light chain: CR-L: SEQ ID NO: 171 Heavy chain: CR-H: SEQ ID NO: 172 In the following figure, AF676 is a commercially available polyclonal antibody preparation purchased from the following link: https: / / www.rndsystems.com / products / human-il-1-racp-il-1-r3-antibody_af676. [Figure 1-7]Sequences (amino acids in single letter code) Complete sequences of variable regions (VR): Heavy chain: VH complete: SEQ ID NOs: 1 to 34 and SEQ ID NOs: 173 and 176 Light chain: VL complete: SEQ ID NOs: 35 to 68 and SEQ ID NOs: 174 and 177 Complementarity determining regions (CDR): Heavy chain: CDRH1: SEQ ID NOs: 69 to 85 and 178 CDRH2: SEQ ID NOs: 86 to 102 and 179 CDRH3: SEQ ID NOs: 103 to 119 and 180 Light chain: CDRL1: SEQ ID NOs: 120 to 136 and 181 CDRL2: SEQ ID NOs: 137 to 153 and 182 CDRL3: SEQ ID NOs: 154 to 170, 175 and 183 Constant regions (CR): Light chain: CR-L: SEQ ID NO: 171 Heavy chain: CR-H: SEQ ID NO: 172 In the following figure, AF676 is a commercially available polyclonal antibody preparation purchased from the following link: https: / / www.rndsystems.com / products / human-il-1-racp-il-1-r3-antibody_af676. [Figure 1-8] Sequences (amino acids in single letter code) Complete sequences of variable regions (VR): Heavy chain: VH complete: SEQ ID NOs: 1 to 34 and SEQ ID NOs: 173 and 176 Light chain: VL complete: SEQ ID NOs: 35 to 68 and SEQ ID NOs: 174 and 177 Complementarity determining regions (CDR): Heavy chain: CDRH1: SEQ ID NOs: 69 to 85 and 178 CDRH2: SEQ ID NOs: 86 to 102 and 179 CDRH3: SEQ ID NOs: 103 to 119 and 180 Light chain: CDRL1: SEQ ID NOs: 120 to 136 and 181 CDRL2: SEQ ID NOs: 137 to 153 and 182 CDRL3: SEQ ID NOs: 154 to 170, 175 and 183 Constant regions (CR): Light chain: CR-L: SEQ ID NO: 171 Heavy chain: CR-H: SEQ ID NO: 172 In the following figure, AF676 is a commercially available polyclonal antibody preparation purchased from the following link: https: / / www.rndsystems.com / products / human-il-1-racp-il-1-r3-antibody_af676. [Figure 2]Human IL-1R3 ELISA 384-microtiter plates were coated with human IL-1R3 protein (0.5 mg / ml, for at least 1 h), representing the human extracellular domain of IL-1R3. After extensive washing steps followed by a blocking step, antibody was added (12.5 μl per well) and incubated for 1 h at room temperature. Unbound antibody was washed away extensively. The amount of bound antibody was determined by incubating the microtiter plate with a peroxidase-conjugated anti-human detection antibody (1 h at room temperature). The peroxidase reaction was initiated by the addition of TMB, and the absorbance was measured at 450 nm / 620 nm. [Figure 3] HEK293 reporter assay. HEK293T / 17-FR cells were stably transfected with the pGL4.32[luc2P / NF-κB-RE / Hygro] vector (Promega) and seeded into 384-well PDL Costar cell culture plates. They were then incubated with antibodies for 30 minutes. Cells were then stimulated with IL-1β for 5 hours, after which NF-κB activity was measured using the Steady-Glo Luciferase Assay Kit (Promega) according to the manufacturer's protocol. [Figure 4] NFκB luciferase reporter assay using the A549 stable expression line. A549-NFkB-RE-Luc stable transfected cells (purchased from Signosis) were cultured for 3 days (1.7E+04 cells / cm3). 384-well, low-flange, white, flat-bottom, polystyrene, TC-treated microtiter plates (Corning) were loaded with 4x104 cells per well. After a 10-hour culture period, cells were incubated with antibody for 1 hour, after which they were stimulated with 10 μl of IL-1β for an additional 5 hours. NFκB modulation was measured using the Steady-Glo™ Luciferase Assay System (Promega) to determine the relative luminescence units of each well relative to unstimulated cells. [Figure 5]Cell Binding Analysis: Binding to IL-1R3-Expressing Cells The humanized anti-IL-1R3 IgG1-LALA antibody was tested for binding to cell lines with different IL-1R3 receptor densities using flow cytometry. The humanized anti-IL-1R3 IgG1-LALA antibody binds to low- and high-IL-1R3-expressing cell lines. The antibody does not bind to mouse NIH-3T3 cells. The experiment was performed according to the method described in Example 4. [Figure 6] Cell Binding Analysis: Cell Binding to the Human IL-1R3 High-Expression Cell Line SK-MEL-30 The EC50 cell binding values ​​of humanized anti-IL-1R3 IgG1-LALA antibodies were determined by binding to the highly IL-1R3-expressing cell line SK-MEL-30 using flow cytometry. The humanized anti-IL-1R3 IgG1-LALA antibodies MAB-16-0030 and MAB-16-0149 exhibited cell binding of 307 and 306 ng / ml, respectively. The experiment was performed according to the method described in Example 4. [Figure 7] Human-IL-1R3 Biochemical ELISA. Binding of the humanized anti-IL-1R3 IgG1-LALA antibody to recombinant human IL-1R3 protein was tested by biochemical ELISA. The exemplified antibodies exhibit EC50 binding values ​​of 16.3 ng / ml and 29.1 ng / ml, respectively. The experiment was performed as described in Example 5. [Figure 8] Inhibition of human IL-1a and IL-1b mediated NfKB signaling in A549-NFkB-RE-Luc cells. Functional neutralization of IL-1a and IL-1b was tested in a cell-based gene reporter assay using A549-NFkB-RE-Luc cells stimulated with 0.1 ng / ml of IL-1a and IL-1b, respectively. The humanized anti-IL-1R3 IgG1-LALA antibody exhibits a superior EC50 value to the goat anti-human IL1-R3 polyclonal antibody AF676 (R&D Systems). The experiment was performed according to the method described in Example 6. [Figure 9]IL-1α and IL-1β Functional Neutralization Assay—Inhibition of Human IL-1a- and IL-1b-Mediated IL-6 Release by A-549 Cells Neutralization of IL-1a- and IL-1b-mediated cellular release of IL-6 by the humanized anti-IL-1R3 IgG1-LALA antibody was tested using A-549 cells. EC50 values ​​show that the humanized anti-IL-1R3 IgG1-LALA antibody is superior to the goat anti-human IL1-R3 polyclonal antibody AF676 (R&D Systems). The experiment was performed according to the method described in Example 7. [Figure 10] IL-33 Functional Neutralization Assay—Inhibition of Human IL-33-Mediated NfkB Signaling in HEK-Blue-IL33™ Cells Neutralization of IL-33-mediated cell signaling by the humanized anti-IL-1R3 IgG1-LALA antibody was tested using IL-33-stimulated gene reporter HEK-Blue-IL33™ cells (InvivoGen). EC50 values ​​show that the humanized anti-IL-1R3 IgG1-LALA antibody is superior to the goat anti-human IL1-R3 polyclonal antibody AF676 (R&D Systems). The experiment was performed according to the method described in Example 8. [Figure 11] IL-36 Functional Neutralization Assay—Inhibition of Human IL-36-Mediated NfkB Signaling in HEK-293 / 17-IF Cells Neutralization of IL-36-mediated cell signaling by humanized anti-IL-1R3 IgG1-LALA antibodies was tested using IL-36g-stimulated gene reporter HEK-293 / 17-IF cells. Representative humanized anti-IL-1R3 IgG1-LALA antibodies exhibit superior EC50 values ​​to goat anti-human IL1-R3 polyclonal antibody AF676 (R&D Systems). The experiment was performed according to the method described in Example 9. [Figure 12]Neutralization of IL-1a-, IL-33-, and IL-36a-Mediated Cellular Cytokine Release Neutralization of IL-1a-, IL-33-, and IL-36a-mediated cellular cytokine release was tested using specific IL-1a-, IL-33-, and IL-36a-dependent cell systems. Inhibition of cytokine release by a representative humanized anti-IL-1R3 IgG1-LALA antibody (MAB-16-0030) according to the present invention was tested and compared with IL-1Ra. The antibody according to the present invention was able to inhibit cytokine release mediated by all three stimuli, whereas IL-1Ra only affected IL-1a-mediated cytokine release. The experiment was performed according to the method described in Example 10. [Figure 13-1] Viability and IL-6 Release of Unstimulated PBMCs Treated with Humanized Anti-IL-1R3 IgG1-LALA Antibody. Antibody binding to immune cells can result in cell depletion and deleterious effects, for example, by directly inducing apoptotic signaling pathways, stimulating excessive cytokine release, or antibody-dependent cellular cytotoxicity (ADCC). To exclude a direct effect of the humanized anti-IL-1R3 IgG1-LALA antibody on PBMC viability, the viability and IL-6 release of PBMCs from three donors were examined after 1, 3, and 5 days of incubation with different concentrations of a representative humanized anti-IL-1R3 IgG1-LALA antibody (MAB-16-0030) according to the present invention. Neither viability nor IL-6 release was affected. These results support that the humanized anti-IL-1R3 IgG1-LALA antibody blocks IL-1R3 function on immune cells without inducing cell depletion or deleterious effects. The experiment was performed according to the method described in Example 11. [Figure 13-2]Viability and IL-6 Release of Unstimulated PBMCs Treated with Humanized Anti-IL-1R3 IgG1-LALA Antibody. Antibody binding to immune cells can result in cell depletion and deleterious effects, for example, by directly inducing apoptotic signaling pathways, stimulating excessive cytokine release, or antibody-dependent cellular cytotoxicity (ADCC). To exclude a direct effect of the humanized anti-IL-1R3 IgG1-LALA antibody on PBMC viability, the viability and IL-6 release of PBMCs from three donors were examined after 1, 3, and 5 days of incubation with different concentrations of a representative humanized anti-IL-1R3 IgG1-LALA antibody (MAB-16-0030) according to the present invention. Neither viability nor IL-6 release was affected. These results support that the humanized anti-IL-1R3 IgG1-LALA antibody blocks IL-1R3 function on immune cells without inducing cell depletion or deleterious effects. The experiment was performed according to the method described in Example 11. [Figure 14-1] Functional Blockade of PBMCs Activated by Different Stimuli To test whether the humanized anti-IL-1R3 IgG1-LALA antibody inhibits the activity of PBMCs stimulated with specific or combined stimuli, PBMCs from 10 donors were stimulated with LPS, heat-inactivated Candida albicans, IL-12 / IL-33, or anti-CD3 / CD28 antibodies. A representative humanized anti-IL-1R3 IgG1-LALA antibody (MAB-16-0030) of the present invention was able to inhibit cytokine release mediated by all stimuli tested. The experiment was performed according to the method described in Example 12. [Figure 14-2] Functional Blockade of PBMCs Activated by Different Stimuli To test whether the humanized anti-IL-1R3 IgG1-LALA antibody inhibits the activity of PBMCs stimulated with specific or combined stimuli, PBMCs from 10 donors were stimulated with LPS, heat-inactivated Candida albicans, IL-12 / IL-33, or anti-CD3 / CD28 antibodies. A representative humanized anti-IL-1R3 IgG1-LALA antibody (MAB-16-0030) of the present invention was able to inhibit cytokine release mediated by all stimuli tested. The experiment was performed according to the method described in Example 12. [Figure 15] Functional Blockade of Immune Cells in Whole Blood Activated by Candida albicans To test whether the humanized anti-IL-1R3 IgG1-LALA antibody inhibits the activation of immune cells in whole blood, whole blood from eight donors was stimulated with heat-inactivated Candida albicans. A representative humanized anti-IL-1R3 IgG1-LALA antibody (MAB-16-0030) according to the present invention was able to inhibit Candida-induced IL-6 cytokine release. The experiment was performed according to the method described in Example 13. [Figure 16-1] Blocking Cytokine Release in a Mixed Lymphocyte Reaction (MLR) The ability of humanized anti-IL-1R3 IgG1-LALA antibodies to block the release of various cytokines was tested in a mixed lymphocyte reaction (MLR) using PBMCs from healthy unmatched donors. A representative humanized anti-IL-1R3 IgG1-LALA antibody (MAB-16-0030) according to the present invention was able to inhibit the release of IFNγ, IL-6, TNF-α, IL-13, IL-17, and IL-10. The experiment was performed according to the method described in Example 14. [Figure 16-2] Blocking Cytokine Release in a Mixed Lymphocyte Reaction (MLR) The ability of humanized anti-IL-1R3 IgG1-LALA antibodies to block the release of various cytokines was tested in a mixed lymphocyte reaction (MLR) using PBMCs from healthy unmatched donors. A representative humanized anti-IL-1R3 IgG1-LALA antibody (MAB-16-0030) according to the present invention was able to inhibit the release of IFNγ, IL-6, TNF-α, IL-13, IL-17, and IL-10. The experiment was performed according to the method described in Example 14. [Figure 16-3]Blocking Cytokine Release in a Mixed Lymphocyte Reaction (MLR) The ability of humanized anti-IL-1R3 IgG1-LALA antibodies to block the release of various cytokines was tested in a mixed lymphocyte reaction (MLR) using PBMCs from healthy unmatched donors. A representative humanized anti-IL-1R3 IgG1-LALA antibody (MAB-16-0030) according to the present invention was able to inhibit the release of IFNγ, IL-6, TNF-α, IL-13, IL-17, and IL-10. The experiment was performed according to the method described in Example 14. [Figure 17] Neutralization of IL-1b-Mediated Signaling in Murine Cells Functional neutralization of IL-1b by anti-mouse IL-1R3 antibodies according to the present invention was tested using mouse NIH-3T3 cells based on an NFkB luciferase gene reporter assay. A representative antibody according to the present invention (MAB-16-0531) showed dose-dependent inhibition of IL-1b-mediated NFkB signaling with an EC50 of 805 ng / ml. The experiment was performed according to the method described in Example 15. [Figure 18] Neutralization of IL-1b-Mediated IL-6 Release from Murine Cells Inhibition of IL-6 release from murine cells by anti-murine IL-1R3 antibodies according to the invention was tested using IL-1b-stimulated NIH-3T3 cells. A representative antibody according to the invention (MAB-16-0531) showed dose-dependent inhibition of IL-6 release with an EC50 of 1560 ng / ml. The experiment was performed according to the method described in Example 16. [Figure 19]Monosodium urate crystal (MSU)-induced mouse peritonitis model. To test the rationale for applying anti-IL-R3 therapy to gout patients, a monosodium urate crystal (MSU)-induced mouse peritonitis model was used. Administration of MSU produces a strong inflammatory response in the peritoneal cavity, inducing the influx of several inflammatory cells (e.g., monocytes and neutrophils), mimicking the classic inflammatory profile of an acute gout response. A representative anti-IL1R3 antibody (MAB-16-0531) according to the present invention or IL-1Ra was administered IP 1 hour before intraperitoneal injection of 3 mg MSU. Measurement of cells in the peritoneal cavity showed that anti-IL-1R3 antibody treatment significantly inhibited MSU-induced infiltration of lymphocytes, monocytes, and granulocytes. The experiment was performed according to the method described in Example 17. [Figure 20] Neutrophil activity and cytokine production in MSU peritonitis. Attenuation of neutrophil activity, including local and systemic markers of inflammation, in MSU peritonitis. (a) Levels of neutrophil protease elastase in IP fluid. (b) Total levels of intracellular elastase in cells from IP fluid. (c) Total levels of intracellular MPO in cells from IP fluid. (d) Local (IP fluid) and systemic cyto- and chemokine concentrations. WB; lysed whole blood, norm.; normalized per mg of protein in spleen. MSU; vehicle + MSU. a-mIL1R3; a-mIL1R3 + MSU. IL-1Ra; IL-1Ra + MSU. Inhibitor-treated group compared to MSU-stimulated group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (Student's t-test, mean+SEM, except for: (b), G CSF, KC(WB) and KC(spleen); Mann-Whitney U test, median with IQR). [Figure 21-1]In vivo model of OVA-induced allergic asthma. a-mIL1R3 antibody reduces OVA-induced cellular influx in vivo. (a) Total WBCs from BAL fluid. (b) Flow-defined cells are different from (a). (c) Histology of mice with thick dots marking group mean (WBCs) using H&E and PAS-stained lung tissue. Comparison with OVA-stimulated + a-mIL1R3 (MAB-16-0531)-treated mice and OVA alone. *p<0.05, **p<0.01, ***p<0.001 (Student's t-test). Mean + SEM. [Figure 21-2] In vivo model of OVA-induced allergic asthma. a-mIL1R3 antibody reduces OVA-induced cellular influx in vivo. (a) Total WBCs from BAL fluid. (b) Flow-defined cells are different from (a). (c) Histology of mice with thick dots marking group mean (WBCs) using H&E and PAS-stained lung tissue. Comparison with OVA-stimulated + a-mIL1R3 (MAB-16-0531)-treated mice and OVA alone. *p<0.05, **p<0.01, ***p<0.001 (Student's t-test). Mean + SEM. [Figure 22-1] Imiquimod psoriasis in vivo model. (a) Induction of IL36α mRNA in skin. (b) Fold change in ear thickness between IMQ and control cream application. (c) Blind visual scoring of n=6 at the end of the study (0; no effect, -4; maximal effect (erythema and scaling)). (d) MPO concentration in skin normalized per mg of protein. (e) Spearman correlation between data from (c) and (d). (e) Granulocyte concentration in whole blood. Comparison between inhibitor-treated and IMQ-stimulated groups. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ((b,f) Mann-Whitney U test; (d,e) Student's test; except mean ± SEM (d) median with IQR). [Figure 22-2]Imiquimod psoriasis in vivo model. (a) Induction of IL36α mRNA in skin. (b) Fold change in ear thickness between IMQ and control cream application. (c) Blind visual scoring of n=6 at the end of the study (0; no effect, -4; maximal effect (erythema and scaling)). (d) MPO concentration in skin normalized per mg of protein. (e) Spearman correlation between data from (c) and (d). (e) Granulocyte concentration in whole blood. Comparison between inhibitor-treated and IMQ-stimulated groups. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ((b,f) Mann-Whitney U test; (d,e) Student's test; except mean ± SEM (d) median with IQR). [Figure 23-1] Imiquimod psoriasis in vivo model: IL36α / β / γ, IL17F and TNFα mRNA levels in skin biopsies of imiquimod-treated mice. [Figure 23-2] Imiquimod psoriasis in vivo model: IL36α / β / γ, IL17F and TNFα mRNA levels in skin biopsies of imiquimod-treated mice. [Figure 24] Effector Cell-Mediated Function of Anti-IL1R3 IgG1 and IgG1-LALA Antibodies The anti-IL1R3 antibody MAB-16-0030, containing either the IgG1-LALA or IgG1 Fc portion, was tested for its activity in Fc receptor-mediated NF-kB signaling in effector cells. Binding of increasing concentrations of IgG1 antibodies to hIL1R3-expressing SK-MEL-30 cells, in contrast to the IgG1-LALA antibody, induces Fc receptor-mediated signaling in Jurkat gene reporter effector cells. DETAILED DESCRIPTION OF THE INVENTION

[0010] The present invention relates to IL-1α, IL-1β, IL-33, IL-36, IL1RA and / or IL1R3, as well as their barriers. These disorders are characterized by unregulated or abnormal expression of members of the IL1R3 signaling pathway, such as The present invention relates to methods for the treatment of medical conditions and / or disorders characterized by:

[0011] More specifically, the present invention relates to anti-IL1R3 antibodies for use in treating IL1R3-mediated inflammatory conditions and / or disorders in a subject.

[0012] Such conditions and disorders include, but are not limited to, inflammatory diseases, immune disorders, fibrotic disorders, eosinophilic disorders, infections, pain, central nervous system disorders, ophthalmologic disorders, genetic systemic inflammatory diseases, and systemic and local inflammatory diseases and cancer-related chronic inflammation.

[0013] definition The term "rabbit" according to the present invention refers to a member of the taxonomic order Lagomorpha. It refers to animals, which includes the families (hares and rabbits) and the Ochotonidae (pikas), preferably the genus Oryctolagus.

[0014] The term "antibody" encompasses various forms of antibody structures, including but not limited to whole antibodies and antibody fragments, so long as they exhibit the properties according to the present invention.

[0015] The term "rabbit monoclonal antibody" according to the present invention refers to a monoclonal antibody produced by immunizing a rabbit and isolated from antigen-producing cells of said rabbit, as well as such antibodies that are further modified, preferably humanized antibodies, chimeric antibodies, fragments thereof, or further genetically engineered and recombinantly produced antibodies, as long as the characteristic properties according to the present invention are retained. Preferably, the antibody is derived from B cells of said rabbit or rabbit hybridoma cells.

[0016] The term "antibody-producing cells" according to the present invention means rabbit B cells, preferably B cells or rabbit hybridoma cells, that produce antibodies.

[0017] "Native antibodies" are usually heterotetrameric glycoproteins composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end followed by several constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Specific amino acid residues are believed to form contact points between the light and heavy chain variable domains.

[0018] "Percent (%) amino acid sequence identity" with respect to a peptide or polypeptide sequence refers to the percentage of amino acid sequence identity of a particular peptide after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. A percent amino acid sequence identity is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a gene or polypeptide sequence. Alignment to determine percent amino acid sequence identity can be accomplished in a variety of ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software.

[0019] The term "VL (or VH) region" is synonymous with VL (or VH) domain.

[0020] The term "Fc receptor" or "FcR" according to the present invention refers to a human receptor that binds to the Fc region of an antibody. FcR binds to IgG antibodies and is a class of receptors that are divided into three subclasses: FcγRI, FcγRII, and FcγRIII. This includes receptors including gene variants and alternative splice forms. FcγRII receptor These receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see review in M. in Daeron, Annu, Rev. Immunol. 15:203-234 (1997)). FcRIIIA (CD16a) mediates ADCC. FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. CHn. Med. 126:330-41 (1995). These and all other FcRs are encompassed herein by the term "FcR." This term also includes the neonatal receptor, FcRn, which is involved in the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), and mediates slower catabolism and consequently a longer half-life.

[0021] As used herein, the term "antibody effector function" or "effector function" refers to the function of the Fc of IgG. Refers to a function conferred by an effector domain (e.g., the Fc region of an immunoglobulin). Such a function can occur, for example, by binding of the Fc effector domain to an Fc receptor on a phagocyte or lytic immune cell or to a component of the complement system. Exemplary effector functions are ADCC, ADCP, and CDC.

[0022] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody and binds to the same antigen as the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0023] An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competition assay; conversely, an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competition assay. Exemplary competition assays are provided herein.

[0024] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" are non-specific cytotoxicity of FcR-expressing cells. ADCC refers to a cell-mediated response in which host cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize antibody bound to a target cell and subsequently cause lysis of the target cell. The primary cells for mediating ADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII, and FcγRIII.

[0025] The terms "antibody-dependent cellular phagocytosis" and "ADCP" refer to the process by which antibody-coated cells are internalized, either in whole or in part, by phagocytic immune cells (e.g., macrophages, neutrophils, and dendritic cells) that bind to the Fc region of immunoglobulin.

[0026] "C1q" is a polypeptide that contains a binding site for the Fc region of immunoglobulins. C1q, together with two serine proteases, C1r and C1s, forms the complex C1, the first component of the complement-dependent cytotoxicity (CDC) pathway. Human C1q can be purchased commercially, for example, from Quidel, San Diego, Calif.

[0027] The "class" of an antibody refers to the type of constant domain or constant region carried by the heavy chain. There are five major antibody classes: IgA, IgD, IgE, IgG, and IgM, and several of these IgA1 and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called a, δ, ε, γ, and μ, respectively.

[0028] An "effective amount" of an agent, e.g., a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic or prophylactic result.

[0029] The term "Fc region" refers to the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term "Fc region" is used herein to define a region. The term includes native sequence Fc regions and variant Fc regions. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0030] A "variant Fc region" is a region that contains at least one "amino acid modification" as defined herein. The term "Fc variant" as used herein refers to a polypeptide comprising modifications to the Fc domain. Fc variants of the present invention are defined by the amino acid modifications that comprise them. Thus, for example, P329G is an Fc variant with a substitution of proline with glycine at position 329 relative to the parent Fc polypeptide, numbering according to the EU index. The identity of the wild-type amino acid may not be clear, in which case the variant described above will be referred to as P329G. For all positions mentioned in the description, the numbering is according to the EU index. EU index or EU index as Kabat or EU numbering scheme refers to the numbering of EU antibodies. (Edelman et al., Proc Natl Acad Sci USA 63 (1969) 78-85, entirely incorporated by reference) The modifications may be additions, deletions or substitutions. Substitutions may be made to replace naturally occurring Variants may include naturally occurring and non-naturally occurring amino acids. Examples include U.S. Patent No. 6,586,207; WO 98 / 48032; WO 03 / 073238; US 2004 / 0214988(Al); WO 05 / 35727 A2; WO 05 / 74524 A2; Chin, JW et al., Journal of the American Chemical Society 124 (2002) 9026-9027; Chin, JW, and Schultz, PG, ChemBioChem 11 (2002) 1135-1137; Chin, JW, et al., PICAS United States of America 99 (2002) 11020-11024; and Wang, L., and Schultz, PG, Chem. (2002) 1-10, which are incorporated by reference in their entireties.

[0031] The term "Fc region-containing polypeptide" refers to a polypeptide such as an antibody or immunoadhesin (see definition below) that comprises an Fc region.

[0032] The term "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc region of an antibody. FcRs that bind IgG antibodies (gamma receptors) include allelic variants and alternatively spliced ​​forms of receptors of the FcγRI, FcγRII, and FcγRIII subclasses. FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibitory receptor"), which have similar amino acid sequences that differ primarily in the cytoplasmic domains of their receptors. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (Daeron, M. Annu. Rev. Immunol. 15 (1997) 203 (See reviews at pp. 1991-234.) FcRs have been reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9 (1991) 457-492; Capel et al., Immunomethods 4 (1994) 25-34; and de Haas et al., J. Lab. Clin. Med. 126 (1995) 330-41. Other FcRs, including those identified in the future, are encompassed by the term "FcR" herein. The term also includes the neonatal receptor, FcRn, involved in the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117 (1976) 587 and Kim et al., J. Immunol. 24 (1994) 249).

[0033] As used herein, "IgG Fc ligand" refers to any biologically derived molecule, preferably a polypeptide, that binds to the Fc region of an IgG antibody to form an Fc / Fc ligand complex. Fc ligands include, but are not limited to, FcγR, FcγR, FcγR, FcRn, Clq, C3, mannan-binding lectin, mannose receptor, staphylococcal protein A, streptococcal protein G, and viral FcγR. Fc ligands also include Fc receptor homologs (FcRH), a family of Fc receptors that are homologous to FcγR (Davis et al., Immunological Reviews 190 (2002) 123-136, see entirety). (Incorporated by reference). Fc ligands may include as yet undiscovered molecules that bind to Fc. Particular IgG Fc ligands are FcRn and Fc gamma receptors. As used herein, "Fc ligand" refers to an antibody that binds to an Fc receptor. Any biological molecule, preferably a molecule of any biological origin, that binds to the Fc region of the host to form an Fc / Fc ligand complex. The term "polypeptide" refers to a polypeptide.

[0034] As used herein, "Fc gamma receptor," "FcγR," or "Fc gamma R" refers to a receptor that binds to the Fc region of an IgG antibody. Any member of the family of proteins that bind to the FcγR region and are encoded by the FcγR genes In humans, this family includes FcγRI (CD64), including isoforms FcγRIA, FcγRIB, and FcγRIC; FcγRII (CD32), including isoforms FcγRIIA (including allotypes H131 and R131), FcγRIIB (including FcγRIIB-1 and FcγRIIB-2), and FcγRIIc; and isoforms FcγRIIIA (including allotypes VI58 and F158) and FcγRIIIb (including allotypes FcγRIIIb). FcγRs include, but are not limited to, FcγRIII (CD16), including FcγRIII types FcγRIIB-NA1 and FcγRIIB-NA2 (Jefferis et al., Immunol Lett 82 (2002) 57-65, entirely incorporated by reference), as well as any undiscovered human FcγR or FcγR isoform or allotype. The mouse FcγR may be derived from any organism, including, but not limited to, human, mouse, rat, rabbit, and monkey. Mouse FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as any unidentified mouse FcγR or FcγR. Includes γR isoforms or allotypes.

[0035] As used herein, "FcRn" or "neonatal Fc receptor" refers to a receptor that binds to the Fc region of an IgG antibody and binds to FcRn FcRn refers to a protein at least partially encoded by a gene. FcRn can be derived from any organism, including, but not limited to, human, mouse, rat, rabbit, and monkey. As known in the art, a functional FcRn protein comprises two polypeptides, often referred to as a heavy chain and a light chain. The light chain is beta-2-microglobulin, and the heavy chain is encoded by the FcRn gene. Unless otherwise specified, FcRn or FcRn protein herein refers to a complex of beta-2-microglobulin and the FcRn heavy chain.

[0036] "Immunoconjugate" means an antibody conjugated to one or more cytotoxic agents, such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin, another antibody, or a radioisotope.

[0037] An "antibody fragment" comprises a portion of a full-length antibody, preferably the variable region thereof or at least the antigen-binding site thereof. Examples of antibody fragments include diabodies, Fab fragments, and single-chain antibody molecules. scFv antibodies are described, for example, in Huston, JS, Methods in Enzymol. 203 (1991) 46-88.

[0038] The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to a preparation of antibody molecules of a single amino acid composition.

[0039] The term "humanized antibody" or "humanized version of an antibody" also refers to antibodies in which both the heavy and light chains have been humanized as a result of antibody engineering. Humanized chains typically have the amino acid sequence of the V region. The amino acid sequence is altered and, when analyzed as a whole, is more closely homologous to the human germline sequence than to the germline sequence of the species of origin. The assessment of humanization is based on the resulting amino acid sequence, not the methodology itself.

[0040] As used herein, the term "specifically binding to a target or anti-target antibody" refers to an antibody that specifically binds to a target in an ELISA. ELISA refers to the binding of antibodies to their respective antigens (targets) measured by solid support. The method preferably includes coating a support with each antigen, adding the antibody under conditions that allow the antibody to form an immune complex with the respective antigen or protein, and detecting the immune complex by measuring the optical density (OD) using a secondary antibody that binds to the antibody according to the present invention and peroxidase-mediated color development.

[0041] The term "antigen" according to the present invention refers to an antigen used for immunization or a protein comprising said antigen as part of its protein sequence. For example, for immunization, a fragment of the extracellular domain of the protein (e.g., the first 20 amino acids) can be used, while for detection / assays etc., the extracellular domain of the protein or the full-length protein can be used.

[0042] As used herein, the terms "specifically bind" or "specifically recognized" mean that an antibody exhibits considerable affinity for an antigen and preferably does not exhibit significant cross-reactivity.

[0043] "Appreciable" binding affinity requires at least 10 -7 M, specifically at least 10 -8 M, more specifically at least 10 -9 M, or more specifically at least 10 -10 Binding with an affinity of M is included.

[0044] An antibody that "does not exhibit significant cross-reactivity" is one that does not appreciably bind to other undesired proteins. Antibodies specific for an epitope according to the present invention do not significantly cross-react with other epitopes on, for example, IL-1R3. Specific binding can be determined by any art-recognized means for determining such binding, such as competitive binding assays (e.g., ELISA).

[0045] All protein terms used herein refer to human proteins. Where a protein from another species is meant, this is specifically mentioned.

[0046] As used herein, the term "IL-1 alpha" refers to human IL-1 (UniProtKB P01583). As used herein, the term "IL-1 beta" refers to human IL-1 beta (UniProtKB P01584). IL-1 acts as a thymic regulator by inducing IL-2 release, B cell maturation and proliferation, and fibroblast growth factor activity. It stimulates cell proliferation. IL-1 protein is involved in the inflammatory response and has been identified as an endogenous pyrogen (UniProtKB).

[0047] As used herein, the term "IL-33" refers to human IL-33 (UniProtKB O95760), which binds to the IL1RL1 / ST2 receptor and, through this, sequentially activates the NF-kappa B and MAPK signaling pathways in target cells. This refers to cytokines that signal and activate the immune system (UniProtKB).

[0048] As used herein, the term "IL-36" refers to human IL-36 alpha (UniProtKB Q9UHA7, IL-36 base IL-36 is a cytotoxic molecule that binds to the IL1RL2 / IL-36R receptor and, through this, mediates the expression of IL-36 in target cells involved in the pro-inflammatory response. signaling cytokines that in turn activate the NF-kappa B and MAPK signaling pathways. IL-36 is thought to be involved in skin inflammatory responses by acting on keratinocytes, dendritic cells, and indirectly on T cells to drive tissue infiltration, cell maturation, and cell proliferation (UniProtKB).

[0049] As used herein, the term "NFkB" refers to human nuclear factor NF-kappa B, which is involved in the synthesis of p105. It consists of a p100 subunit (P19838) and a p100 subunit (Q00653).

[0050] "Inhibition of NFkB" is measured in the present invention as the inhibition of NFkB-dependent luciferase gene expression in human cells. Such methods are described, for example, in Windheim M. et al., Mol. Cell. Biol. 28 (2008) 1783-1791; Huang J. et al., PNAS USA 94 (1997) 12829-12832; Xiaoxia L. et al., Mol. Cell. Biol. 19 (1999) 4643-4652. When referring to murine NFkB in this specification, this is explicitly mentioned.

[0051] As used herein, the term "variable region (or domain) of an antibody according to the present invention" (light chain variable region (VL) and heavy chain variable region (VH)) refers to each of a pair of light and heavy chain regions that are directly involved in the binding of an antibody to an antigen. The variable light and heavy chain regions have the same overall structure, and each region is made up of four framework regions whose sequences are widely conserved and connected by three complementarity-determining regions (CDRs). The antibodies according to the present invention comprise a VH region and a VL region or portions thereof, which together are sufficient for specific binding to their respective antigens.

[0052] The term "antigen-binding portion of an antibody," as used herein, refers to the amino acid residues of an antibody that are responsible for antigen binding. The antigen-binding portion of an antibody preferably comprises amino acid residues from the "complementarity-determining regions" or "CDRs." CDR sequences are defined and described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of These residues are numbered according to the numbering system of the FRs or CDRs of the variable regions. It may contain fewer or additional amino acids corresponding to the insertion. The Kabat numbering of residues can be determined for a particular antibody by alignment of the homologous regions of the antibody's sequence with the "standard" Kabat numbered sequence.

[0053] As used herein, the phrases "parenteral administration" and "administered parenterally" mean modes of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

[0054] The term "cancer" as used herein includes, for example, lung cancer, non-small cell lung (NSCL) cancer, bronchioloalveolar carcinoma, bone cancer, pancreatic cancer, skin cancer, cancer of the head and neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, gastric cancer, colon cancer, breast cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma tissue, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvis cancer, mesothelioma, hepatocellular carcinoma, bile duct cancer, tumors of the central nervous system (CNS), spinal axis tumors, brain stem glioma, glioblastoma multiforme, astrocytoma, schwannoma, upper respiratory tract cancer, and the like. The cancer may be a thymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, lymphoma, lymphocytic leukemia, refractory versions of any of the above cancers, or a combination of one or more of the above cancers.

[0055] Detailed Description of the Invention As outlined in the introduction to this application, through unregulated expression of members of the IL1R3 pathway. There are several challenges in providing suitable methods for treating conditions of inflammation mediated by immune disorders, fibrotic disorders, eosinophilic disorders, infectious diseases, Methods are provided for treating, inhibiting, or ameliorating inflammatory conditions and / or disorders, including pain, central nervous system disorders, ophthalmologic disorders, genetic systemic inflammatory diseases, and systemic and local inflammatory diseases.

[0056] The present invention provides a method for treating IL1R3-associated leukemia, comprising administering to a subject a therapeutically effective amount of an antagonistic IL1R3 antibody. and a method for treating, inhibiting, or ameliorating inflammatory conditions and / or disorders in a subject in need thereof. Regarding.

[0057] In particular, the present invention encompasses anti-IL1R3 antibodies for use in treating IL1R3-mediated inflammatory conditions and / or disorders in a subject. Such inflammatory conditions and / or disorders include inflammatory conditions, immune disorders, fibrotic disorders, eosinophilic disorders, and the like. The inflammatory disease may be selected from the group consisting of: injury, infection, pain, central nervous system disorders, ophthalmologic disorders, genetic systemic inflammatory diseases, and systemic and local inflammatory diseases.

[0058] In one aspect of the invention, the inflammatory disorder to be treated is an IL-1 dependent disease, such as Schnitzler's syndrome, Behçet's disease, secondary amyloidosis, Henoch-Schönlein purpura, idiopathic relapsing pericarditis, systemic-onset juvenile idiopathic arthritis, adult-onset Still's disease, macrophage activation syndrome, Sweet's syndrome / neutrophilic dermatosis, neutrophilic pancreatitis, and inflammatory bowel disease. Histiocytosis, Erdheim-Chester / Histiocytosis, SAPHO, PFAPA, Multicentric Castleman The primary inflammatory cause may be systemic or localized, such as inflammatory bowel disease, Jessner-Kanoff disease, primary Sjögren's fatigue syndrome, Kawasaki disease, chronic granulomatous colitis, hidradenitis suppurativa, autoimmune inner ear disease, or severe traumatic brain injury, or genetic systemic inflammatory disorders such as familial Mediterranean fever (FMF), CAPS, TRAPSa, HIDS, PAPA, PASH, DIRA, Blau syndrome / granulomatous arthritis, mevalonate kinase deficiency, Majeed syndrome, or NLRP12 autoinflammatory syndrome.

[0059] In a preferred embodiment of the invention, the inflammatory condition is selected from the group of COPD, inflammatory skin diseases, psoriasis, generalized pustular psoriasis (GPP), inflammatory bowel disease (IBD), asthma, atopic dermatitis, idiopathic pulmonary fibrosis, peritonitis, rheumatoid arthritis (RA), or metabolic rheumatic disorders associated with hyperuricemia.

[0060] In a preferred embodiment, the inflammatory condition is a metabolic rheumatic disorder associated with hyperuricemia, which may be selected from the group consisting of gout, pseudogout, drug-induced gout, and chronic active (refractory) gout.

[0061] For the treatment of metabolic rheumatic disorders according to one aspect of the present invention, an IL1R3 antagonist is The IL1R3 antagonist antibody is administered in combination with a therapeutic agent for the treatment of gout. They may be administered in combination.

[0062] It may be administered simultaneously with a therapeutic agent for the treatment of gout, but it may also be administered sequentially with a therapeutic agent for the treatment of gout.

[0063] Another aspect of the present invention includes a method for treating an inflammatory condition and / or disorder, wherein the inflammatory condition is a cancer-associated chronic inflammation.

[0064] The present invention also relates to anti-IL1R3 antibodies for the treatment of inflammatory conditions and / or disorders, wherein the inflammatory condition is cancer associated with chronic inflammation.

[0065] As used herein, the term "cancer" refers to, for example, lung cancer, non-small cell lung (NSCL) cancer, bronchioloalveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, gastric cancer, colon cancer, breast cancer, uterine cancer, fallopian tube cancer, endometrial cancer, and cervical cancer. , cancer of the vagina, cancer of the vulva, Hodgkin's disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, cancer of the renal pelvis, mesothelioma, hepatocellular carcinoma, biliary tract cancer, neoplasms of the central nervous system (CNS), spinal axis tumors, brain stem gliomas, multiple The cancer may include glioblastoma, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, lymphoma, lymphocytic leukemia, refractory versions of any of the above cancers, or a combination of one or more of the above cancers.

[0066] Preferably, the antibody according to the present invention is effective against pancreatic cancer, liver cancer, lung cancer (associated with inflammation due to asbestos, infection, smoking, silica), non-small cell lung cancer, colon cancer / colon-associated cancer (inflammation), and the like. It is used to treat chronic inflammation associated with cancer selected from the group consisting of: gastric cancer (associated with chronic cholecystitis), stomach cancer, gastric cancer, chronic gastritis associated with gastric cancer, estrogen receptor positive breast cancer, head and neck squamous cell carcinoma, mesothelioma, gallbladder cancer (associated with chronic cholecystitis), ovarian cancer, bladder cancer, prostate cancer, prostate cancer associated with E. coli infection, thyroid cancer, Hodgkin's disease, MALT lymphoma, salivary gland cancer, melanoma, endometrial cancer associated with endometriosis, and esophageal cancer associated with Barrett's esophagitis.

[0067] Preferably, the cancer is breast cancer, colon cancer, lung cancer, pancreatic cancer, liver cancer, non-small cell lung cancer, rectal cancer, stomach cancer, gastric cancer, estrogen receptor positive breast cancer, head and neck squamous cell carcinoma, mesothelioma, gallbladder cancer, ovarian cancer, bladder cancer, prostate cancer, thyroid cancer, Hodgkin's disease, MALT lymphoma, salivary gland cancer, or melanoma.

[0068] The antibodies of the invention may be used to treat chronic inflammation associated with cancer, as some tumors are caused or promoted by cells in the tumor microenvironment that secrete pro-inflammatory cytokines such as IL-1α, IL-1β, IL-33, and IL-36. In some instances, the expression of such cytokines leads to the development of tumor resistance.

[0069] Thus, in one aspect of the invention, the antibodies are used to treat a subject, the subject comprising a tumor, such as a solid tumor, and tumor resistance to cytotoxic, cytostatic or targeted / immunotherapy. Preferably, the subject is a human subject, for example, a cancer patient.

[0070] The simultaneous or sequential use of cytokine inhibitors and anti-cancer compounds can significantly improve the response rate of such treatments or break tumor resistance.

[0071] Accordingly, the present invention also encompasses methods of treating a subject, wherein the subject is suffering from one or more cytotoxic These tumors are characterized by resistance or an inadequate response to treatment with cytostatic, cytostatic, or targeted anticancer drugs.

[0072] In one aspect of the invention, the IL1R3 antibody has one or more cytotoxic, cytostatic or targeting properties. It is administered in combination with targeted anti-cancer drugs.

[0073] In one aspect of the invention, the IL1R3 antagonist antibody has one or more of cytotoxicity, cell proliferation, In another embodiment, the IL1R3 antagonist is administered simultaneously with a growth-suppressing or targeted anti-cancer drug. The antibody is sequentially combined with one or more cytotoxic, cytostatic or targeted anti-cancer drugs. It is administered.

[0074] In the latter case, the antibody may be coupled to one or more cytotoxic, cytostatic, or targeted anti-cancer agents. Preferably, it is administered after treatment.

[0075] Cytotoxic or cytostatic anti-cancer agents according to the present invention may be taxanes, anthracyclines, alkylating agents, histone deacetylase inhibitors, topoisomerase inhibitors, kinase inhibitors, nucleotide analogs, peptide antibiotics, and platinum-based agents.

[0076] Preferably, the targeted anti-cancer agent is used in targeted therapy and is selected from one or a combination of the following: anti-EGFR compounds such as cetuximab, gefitinib, erlotinib, lapatinib, panitumumab, and anti-HER2 compounds such as trastuzumab, ado-trastuzumab, emtansine, pertuzumab.

[0077] More preferably, the targeted anti-cancer drug is a targeted checkpoint inhibitor. These include anti-PD1 compounds such as pembrolizumab and nivolumab, atezolizumab, avelumab, and the like. The compounds may be, but are not limited to, anti-PDL1 compounds such as ipilimumab, durvalumab, and anti-CTLA-4 compounds such as ipilimumab and tremelimumab.

[0078] The present invention provides significantly improved response rates to targeted cancer therapy because broad-spectrum inhibition of cytokine-induced signaling is achieved. Such activity in cancer conditions is achieved not through direct cancer cell elimination activity (as achieved by some prior art compounds), but through inhibition of cancer-associated inflammation by modulation of the IL1R3 signaling pathway.

[0079] The antibodies of the present invention offer a highly advantageous activity profile, as they allow for the effective inhibition of cancer-associated chronic inflammation, and at the same time do not affect the viability of target cells expressing IL-1R3, thereby avoiding undesirable side effects.

[0080] Thus, the present invention offers several advantages over other IL-1 family targeted therapies. The commercially available compound Kineret (IL-1Ra) has a short half-life, resulting in very frequent (daily) treatment intervals. Antibodies targeting a single cytokine (such as Ilaris) result in unwanted signaling / activation of other IL-1 family cytokines. Furthermore, the IL-1 family These cytokines are known to induce both pro-inflammatory and IL-1R3-independent anti-inflammatory signaling, both of which are interfered with when targeting the cytokine or its alpha chain receptor. In contrast, the antibodies of the present invention combine the advantages of reduced treatment intervals, simultaneous inhibition of different IL-1 family cytokine-mediated signaling, and specificity with respect to blocking pro-inflammatory signaling pathways. The antibodies described herein are preferably monoclonal antibodies with high affinity, high specificity, and potent neutralizing activity for IL-1R3. Accordingly, the present invention also encompasses IL-1R3 antibodies with high affinity and specificity for IL-1R3, potent IL-1R3 neutralizing activity, and improved stability.

[0081] In a preferred embodiment of the present invention, the antibody has reduced effector function. Preferably, the antibody of the present invention exhibits reduced or no Fcγ receptor signaling. More preferably, the antibody does not induce ADCC.

[0082] Another aspect of the invention is an anti-IL1R3 antibody that contains at least the amino acid substitutions L234A and L235A in the human IgG1 Fc region, or S228P and L235E in the human IgG4 Fc region, or the corresponding substitutions in another organism. The aim is to include functional mutations that

[0083] In one embodiment of the present invention, the anti-IL1R3 antibody is a) a heavy chain variable region (VH) comprising complementarity determining regions including CDR-H1, CDR-H2, and CDR-H3; wherein the CDR-H1 region is an amino acid sequence selected from the group consisting of SEQ ID NOs: 69 to 85 and 178. Contains columns, The CDR-H2 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 86 to 102 and 179. , and The CDR-H3 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 103 to 119 and 180. Hmm, as well b) a light chain variable region (VL) comprising complementarity determining regions including CDR-L1, CDR-L2, and CDR-L3; wherein the CDR-L1 region is an amino acid sequence selected from the group consisting of SEQ ID NOs: 120 to 136 and 181. Contains an array, The CDR-L2 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 137 to 153 and 182. and The CDR-L3 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 154 to 170, 175, and 183. Includes.

[0084] In one embodiment, the antibody of the invention comprises a substitution at position 2 of CDR-L3. It may be a tein to serine substitution.

[0085] In one aspect of the invention, the subject is a human subject and the antibody is a) a heavy chain variable region (VH) comprising complementarity determining regions including CDR-H1, CDR-H2, and CDR-H3; wherein the CDR-H1 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 69 to 85; The CDR-H2 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 86 to 102, and the CDR-H3 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 103 to 119; and b) a light chain variable region (VL) comprising complementarity determining regions including CDR-L1, CDR-L2, and CDR-L3; wherein the CDR-L1 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 120 to 136; The CDR-L2 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 137 to 153; and The CDR-L3 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 154 to 170 and 175. Contains arrays, Includes.

[0086] In another aspect of the invention, the subject is a mouse and the antibody is a) a heavy chain variable region (VH) comprising complementarity determining regions including CDR-H1, CDR-H2, and CDR-H3; wherein the CDR-H1 region comprises the amino acid sequence of SEQ ID NO: 178; the CDR-H2 region comprises the amino acid sequence of SEQ ID NO: 179; and the CDR-H3 region comprises the amino acid sequence of SEQ ID NO: 180; and b) a light chain variable region (VL) comprising complementarity determining regions including CDR-L1, CDR-L2, and CDR-L3; wherein the CDR-L1 region comprises the amino acid sequence of SEQ ID NO: 181; the CDR-L2 region comprises the amino acid sequence of SEQ ID NO: 182; and The CDR-L3 region comprises the amino acid sequence of SEQ ID NO: 183. Includes.

[0087] In another embodiment of the invention, the antibody comprises the VH region of SEQ ID NOs: 1-34, 173 and 176. and a light chain variable (VL) region that is at least 90% identical to a VL region selected from the group consisting of SEQ ID NOs: 35 to 68, 174, and 177.

[0088] In one embodiment of the invention, the antibody is a humanized antibody. In such embodiments, the subject is a human subject, and the antibody comprises a heavy chain variable (VH) region that is at least 90% identical to a VH region selected from the group consisting of the VH regions of SEQ ID NOs: 1-34, and 173, and a light chain variable VL region selected from the group consisting of the VL regions of SEQ ID NOs: 35-68, and 174. The VL region is at least 90% identical to the VL region of the target gene.

[0089] In another embodiment, the subject is a mouse and the antibody has a heavy chain variable (VH) sequence of SEQ ID NO: 176. a VH region that is at least 90% identical to the light chain variable (VL) region of SEQ ID NO: 177; The VL regions are at least 90% identical.

[0090] In one embodiment, the antibody of the present invention is an antibody that specifically binds to IL-1R3, or a fragment or derivative of the antibody comprising at least a portion of the antibody that is sufficient to confer IL-1R3 specific binding, and is selected from the group consisting of the VH regions of SEQ ID NOs: 1 to 34, 173 and 176. The antibody may comprise a heavy chain variable (VH) region that is at least 60% identical, preferably at least 70% identical, more preferably at least 80% identical, more preferably at least 90% identical to a VH region of the antibody.

[0091] In one embodiment, the antibody shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence selected from the group of VH sequences according to the invention. The heavy chain variable region (VH) sequence has a sequence identity of:

[0092] In certain embodiments, a VH having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity has substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, such that the antibodies retain the ability to specifically bind to their respective antigens in accordance with the present invention.

[0093] The present invention also relates to an antibody of the present invention that specifically binds to IL-1R3, or a fragment or derivative of the antibody comprising at least a portion of the antibody sufficient to confer specific binding to IL-1R3, and which comprises a light chain variable (VL) region that is at least 60% identical, preferably at least 70% identical, more preferably at least 80% identical, more preferably at least 90% identical to a VL region selected from the group consisting of the VL regions of SEQ ID NOs: 35 to 68, 174, and 177.

[0094] The antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of the light chain variable region (VL) sequence of the present invention. It may contain a VL that:

[0095] In certain embodiments, VH sequences with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity have substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, such that the antibodies retain the ability to specifically bind to their respective antigens according to the present invention.

[0096] In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, or and / or deleted. In certain embodiments, the substitution, insertion or deletion is The present invention also includes affinity-matured antibodies, which can be produced by methods known to those skilled in the art. Marks et al., Bio / Technology 10:779-783 (1992) discloses that: describes affinity maturation by VH and VL domain shuffling. Or random mutagenesis of framework residues is described in: Barbas et al., Proc Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al., Gene 169:147-155 (1995); Yelton et al., J. Immunol. 1 55:1994-2004 (1995); Jackson et al., J. Immunol. 1 54(7):3310-9 (1995); and Hawkins et al., J. Mol. Biol. 226:889-896 (1992), and WO2010108127.

[0097] In certain embodiments, a total of 1 to 10 amino acid substitutions are made in each of the VH or VL sequences, In one embodiment, the antibody of the invention has an insertion and / or deletion at position 90 of the VH or VL sequence. Preferably, the amino acid at position 90 is substituted with serine. This substitution is preferably at position 90 of the light chain variable region (VL). In a preferred embodiment, the cysteine ​​at position 90 of SEQ ID NO: 62 is replaced with serine. However, antibodies of the present invention may also be used without the amino acid at position 90. The VL and VH sequences of the antibodies of the present invention may contain additional mutations at different positions, but are not limited to amino acid substitutions, and may include any substitution, deletion, or insertion that results in a functional antibody having the properties of the antibodies of the present invention. Thus, the VL and VH sequences of the antibodies of the present invention may contain additional mutations at different positions. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside of the CDRs (i.e., the FR ) occurs.

[0098] In other embodiments, the substitutions, insertions, or deletions occur in regions within the CDRs. In one embodiment, the antibody of the invention comprises a substitution at position 2 of CDR-L3. In one embodiment, the substitution is in SEQ ID NO: 164.

[0099] The present invention also relates to an antibody of the present invention that specifically binds to IL-1R3, or a fragment or derivative of the antibody comprising at least a portion of said antibody sufficient to confer IL-1R3 specific binding, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-34, 173 and 176. (VH)

[0100] Preferably, the heavy chain variable region (VH) sequence is SEQ ID NO: 1, or SEQ ID NO: 2, or SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: No. 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, or SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or alternatively SEQ ID NO: 34, or 173 and 176.

[0101] The present invention further relates to a method, wherein the antibody is an antibody of the present invention that specifically binds to IL-1R3, or a fragment or derivative of the antibody comprising at least a portion of the antibody sufficient to confer IL-1R3 specific binding, and comprises a light chain variable region (VL) comprising an amino acid sequence selected from the group of SEQ ID NOs: 35-68, 174 and 177.

[0102] Even more preferably, the light chain variable region (VL) sequence is SEQ ID NO:35, or SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, or alternatively SEQ ID NO:68, or 174 or 177.

[0103] The antibody of the invention that specifically binds to IL-1R3, or a fragment or derivative thereof comprising at least a portion of said antibody sufficient to confer IL-1R3 specific binding, is also designated MAB-15-0139. M AB-15-0097, MAB-15-0135, MAB-15-0133, MAB-15-0107, MAB-15-0128, MAB-15-0116, MAB-16-0004, MAB-16-0009, MAB-16-0028, MAB-16-0031, MA The present invention also includes a VH region and a VL region comprising the CDR1, CDR2, and CDR3 regions, respectively, of an antibody selected from the group consisting of MAB-16-0043, MAB-16-0049, MAB-16-0045, MAB-16-0040, MAB-16-0036, MAB-16-0046, MAB-16-0030, MAB-16-0021, MAB-16-0019, MAB-16-0015, MAB-16-0027, MAB-16-0048, MAB-16-0041, MAB-16-0149, MAB-16-0150, and MAB-16-0531.

[0104] In one embodiment, an antibody of the invention that specifically binds to IL-1R3, or a fragment or derivative of the antibody comprising at least a portion of said antibody sufficient to confer IL-1R3 specific binding, comprises SEQ ID NOs: 1 and 35, or SEQ ID NOs: 2 and 36. Antibodies according to the invention also include those represented by SEQ ID NOs: 3 and 37, or SEQ ID NO: 4 and 38, or SEQ ID NO: 5 and 39, or SEQ ID NO: 6 and 40, or SEQ ID NO: Nos. 7 and 41, or SEQ ID NOs. 8 and 42, or SEQ ID NOs. 9 and 43, or SEQ ID NOs. 10 and 44, or may comprise SEQ ID NOs: 11 and 45, or SEQ ID NOs: 12 and 46. Alternatively, the antibody according to the invention comprises SEQ ID NOs: 13 and 47, or SEQ ID NOs: 14 and 48, or SEQ ID NOs: 15 and 49, or SEQ ID NOs: 16 and 50, or SEQ ID NOs: 17 and 51, or SEQ ID NOs: 18 and 52, or SEQ ID NOs: 19 and 53, or SEQ ID NOs: 20 and 54, or SEQ ID NOs: 21 and 55, or SEQ ID NOs: 22 and 56, or SEQ ID NOs: 23 and 57, or SEQ ID NOs: 24 and 58, or SEQ ID NOs: 25 and 59, or SEQ ID NOs: 26 and 60, or SEQ ID NOs: 27 and 61.

[0105] Alternatively, the antibody according to the invention may be selected from the group consisting of SEQ ID NOs: 28 and 62, or SEQ ID NOs: 29 and 63, or SEQ ID NOs: 30 and 64, or SEQ ID NOs: 31 and 65, or SEQ ID NOs: 32 and 66, or SEQ ID NOs: 33 and 67, or SEQ ID NOs: 34 and 68, or SEQ ID NOs: 173 and 54, or SEQ ID NOs: 28 and 174, or SEQ ID NO: 176. and 177.

[0106] Most preferably, the antibodies of the invention that specifically bind to IL-1R3, or fragments or derivatives of the antibodies comprising at least a portion of said antibodies sufficient to confer IL-1R3 specific binding, comprise the constant region sequences CR-H (SEQ ID NO: 172) and CR-L (SEQ ID NO: 171), and a VH region selected from the group of SEQ ID NOs: 1-34, 173, and 176, and a VL region selected from the group of SEQ ID NOs: 35-68, 174, and 177. Includes the area.

[0107] Antibodies of the invention that specifically bind to IL-1R3, or fragments or derivatives of the antibodies that contain at least a portion of said antibodies sufficient to confer IL-1R3 specific binding, include the constant region sequences CR-H (SEQ ID NO: 172) and CR-L (SEQ ID NO: 171), as well as MAB-15-0139, MAB-15-0106, MAB-15-0108, MAB-15-0110, MAB-15-0117, MAB-15-0121, MAB-15-0140, MAB-15-0115, MAB-15-0125, and the like. , MAB-15-0119, MAB-15-0109, MAB-15-0097, MAB-15-0135, MAB-15-0133, MAB-15-0107, MAB-15-0128, MAB -15-0116, MAB-16-0004, MAB-16-0009, MAB-16-0028, MAB-16-0031, MAB-16-0043, MAB-16-0049, MAB-16- 0045, MAB-16-0040, MAB-16-0036, MAB-16-0046, MAB-16-0030, MAB-16-0021, MAB-16-0019, MAB-16-0015, MAB-16-0027, MAB-16-0048, MAB-16-0041, MAB-16-0149 and MAB-16-150, and MAB-16-0531. Also included are VH and VL regions, each containing its respective CDR1, CDR2 and CDR3 regions.

[0108] According to preferred therapeutic applications of the antibodies of the invention, the effector functions (such as ADCC) of the antibodies of the invention are reduced or absent: in contrast to other antibodies of the prior art, such as CAN04 (e.g., WO 2015 / 132602 A1), the antibodies of the invention avoid undesired depletion of immune cells.

[0109] Preferably, antibodies according to the invention exhibit reduced or no Fcγ receptor signaling.

[0110] Therefore, the present invention also provides a method for producing a human IgG1 Fc region comprising at least L234A and L235A of the human IgG1 Fc region, or Contains amino acid substitutions of S228P and L235E in the human IgG4 Fc region, or functionally equivalent mutations The present invention relates to the antibody.

[0111] In one embodiment according to the invention, the antibody is a humanized IgG1 LALA It is an antibody. In another embodiment, the antibody is a murine IgG2a LALA It is an antibody.

[0112] In one embodiment according to the invention, the antibody inhibits IL-1R3-induced NFkB activity.

[0113] In another embodiment, an antibody of the invention that specifically binds to IL-1R3, or a fragment or derivative of the antibody comprising at least a portion of said antibody sufficient to confer IL-1R3 specific binding, is selected from the group consisting of antibodies MAB-15-0139, MAB-15-0106, MAB-15-0108, MAB-15-0110, MAB-15-0117, MAB-15-0121, , MAB-15-0140, MAB-15-0115, MAB-15-0125, MAB-15-0119, MAB-15-0109, MAB-15-0097, MAB-15-0135, MAB-15-0133, MA B-15-0107, MAB-15-0128, MAB-15-0116, MAB-16-0004, MAB-16-0009, MAB-16-0028, MAB-16-0031, MAB-16-0043, MAB-1 and MAB-16-0049, MAB-16-0045, MAB-16-0040, MAB-16-0036, MAB-16-0046, MAB-16-0030, MAB-16-0021, MAB-16-0019, MAB-16-0015, MAB-16-0027, MAB-16-0048, MAB-16-0041, MAB-16-0149, MAB-16-150, and MAB-16-0531.

[0114] The antibodies according to the present invention have the advantage of being very potent in binding to their targets. They exhibit strong binding ability to the antigen IL1R3, but not to other receptors. The binding properties of antibodies can be determined by enzyme-linked immunosorbent assay (ELISA) and cell binding assay ( flow cytometry) and are illustrated in Figures 2, 6, and 7.

[0115] Preferred antibodies according to the invention exhibit a half maximal effective concentration (EC50) of less than 30 ng / ml, preferably less than 20 ng / ml. In other embodiments, they exhibit an EC50 of less than 15 ng / ml, 10 ng / ml or less than 5 ng / ml. Preferred antibodies according to the invention exhibit an EC50 of 16.3 ng / ml in biochemical ELISA experiments (see Figure 7).

[0116] The antibodies according to the invention also showed that human IL1R3 binds to it in experiments where it is expressed in different cell lines. Although the antibody shows very strong binding to these antigens, it binds to cell lines that do not express human IL1R3. (e.g., NIH-3T3, see Figure 5).

[0117] In the IL1R3 highly expressing cell line SK-MEL-30 (see Figure 6, Example 4), the antibody preferably exhibits an EC50 of less than 400 ng / ml, more preferably less than 350 ng / ml or less than 310 ng / ml.

[0118] In a preferred embodiment encompassed by the present invention, the antibodies according to the present invention inhibit NFkB activity stimulated by IL-1 alpha and / or IL-1 beta. Figures 3, 4 and 8 illustrate the potent inhibitory activity of the antibodies according to the present invention.

[0119] In one embodiment, an antibody of the invention that specifically binds to IL-1R3, or a fragment or derivative of the antibody comprising at least a portion of the antibody sufficient to confer IL-1R3 specific binding, inhibits IL-1 alpha stimulated NFkB activity.

[0120] In another embodiment, an antibody of the invention that specifically binds to IL-1R3, or a fragment or derivative of the antibody comprising at least a portion of the antibody sufficient to confer IL-1R3 specific binding, inhibits IL-1beta-stimulated NFkB activity.

[0121] The antibodies according to the invention preferably inhibit IL-1beta stimulated NFkB activity in HEK293T / 17-FR cells with an EC50 of less than 100ng / ml, preferably less than 95ng / ml, 85ng / ml, 75ng / ml, 65ng / ml, 55ng / ml, 45ng / ml, 35ng / ml, 25ng / ml, 20ng / ml, and most preferably less than 15ng / ml. (See Figure 3 for an example.)

[0122] The antibodies of the present invention inhibit IL-1 alpha stimulation in A549-NFkB-RE-Luc cells with an EC50 of less than 1000 ng / ml, preferably less than 500 ng / ml, 300 ng / ml, 200 ng / ml, and most preferably less than 100 ng / ml. It inhibits NFkB activity (see, for example, Figure 8).

[0123] The antibodies of the present invention stimulate IL-1 beta in A549-NFkB-RE-Luc cells with an EC50 of less than 700 ng / ml, preferably less than 600 ng / ml, 300 ng / ml, 200 ng / ml, 100 ng / ml, and most preferably less than 50 ng / ml. It inhibits NFkB activation induced by stimulation (see, for example, Figure 8).

[0124] The present invention also provides IL-1β / IL-1R1 / IL-1RAcP, IL-1α / IL-1R1 / IL-1RAcP IL-33 / ST2 / IL-1RAc P, and / or IL-36 / IL-36R / IL-1RAcP. The present invention also encompasses the above-mentioned antibodies that inhibit NFkB activity.

[0125] Furthermore, in A549-NFkB-RE-Luc cell lysate, the antibody according to the invention, at a concentration of 10 μg / ml (rabbit IgG isotype has a molecular weight of 150 KD), inhibits NFkB expression stimulated with 0.1 ng / ml human IL-1 alpha, human IL-1 beta, IL-33 and / or IL-36 (for molecular weights see UniProtKB / Swiss-Prot) by more than 50%, preferably more than 70%, preferably more than 80%, more preferably more than 90%, more preferably more than 95% compared to the same assay without the antibody according to the invention (Steady-Glo™ Luciferase Assay System; Promega; Catalog number E2510).

[0126] In one embodiment, the antibody according to the invention is expressed in HEK 293T / 17 cells (HEK293T / 17-FR cells transfected with luciferase under the control of an NF-kB reporter gene), HEK-Blue-IL33 TM Inhibits IL-1 alpha, IL-1 beta, IL-33, and / or IL-36 stimulated luciferase activity in cells (Invivogen) or HEK-293 / 17-IF cells, respectively.

[0127] Preferably, the IL-1 alpha stimulated luciferase activity is inhibited by at least 50%, preferably at least 70%, preferably at least 80%, preferably at least 90%, more preferably at least 95%. Preferably, the IL-1 alpha stimulated luciferase activity is inhibited by 95%.

[0128] Preferably, the IL-1beta stimulated luciferase activity is inhibited by 50% or more, preferably 70% or more, preferably 80% or more, preferably 90% or more, more preferably 95% or more. Preferably, the IL-1beta stimulated luciferase activity is inhibited by 95%.

[0129] Preferably, the IL-33 stimulated luciferase activity is 50% or more, preferably 70% or more. Preferably, the IL-33-stimulated luciferase activity is inhibited by 95% or more, preferably by 80% or more, preferably by 90% or more, more preferably by 95% or more.

[0130] Preferably, the IL-36-stimulated luciferase activity is 50% or more, preferably 70% or more. Preferably, the IL-36-stimulated luciferase activity is inhibited by 95%.

[0131] Furthermore, the antibodies according to the present invention inhibit human IL-1a and IL-1b mediated IL-6 release and are superior to polyclonal antibodies. This potent inhibitory activity is shown and exemplified in Figure 9. In these experiments, the EC50 values ​​show that the humanized anti-IL-1R3 IgG1-LALA antibody is superior to the goat anti-human IL1-R3 polyclonal antibody AF676 (R&D Systems). In a preferred embodiment, the antibody inhibits human IL-α-mediated IL-6 release with an EC50 of less than 2500 ng / ml. , preferably less than 1500 ng / ml, less than 1000 ng / ml, less than 600 ng / ml, less than 400 ng / ml, or less than 300 ng / ml. It is also preferred that the antibodies of the invention inhibit human IL-β-mediated IL-6 release with an EC50 of less than 500 ng / ml, preferably less than 400 ng / ml, less than 300 ng / ml, less than 200 ng / ml, or less than 150 ng / ml.

[0132] In another embodiment of the invention, the antibody inhibits human IL-33-mediated NfkB signaling. Figure 10 illustrates the inhibitory activity of selected antibodies of the invention in HEK-Blue-IL33™ cells and demonstrates their superiority over polyclonal antibodies. In a preferred embodiment of the invention, the antibody inhibits human IL-33-mediated NfkB signaling with an EC50 of less than 20,000 ng / ml, preferably less than 10,000 ng / ml. is less than 18000ng / ml, less than 3000ng / ml, less than 1000ng / ml, less than 500ng / ml, or less than 400ng / ml.

[0133] The antibodies of the present invention can also inhibit human IL-36-mediated NfkB signaling (FIG. 11). Preferably, they inhibit human IL-36-mediated NfkB signaling with an EC50 of less than 100 ng / ml. or less than 50 ng / ml, less than 40 ng / ml, less than 30 ng / ml, less than 20 ng / ml, or less than 15 ng / ml. Surprisingly, the present inventors have found that antibodies according to the present invention inhibit cytokine release mediated by a variety of different stimuli. For example, the antibodies inhibit cytokine release mediated by IL-1a, IL-33, and IL-36a. The results for selected antibodies are shown in Figure 12. For example, antibody MAB-16-0030 inhibits cytokine release mediated by all three stimuli, while IL-1Ra only affects cytokine release mediated by IL-1a.

[0134] Diseases associated with acute or chronic inflammation are maintained or established by the actions of multiple cytokines, either simultaneously or sequentially. Early "alarmins" such as IL-1a and IL-33 can induce other cytokines, including IL-1b and IL-36, to establish a strong inflammatory environment. Therefore, simultaneous inhibition of signaling mediated by multiple cytokines exerts effective control of the inflammatory process. Inhibition of multi-cytokine signaling via blockade of the IL1R3 receptor This is an important aspect of the antibodies of the present invention.

[0135] Antibody binding to immune cells can result in cellular depletion and deleterious effects, for example, by directly inducing apoptotic signaling pathways, stimulating excessive cytokine release, or antibody-dependent cellular cytotoxicity (ADCC).

[0136] Importantly, the antibodies according to the invention do not affect the viability of immune cells, for example they do not affect the viability of human peripheral blood mononuclear cells (PBMCs) and do not induce the release of IL-6 in PBMCs (see Figure 13).

[0137] The antibodies according to the invention not only inhibit the functional activity of cytokine release in the different cell lines mentioned above, but also in PMBCs or whole blood cells from donors. The antibodies inhibit cytokine release mediated by various specific or combined stimuli, such as LPS, heat-inactivated Candida albicans, IL-12 / IL-33, or anti-CD3 / CD28 antibody-stimulated PBMCs (see Figures 14 and 15).

[0138] Additionally, in one embodiment, the anti-IL-1R3 IgG1-LALA antibody according to the present invention can inhibit the release of IFNg, IL-6, TNF-α, IL-13, IL-17, and IL-10 in a mixed lymphocyte reaction (see Figure 16).

[0139] The method of the present invention also includes administering to a patient a pharmaceutically effective amount of an antibody, or a derivative or fragment thereof, according to the present invention in the form of a pharmaceutical composition. Such pharmaceutical compositions, in accordance with the present invention, may comprise a pharmaceutically acceptable carrier and a therapeutically effective amount of an antibody of the present invention that specifically binds to IL-1R3, or a fragment or derivative of the antibody comprising at least a portion of the antibody sufficient to confer IL-1R3 specific binding.

[0140] As used herein, "pharmaceutical carrier" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).

[0141] The compositions of the present invention can be administered by various methods known in the art. As will be recognized by those skilled in the art, the route and / or mode of administration will vary depending on the desired results. To administer the compounds of the present invention by a particular route of administration, the compounds may need to be coated with or co-administered with a material to prevent inactivation. For example, the compounds may be administered to a subject in an appropriate carrier, such as liposomes or a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Pharmaceutical carriers may be administered in the form of sterile, injectable These media and agents include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of such ready-to-use solutions or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art.

[0142] As used herein, the phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

[0143] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the presence of microorganisms can be ensured both by the above-mentioned sterilization procedures and by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like, in the compositions. In addition, prolonged absorption of injectable pharmaceutical forms can be brought about by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin. Regardless of the route of administration selected, the compounds and / or pharmaceutical compositions of the present invention, which may be used in a suitable hydrated form, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art. The actual dosage level of the active ingredient in the pharmaceutical compositions of the present invention can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors, including the activity of the particular composition of the invention employed, the route of administration, the time of administration, the excretion rate of the particular compound employed, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, overall health and prior medical history of the patient being treated, as well as factors well known in the medical arts.

[0144] In another aspect, the invention relates to an antibody of the invention that specifically binds to the mouse IL-1R3 receptor, or a fragment or derivative of said antibody, wherein the antibody has at least one VH region of SEQ ID NO: 176. a heavy chain variable (VH) region that is at least 90% identical to the VL region of SEQ ID NO: 177; They contain identical light chain variable (VL) regions.

[0145] Preferably, the antibody comprises a heavy chain variable region (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the group of VH sequences of the invention.

[0146] In certain embodiments, VH sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the sequences herein, such that the antibodies retain the ability to specifically bind to their respective antigens according to the invention.

[0147] In another embodiment of the invention, the antibody comprises a light chain variable region (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the group of VL sequences according to the invention.

[0148] In certain embodiments, VL sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the sequences herein, such that the antibodies retain the ability to specifically bind to their respective antigens according to the invention.

[0149] The antibody of the present invention that specifically binds to the mouse IL-1R3 receptor, or a fragment or derivative of said antibody, preferably has reduced or no effector function.

[0150] Preferably, antibodies according to the invention exhibit reduced or no Fcγ receptor signaling. It is even more preferred that they do not induce ADCC.

[0151] In one embodiment, the antibody of the present invention, or a fragment or derivative thereof, that specifically binds to the mouse IL-1R3 receptor is a mouse IgG2a with the mutations L234A and L235A in the Fc portion. (amino acid positions according to the EU numbering index).

[0152] Another aspect of the present invention relates to an antibody of the present invention, or a fragment or derivative thereof, that specifically binds to the mouse IL-1R3 receptor, for use in preclinical studies. Such studies can be carried out in animal models, preferably murine disease models. Such model systems include the monosodium urate crystal (MSU)-induced mouse peritonitis model, serum Migration-induced rheumatoid arthritis, collagen-induced arthritis, antibody-induced arthritis, collagen antibody-induced arthritis, K / BxN antibody migration arthritis, imiquimod-induced inflammatory skin disease / psoriasis, epidermolysis bullosa acquisita model, thioglycollate-induced peritonitis, immune complex-induced peritonitis, bleomycin-induced pulmonary fibrosis, xenograft psoriasis, cigarette smoke-induced pulmonary inflammation in COPD, tracheal instillation of elastase in COPD, fluorescein isothiocyanate (FITC)-induced lung injury / fibrosis, radiation-induced irritation fibrosis, silica-induced pulmonary fibrosis, asbestos fiber-induced pulmonary fibrosis, DSS-induced colitis, Trini Benzene sulfonic acid-induced colitis, oxazolone-induced colitis, adoptive transfer colitis, house dust mite (HDM), cockroach, or Alternaria alternata-induced asthma, ovalbumin, Papain-induced asthma, papain-induced lung inflammation, skin sensitization atopic dermatitis model (ovalbumin, house dust mite (HDM), hapten, Staphylococcus aureus), immunodeficient or human These include xenograft or patient-derived xenograft tumor growth models using immunized mice, transplantation of syngeneic tumors, chemically induced skin tumor models, and DSS-induced rectal tumor models. Not limited to.

[0153] Preferably, the antibody is used in a monosodium urate crystal (MSU)-induced mouse peritonitis model. can be. [Example]

[0154] The following examples are used in conjunction with the figures and tables to illustrate the present invention.

[0155] Example 1: Human-IL-1R3 biochemical ELISA Assay principle: NUNC Maxisorp 384-well microtiter plates were coated with Fc-tagged human IL-1R3. After the blocking process, specific antibodies from the B cell supernatant bind to the antigen, and It is then detected with a peroxidase-conjugated antibody. Samples are tested at a 1:2 dilution. material: Plate: 384-well NUNC Maxisorp Plate; Catalog Number 464718 Protein: Fc-tagged human IL-1R3 (concentration 1.5 mg / ml; assay concentration 0.5 μg / ml) Standard Ab: P013-02 (concentration 1mg / ml; starting assay concentration 2µg / ml) Detection Ab: Anti-rabbit IgG, peroxidase-conjugated species-specific whole antibody (donkey origin) (ECL); GE; Cat. No. NA9340; Assay dilution: 1:5000 PBS: Boxed buffer, premixed PBS buffer, 10x; Roche Applied Science Catalog Number 11666789001 BSA: Bovine serum albumin fraction V derived from bovine serum; Roche Applied Science; Catalog no. No. 10735086001 Tween 20: Tween 20; Carl Roth; Catalog Number 9127.2 TMB: TMB solution; Life Technologies; Catalog No. SB02 HCl: 1M Titripur Hydrochloric Acid; Merck; Cat. No. 1090571000 ELISA buffer: PBS, 0.5% BSA, 0.05% Tween Washing buffer: PBS, 0.1% Tween Blocking buffer: PBS, 2% BSA, 0.05% Tween procedure: 1. Add 12.5 μL of Fc-tagged human IL-1R3 (0.5 μg / ml) in PBS to a 384-well NUNC Maxisorp plate and incubate at room temperature for 1 hour. 2. Wash three times with 90 μL of wash buffer. 3. Add 90 μL of blocking buffer to each well and incubate at room temperature for 1 hour. do. 4. Wash three times with washing buffer. 5. Add 12.5 μL of antibody-containing Elisa buffer and incubate at room temperature for 1 hour. 6. Wash three times with wash buffer. 7. Add 12.5 μL of ELISA buffer containing 1:5000 POD antibody and incubate at room temperature for 1 hour. To do. 8. Wash six times with wash buffer. 9. Add 15 μL TMB. 10. After sufficient progress, add 15 μL HCl. 11. Read absorbance at 450nm / 620nm.

[0156] Example 2: hIL-1R3 receptor-inhibiting hIL-1R3 specificity in luciferase reporter experiments Characterization of specific antibodies Assay principle: 293T / 17-FR cells expressing NF-kB-RE firefly luciferase reporter were transfected with Poly-D-Lysin- After stimulation with IL-1b, 293T / 17-FR lysates were tested for activated NF-kB using the Steady-Glo Luciferase Assay Kit. The antibody binds to hIL-1R3 and inhibits NF-kB activity, which is indicated by a low signal. material: Plates: Cell Plates: 384-well PDL Costar cell culture plates; Catalog No. 3844 Assay Plate: 384-well lumitrac white plate; Corning; Catalog Number 3572; Cells: 293T / 17-FR; Assay concentration: 250,000 cells / ml Protein: IL-1b (concentration 0.03 mg / ml; assay concentration 115 pg / ml; working concentration 230 pg / ml) Standard Ab: P013_06 (concentration 0.2 mg / ml; starting working concentration 6 μg / ml) Kit: Steady-Glo Luciferase Assay System; Promega; Catalog No. E2510 Cell culture medium: DMEM medium; PAN Biotech; Catalog number P04-04510 FCS: Fetal calf serum, HyClone; Thermo Scientific; Cat. No. St30070.03 293T / 17-FR medium: DMEM medium, 10%FCS, (+20μg / ml hygromycin-B) procedure: 1. Cell culture procedure: Use trypsin / EDTA (incubate for 30 seconds at room temperature) every Monday (seeding: 5x10 6 cells / T175 flask) and Friday (seeding: 3x10 6 Cells / T175 flask) to confluency Split 293T / 17-FR cells. 2. In a 384-well PDL plate (Corning cat#3844), add 0.25x10 cells in 25µl DMEM + 10% FCS. 6 cells / ml) and incubate overnight at 37°C, 5% CO2. 3. Aspirate the medium and add 12.5 μL of antibody-containing conditioned medium or simply conditioned medium. Incubate at 37°C, 5% CO for 30 minutes (program: 3 aspirations and sample transfers). 4. Add 12.5 μl of DMEM + 10% FCS containing IL-1b and incubate for 5 hours at 37°C, 5% CO2. 5. Equilibrate the cultured cells at room temperature for 10 minutes. 6. Add 25 μL of Steady-Glo reagent and mix several times with a pipette. 7. Leave for 5 minutes and then transfer 45 μL of the supernatant to a 384-well lumitrac white plate (Corning Cat# 3572 ) to 8. Measure luminescence with a Tecan Reader: Integration time: 0.5 seconds

[0157] Example 3: Inhibition of NFκB expression in A549-NFκB-RE-Luc stably transfected cells after IL-1(α / β) stimulation Assay principle: A549-NFκB-RE-Luc stable transfected cells (Signosis) are pipetted into a 384-well plate and incubated overnight. On day 2, anti-IL1R3 antibody is bound to the A549-NFκB-RE-Luc stable transfected cells and stimulated by the addition of IL-1 (α or β). This results in transcription of the luciferase gene by activation of the NFκB signaling pathway and cell lysis. It can be measured by adding luciferin to the solution. The antibodies are tested to see if they can inhibit activation of the NFkB pathway and consequently reduce the luminescent signal. material: Plate: 384-well low flange white flat bottom polystyrene TC treated microplate, sterilized Corning Catalog Number 3570 Protein: IL-1α (P026_09); recombinant human IL-1 alpha / IL-1F1; 10 μg / mL; R&D Systems; Catalog No. 200-LA-002 IL-1β (P026_10); recombinant human IL-1 beta / IL-1F2; 25 μg / mL; R&D Systems; Catalog No. 200-LB-005 Standard Ab: MAB-15-0115; MAB Discovery GmbH; 2.51 mg / ml; working concentration 10 μg / ml Cells: A549-NFκB-RE-Luc stably transfected cells; Signosis; Catalog No. SL-0014 Culture medium: DMEM; PAN; Catalog number P04-04510 FCS: Fetal Bovine Serum South Africa Low IgG; PAN; Catalog Number 1552-P120909 Penicillin / Streptomycin: 10,000 U penicillin / ml; 10 mg streptomycin / ml; PAN Biotech; Catalog No. P06-07100 Detachment agent: Trypsin-EDTA 1x; PAN; Catalog No. P10-023100 (4 mL for T175 / 2 mL for T75; ~8 min 37°C) Cell culture medium: DMEM, 10% FCS, 1% penicillin / streptomycin Detection kit: Steady-Glo TM Luciferase Assay System; Promega; Catalog No. E2510 procedure: 1. Culture A549-NFκB-RE-Luc stably transfected cells in cell culture medium (1.7E+04 cells / cm for 3 days) 2 2.28E+04 cells / cm in 2 days 2 ) should not be cultured for more than 10 passages. 2. Inoculate 40,000 A549-NFκB-RE-Luc stable transfected cells in 25 µL of medium per well (concentration = 1.6 x 10 6 cells / mL) into white cell culture flat-bottom 384-well plates. Incubate overnight at 37°C / 5% CO2. 3. Aspirate the medium from the plate and use the CyBio pipetting robot to dispense 10 μL of sample. Add the pull or standard in medium to the plate (program: "Medium removal and sample transfer" in folder P026 / NFκB). Incubate for 1 hour at 37°C / 5% CO2. 4. Using the CyBio pipetting robot, dispense 10 μL of medium containing IL-1 (α or β) into the plate. Add the NFκB to the solution (Program: "Translocation from reservoir" in folder P026 / NFκB) (working concentration: 0.2 ng / mL; assay concentration: 0.1 ng / mL) and incubate for 5 hours at 37°C / 5% CO2. Before performing step 4, dissolve the Steady-Glo substrate in Steady-Glo buffer according to the Steady-Glo protocol and allow this solution and the assay plate to equilibrate to room temperature. 5. Add 20 μL of Steady-Glo mix and mix thoroughly to ensure proper cell lysis. Incubate at room temperature for 10 minutes. 6. Using a microplate reader set to an integration time of 500 ms, measure the relative Determine the luminescence units (program: Lumineszenz-384).

[0158] Example 4: Cell Binding Assay A549 and NIH-3T3 cells were cultured in DMEM + 10% FCS. HEK-293 cells were cultured in DMEM + 15% FCS, and SK-MEL-30 cells were cultured in RPMI + 10% FBS. Cells were harvested using Accumax (Sigma), washed with PBS, and resuspended in staining buffer (BD Pharmingen). Anti-IL-1R3 antibody was used for staining. For EC50 SK-MEL-30 cell binding assays, cells were incubated with the dye at a concentration of 10 μg / ml starting at 20 μg / ml. The cells were washed with staining buffer and incubated with Alexa-488-conjugated goat anti-human secondary antibody (Dianova) for 30 minutes at 4°C. The cells were washed with staining buffer and resuspended in buffer containing 1:100 diluted DRAQ7 (Abcam) dead cell stain. The BD Accuri C6 Sampler flow cytometer was used. Cells were analyzed using a cytometer. Fit curves and EC50 calculations were performed using Excel (Microsoft) and XLfit (IDBS).

[0159] Example 5: Biochemical human IL-1R3 ELISA Nunc 384-well Maxisorp plates were incubated with 0.25µg / ml of ATP in PBS for 60 minutes at room temperature. Plates were coated with recombinant Fc-tagged hIL-1R3 (Ser21-Glu359). The cells were washed three times with PBS 0.1% Tween and then blotted with PBS, 0.2% BSA, 0.05% Tween at room temperature for 60 minutes. After washing three times with wash buffer, antibodies were added in ELISA buffer (PBS, 0.5% BSA, 0.05% Tween) at concentrations ranging from 6 to 0.03 μg / ml (1:3 dilution series) and incubated at room temperature for 60 minutes. The plate was washed three times with washing buffer and then peroxidase-conjugated Anti-human IgG species-specific F(ab)2 fragments (goat, AbD Serotec) were diluted 1:5000 in ELISA buffer and incubated for 60 min at room temperature. The plate was washed six times with wash buffer before adding TMB substrate solution (Invitrogen, 15 μl / well). After a 5 min incubation, stop solution (1 M HCl, 15 μl / well) was added and the plate was read using a Tecan M1000 plate reader. The absorbance (450 nm / 620 nm) was measured. The fitting curve and EC50 calculation were performed using Excel (Microsoft ) and XLfit (IDBS).

[0160] Example 6: IL-1α and IL-1β functional neutralization assay A-549-NFκB-RE-Luc (Signosis) was cultured in DMEM, 10% FCS, and 1% Pen / Strep for 5 days, and then 25 μL of the culture medium was placed in a 384-well white, flat-bottom polystyrene tissue culture-treated microplate (Corning). Plates were seeded at a cell density of 40,000 cells / well in medium. Cells were incubated overnight at 37°C / 5% CO2. The medium was removed by aspiration, and monoclonal or polyclonal (goat anti-human IL-1R3, AF676, R&D Systems) antibodies were added at various concentrations to 10 μL of medium and incubated for 60 minutes at 37°C / 5% CO2. Recombinant human IL-1α or IL-1β (R&D Systems) proteins were added to 10 μL of medium at a final concentration of 0.1 ng / ml, and the plates were incubated for 5 hours at 37°C / 5% CO2. 20 μL Steady-Glo TM (Promega) solution was added to each well, mixed thoroughly, and The plates were incubated at room temperature for 10 minutes and then developed using a Tecan M1000 plate reader. The light was measured. The fitting curve and EC50 calculation were performed using Excel (Microsoft) and XLfit (IDBS). I used it.

[0161] Example 7: IL-1α and IL-1β functional neutralization assay - A-549 IL6-release assay A549 cells were seeded in 384-well clear cell culture-treated plates (Corning) at a density of 6,000 cells / well in 25 μl of DMEM, 10% FCS, 1% Pen / Strep medium. Cells were incubated overnight at 37°C / 5% CO2. The medium was removed by aspiration, and monoclonal or polyclonal (goat anti-human IL-1R3, AF676, R&D Systems) antibodies were added at various concentrations to 12.5 μl of medium and incubated for 3 hours at 37°C / 5% CO2. Recombinant human IL-1α or IL-1β (R&D Systems) proteins were added to 12.5 μl of medium at a final concentration of 0.1 ng / ml, and the plates were incubated for 48 hours at 37°C / 5% CO2. Secreted human IL-6 levels in the cell supernatants were measured according to the manufacturer's instructions. The IL-6 activity was measured using the DuoSet Human IL-6 ELISA Kit (R&D Systems, Cat. No. DY206-05) according to the method described above. Fit curves and EC50 calculations were performed using Excel (Microsoft) and XLfit (IDBS). I used it.

[0162] Example 8: IL-33 functional neutralization assay HEK-Blue TM IL-33 cells (InvivoGen) were cultured in DMEM, 10% FCS for 5 days, and then 15 μL of medium was added. Cells were seeded at a density of 25,000 cells / well in 384-well clear, flat-bottom, tissue culture-treated microplates (Corning) at 100°C. Various concentrations of monoclonal or polyclonal (goat anti-human leukocyte antigen) antibodies were used. Anti-IL-1R3 (AF676, R&D Systems) antibody was added to 5 μL of medium, and the plate was incubated at 37°C / 5% CO for 60 minutes. Recombinant human IL-33 (R&D Systems) protein was added to 5 μL of medium. to a final concentration of 5 ng / ml and the plate was incubated overnight at 37°C / 5% CO2. The cell supernatant was transferred to a clear, flat-bottom polystyrene tube containing 20 μL of 2x QUANTI-Blue reagent (InvivoGen). NBS TM The plates were incubated at 37°C for 45 minutes and the optical density was measured at 655 nm using a Tecan M1000 plate reader. Fit curves and EC50 calculations were performed using Excel (Microsoft) and XLfit (IDBS).

[0163] Example 9: IL-36 functional neutralization assay HEK293 / 17-IF cells (MAB Discovery GmbH) were cultured in DMEM, 10% FCS, and 20 μg / ml hygromycin. After 5 days of culture in PBS, cells were seeded into 384-well white flat-bottom, cell culture-treated plates (Corning) at a cell density of 30,000 cells / well in 20 μL of medium. The cells were incubated overnight at 37°C / 5% CO2. The medium was removed by aspiration, and various concentrations of monoclonal or polyclonal (goat anti-human IL-1R3, AF676, R&D Systems) antibodies were added in 10 μL of medium. The plates were incubated for 60 minutes at 37°C / 5% CO2. Recombinant human IL-36g (R&D Systems) protein was added. Protein was added to 10 μl of medium to a final concentration of 15 ng / ml, and the plates were incubated at 37°C / 5% CO2 for 5 hours. TM (Promega) solution to each well and mix thoroughly. The plate was incubated at room temperature for 10 minutes and then read using a Tecan M1000 plate reader. Luminescence was read using a Fluorescence Indicator. Fit curves and EC50 calculations were performed using Excel (Microsoft) and XLfit (IDBS).

[0164] Example 10: Neutralization of IL-1α, IL-33, and IL-36α The function of the anti-IL-1R3 antibody was examined in three different cells containing either IL-1α, IL-33, or IL-36α. The cells were tested to determine their effects on signaling pathways involving three IL-1 receptors (IL-1R1, -R4, or -R6) that depend on IL-1R3 for signaling. The human epithelial lung cell line A549 was stimulated with IL-1α as a model for IL-1-dependent diseases, such as autoinflammatory diseases. The cell line was cultured in a T75 flask (37°C) in complete F-12K medium (10% FCS, 1% Pen / Strep). Cells were cultured at 4°C (27°F, 5% CO2) and split an average of twice per week, not exceeding 15 passages before assay. A549 cells were seeded (50,000 cells / well) into 96-well flat-bottom plates and incubated for 3 hours before preincubation with MAB-16-0030 (20 μg / mL–1 μg / mL) or IL-1Ra (10 μg / mL) for 1 hour. Cells were then stimulated with recombinant human IL-1α (50 pg / mL, Peprotech) for 24 hours, after which supernatants were collected and assayed for IL-6 production (Duoset ELISA, RnD Systems). A human mast cell line (HMC-1) was investigated for IL-33-dependent induction of IL-8 production. Cell lines were cultured in complete Iscove's modified Dulbecco's medium (IMDM, 10% FCS, 1% Pen / Strep) in T75 flasks (37°C, 5% CO2) and split an average of three times per week until the cell density reached 2. * 10 6 The concentration of HMC-1 cells was not allowed to exceed 1 / mL and not allowed to exceed 15 passages before assay. HMC-1 cells were seeded (30,000 / well) in 96-well flat-bottom plates and incubated for 3 hours before preincubation with MAB-16-0030 (20 μg / mL-1 μg / mL) or IL-1Ra (10 μg / mL) for 1 hour. Cells were then stimulated with recombinant human IL-33 (20 ng / mL, RnD Systems) for 24 hours, after which supernatants were collected and assayed for IL-8 production (Duoset ELISA, RnD Systems). did. The effect on IL-36 signaling was investigated using a human keratinocyte cell line (HaCaT). The cell line was cultured in complete DMEM (10% FCS, 1% Pen / Strep) in a T75 flask at 37°C and 5% CO2. HaCaT cells were split on average 3 times per week, no more than 15 passages before each culture. After 3 hours of incubation, cells were preincubated with MAB-16-0030 (20 μg / mL-1 μg / mL) or IL-1Ra (10 μg / mL) for 1 hour. Cells were then stimulated with recombinant human IL-36α (50 ng / mL, RnD Systems) for 24 hours, after which supernatants were harvested and assayed for IL-8 production (Duoset ELISA, RnD Systems).

[0165] Example 11: PBMC viability and IL-6 release The effect of the anti-hIL-1R3 antibody MAB-16-0030 on the viability of unstimulated PBMCs (500,000 / well) from three healthy donors was tested using a conventional MTT reduction assay. Briefly, PBMCs (200 μL) were incubated with medium alone or MAB-16-0030 (20 μg / mL). After 1, 3, and 5 days, PBMCs were incubated with MTT (20 μL) for 2 hours, after which the absorbance at 570 nM was measured in an ELISA reader. Using the known linearity between absorbance and viable cell converting MTT, the number of viable cells was calculated using medium alone as a control, which was set at 100%. The supernatants of PBMCs incubated under the same conditions and from the same donors on the same day of MTT analysis were were harvested and then assayed for IL-6 production (Duoset ELISA, RnD Systems ), any possible stimulatory effects of MAB-16-0030 alone were assessed.

[0166] Example 12: Functional Blocking of PBMCs Freshly isolated PBMCs from healthy donors were used to evaluate the effect of MAB-16-0030 on human cells stimulated with various antigens. For all stimulations, 500,000 PBMCs / well were used. Experiments were performed using 100 μL of medium and stimulated in a total volume of 200 μL. Cells were seeded and incubated in medium for 1 hour before stimulation. Incubation with either MAB-16-0030 (20-0.1 μg / mL) or IL-1Ra (10 μg / mL) was performed. The following stimuli were used: LPS (10 ng / mL, 24 hours, RPMI without FCS), anti-human CD3 / CD28 (1.25 μg / mL; 0.5 μg / mL (eBioscience) for 3 days, RPMI 10% FCS), IL-12 / IL-33 (2 ng / mL; 20 ng / mL (Peprotech; RnD Systems) for 3 days, RPMI 10% FCS), or heat-inactivated Candida albicans. Rubicance (0.5 * 10 6 / mL, 5 days, RPMI 10% FCS). After stimulation, supernatants were collected and assayed for cytokine production using Duoset ELISA (RnD Systems) according to the manufacturer's protocol. .

[0167] Example 13: Functional blocking of immune cells in whole blood Heat-inactivated Candida albicans was used to stimulate whole blood. Freshly collected blood (EDTA tubes) from healthy donors was aliquoted into microcentrifuge tubes (250 μL / tube). The cells were preincubated with medium alone (RPMI, without FCS), MAB-16-0030 (20-0.1 μg / mL), or IL-1Ra (10 μg / mL) for 1 hour, and then incubated with Candida albicans (0.5 μg / mL). * 10 6 / mL), final Stimulation was performed in a volume of 1 mL. After 24 hours of incubation (37°C, 5% CO2), supernatants were collected and assayed for cytokine production by ELISA (Duoset, RnD Systems).

[0168] Example 14: Mixed Lymphocyte Reaction (MLR) PBMCs from healthy, unmatched donors were mixed at a 1:1 ratio (250,000 / donor) and incubated with either medium alone, MAB-16-0030 (20-1 μg / mL), or IL-1Ra (10 μg / mL) for 5 days (RPMI, 10% FCS). Cytokine production was assayed using the Quansys multiplex platform according to the manufacturer's protocol.

[0169] Example 15: NFkB luciferase gene reporter assay NFkB luciferase reporter NIH3T3 cells (Signosis) were plated in white, cell culture-treated, flat-bottom 384-well plates at 20,000 cells (concentration = 0.8x10) in 25 μL DMEM, 10% FCS, 1% Pen / Strep medium per well. 6 Cells were incubated overnight at 37°C / 5% CO2. The medium was aspirated, and 12.5 μL of antibody solution containing MAB-16-0531 was added to the cells at various concentrations. After 1 hour of incubation at 37°C / 5% CO2, 12.5 μL of mouse IL-1β was added to the medium to achieve a final concentration of 100 μg. The concentration was adjusted to 50 pg / ml. Cells were incubated at 37°C / 5% CO2 for 5 hours. 25 μL of Steady-Glo T M (Promega) solution was added to each well, mixed thoroughly, and the plate was incubated at room temperature for 10 minutes before measuring luminescence using a Tecan M1000 plate reader. Fit curves and EC50 calculations were obtained using Excel (Microsoft) and XLfit (IDBS).

[0170] Example 16: NIH-3T3 IL6 release assay NIH 3T3 cells were plated in cell culture-treated, flat-bottom 384-well plates at 12,500 cells per well (concentration = 0.83 x 10) in 15 μL DMEM + 1% FCS medium. 6Cells were incubated at 37°C / 5% CO2 for 2 hours. 10 μL of antibody MAB-16-0531 was added to the cells at various concentrations in the medium. After 1.5 hours of incubation at 37°C / 5% CO2, 25 μL of human IL-1β was added to the medium. The final concentration was 50 pg / ml. Cells were incubated overnight at 37°C / 5% CO2. Secreted mouse IL6 in the culture supernatant was analyzed by ELISA (DuoSet ELISA; R&D Systems; Quantification was performed using Excel (Microsoft) and XLfit (IDBS).

[0171] Example 17: Monosodium urate crystal (MSU)-induced peritonitis model in mice Three mg of MSU (Invitrogen) was injected intraperitoneally (IP) per mouse as a stimulator, and anti-IL-1R3 (MAB-16-0531, 500 μg / mouse) or IL-1Ra (10 mg / kg) was used for inhibition. Saline was used as a control for both stimulation and inhibition. Four mice were used as control mice (saline (inhibition) + saline (stimulation)), MSU These included 8 mice with only saline (saline + MSU), 8 mice with MAB-16-0531 (500 μg / mouse + MSU), and 8 mice with IL-1Ra (10 mg / kg + MSU). MAB-16-0531 and IL-1Ra were injected IP 1 hour before MSU challenge. Mice were placed in a comfortable restroom 6 hours after MSU or saline injection. The animals were sacrificed. Blood was collected in a microcentrifuge tube containing EDTA, and ascites was collected by lavage using 10 mL of ice-cold PBS. Bone marrow cells were isolated, and organs were immediately frozen in liquid nitrogen. The number of cells in the ascites was counted (HESKA HemaTrue).

[0172] Example 18: Neutrophil activity and cytokine production in MSU peritonitis Neutrophils are the most abundant cell type in gout, and their activity is associated with elastase production. The IP fluid and IP cell lysate from Example 17 were analyzed for neutrophil elastase marker levels and MPO levels. IP lysates were prepared with 0.5% TritonX. Cytokines were measured in both the IP fluid and the IP cell lysate. If the amount of cytokines in the IP fluid was low, 8 mL of the fluid was concentrated with a pre-boiled (15 min) dialysis membrane (MWCO 3.5 kDa, Spectra / Por3 dialysis membrane, Spectrumlabs) soaked in polyethylene glycol (MN6-8000, Sigma Aldrich) as a hygroscopic solution at 4°C (final range: 4.7-10.9x concentrated). The initial concentration was divided by the dialysis concentration factor. Spleen and whole blood lysates were assayed for cytokine levels and normalized to the protein concentration of the lysates. Cytokine concentrations were determined using Duoset ELISA (R&D Systems) or multiplex assays (Quansys Biosciences). Protein levels in spleen lysates and IP fluids were measured using a protein assay staining reagent (BioRad) and BSA as a standard, and standard blood phosphatase assay (BioRad). As shown in Figure 20a-c, treatment with the a-mIL1R3 antibody MAB-16-0531 significantly reduced MSU-induced intracellular elastase and elastase in the IP fluid. IL-6, G-CSF, KC, and CCL-2 levels were strongly reduced in the IP fluid of MAB-16-0531 mice. Systemically, MPO and KC were also reduced in spleen lysates, and IL-6 and G-CSF were reduced in plasma (Figure 20d). ).

[0173] Example 19: OVA-induced allergic asthma in vivo model The OVA-induced allergic asthma model is applied as an in vivo model of IL-33 dependence. Wild-type 6-week-old C57BL / 6 male mice (Jackson Laboratories) were immunized with OVA (15 μg / 100 μL, Sigma-Aldrich) mixed 1:1 with Imject Alum adjuvant (100 μL, ThermoFischer). Mice were IP sensitized with MAB. Mice were injected on days 1, 14, and 21. On days 25-28, mice were injected with MAB. Mice were IP-injected with either 16-0531 (500 μg / mouse, MAB Discovery) or mouse IgG2a-LALA control (500 μg / mouse, MAB Discovery, groups "vehicle" and "OVA"). Intratracheal instillation of OVA (50 μg / mouse, 50 μL) was performed 30 minutes after injection under short-term carbon dioxide anesthesia on days 26-28. Bronchoalveolar lavage (BAL) was performed by inserting a catheter into the trachea and lavaging with 3 x 1 ml of lavage fluid. Airway lavage was performed, followed by cell phenotyping using flow cytometry. Flow cytometry using a mouse Fc block (BD Biosciences) was performed using the following monoclonal antibodies (mAbs) against mouse targets: PE-Cy7 CD11c (N418), PerCP-Cy5.5 CD11b (M1 / 70), FITC-Ly6G (1A8-Ly6G) (all from eBioscience); PE-Siglec-F (E250-4440) (from BD Biosciences) (stained in DPBS containing 10 mg / ml BSA and 0.1 mg / ml NaN). FlowJo software (Treestar) was used to analyze the cells on a Canto II flow cytometer. BAL cells were counted by trypan blue exclusion. Following BAL, lungs were inflated with pre-warmed 3% low-melting-point agarose in DPBS, cooled, and then infused overnight in phosphate-buffered saline. The lungs were then transferred to marine. The lungs were embedded in paraffin, sectioned, and stained with H&E and PAS. Histology was assessed using a Leica DM2000 LED microscope and LAS V4.12 software. Figures 21a-b MAB-16-0531 treatment significantly reduced total BAL white blood cell count (WBC), eosinophils, and neutrophils. In Figure 21c, HE staining of lung sections confirms the reduction in pathology with MAB-16-0531 treatment, and PAS staining shows a reduction in the amount of mucus-producing goblet cells in the airways (PAS).

[0174] Example 20: Imiquimod-induced psoriasis in vivo model IL-36 plays a prominent role in skin inflammation, and mutations in IL-36Ra are associated with human pustular psoriasis. Therefore, application of anti-IL1R3 therapy to skin inflammatory diseases may be beneficial in preventing imiquimod-induced psoriasis. An in vivo psoriasis model was used. The backs of WT 12-week-old C57BL / 6 mice (Jackson Laboratories) were shaved and treated with a depilatory cream (Nair). 75 mg of IMQ (Aldara 5% IMQ) or control cream (Vaseline cream) was applied to the back skin (IMQ; IMQ (n = 10) and a-mIL1R3 (n = 9) groups, control cream; vehicle group (n = 3)) and 1 day after treatment. IP injections were given every other day (days 1, 3, and 5) for 1-5 days. The vehicle and IMQ groups received a mouse IgG control (20 mg / kg, MAB Discovery), and the treatment group received MAB-16-0531 (20 mg / kg, MAB Discovery). The ears were treated with a control cream (all 100 mg / kg, MAB Discovery) on the left ear. 5 mg Vaseline cream) and the appropriate irritant cream (vehicle; 5 mg Vaseline cream) on the right ear. IMQ cream, IMQ, and a-mIL1R3 (5 mg IMQ cream) were applied simultaneously. Body weight was monitored daily. Mice were euthanized on day 6 and the left and right ears were examined using a gauge. The thickness of the spleen was measured. Whole blood cell counts were assessed using a HemaTrue analyzer (HESKA). Photographs of the dorsal skin were visually scored (erythema and scaling, The scores ranged from 0 (no response) to 4 (maximally affected). RNA was isolated from skin punch biopsies. RNA was extracted from the lysate and 0.8 μg RNA (NanoDrop) (High Capacity cDNA Reverse Transcription Kit) was used. cDNA was generated from SYBR Green master mix (Applied Biosystems). 50 ng cDNA was used in real-time qPCR reactions using SYBR Green master mix (Applied Biosystems) and 0.1 μM cytokine-specific primers. GAPDH was used as a reference gene, and the ratios were analyzed. Primer efficiencies were calculated using the Pfaffl method. Myeloperoxidase (MPO) levels were measured in freshly obtained skin biopsies by ELISA. MPO levels were normalized to total protein measurements (determined by the Bradford method). Ta. Figures 22 and 23 show that treatment with MAB-16-0531 significantly reduces imiquimod-induced skin inflammation. This was also associated with decreased skin granulocyte infiltration, MPO levels, and IL17F mRNA expression. is doing.

[0175] Example 21: Activation of Fc receptor-mediated effector functions by IgG1 and IgG1-LALA anti-IL1R3 antibodies Humanized anti-IL1R3 antibodies inhibit IgG-1 and IL-1R3 in eliciting Fc-mediated effector cell functions such as ADCC. To test the activity of the IgG1-LALA version, MAB-16-0030 was tested against either the IgG1 or IgG1-LALA antibody. hIL1R3-expressing target cells, SK-MEL-30 cells, were seeded in 384-well tissue culture-treated plates at a density of 2500 cells / well in 25 μl RPMI medium containing 10% FCS. 24 hours after seeding After incubation, 4000 effector cells / well (ADCC Bioassay Effector Cells, Jurkat, Promega Cat. #G701A) were added to RPMI medium containing 4% low IgG-FCS. Antibodies were then added to final concentrations ranging from 10,000 to 0.002 ng / ml, and the plate was incubated at 37°C, 5% CO2 for 6 hours. The activity of NF-kB signaling in luciferase gene reporter Jurkat cells was measured using the manufactured ELISA kit. Measurements were made using a Tecan M1000 microplate reader according to the manufacturer's instructions (Bio-Glo luciferase assay). "Fold of induction" values ​​represent RLU (antibody treated - background) / RLU (no antibody control - background). Fit curves and EC50 calculations were obtained using Excel (Microsoft) and XLfit (IDBS). Figure 24 As shown in Figure 1, only the IgG1 version of MAB-16-0030 inhibited the effector Jurkat reporter cells induced NF-kB signaling, whereas the IgG1-LALA version abolished effector cell activation.

Claims

1. 1. Use of an antibody or antigen-binding fragment thereof that specifically binds to IL-1R3 in the manufacture of a medicament for treating an IL1R3-mediated inflammatory condition and / or disorder in a subject, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable (VH) region that is at least 95% identical to a VH region comprising the amino acid sequence of SEQ ID NO:28 and a light chain variable (VL) region that is at least 95% identical to a VL region comprising the amino acid sequence of SEQ ID NO:174, wherein the antibody or antigen-binding fragment thereof comprises: a) a complementarity determining region comprising CDR-H1, CDR-H2, and CDR-H3, wherein the CDR-H1 region comprises the amino acid sequence of SEQ ID NO: 79, the CDR-H2 region comprises the amino acid sequence of SEQ ID NO: 96, and the CDR-H3 region comprises the amino acid sequence of SEQ ID NO: 113; and b) a complementarity determining region comprising CDR-L1, CDR-L2 and CDR-L3, wherein the CDR-L1 region comprises the amino acid sequence of SEQ ID NO: 130, the CDR-L2 region comprises the amino acid sequence of SEQ ID NO: 147, and the CDR-L3 region comprises the amino acid sequence of SEQ ID NO: 175; Including, use.

2. 2. The use of claim 1, wherein the IL1R3-mediated inflammatory condition and / or disorder is selected from the group consisting of neutrophilic inflammatory disorders, fibrotic disorders, eosinophilic disorders, infectious diseases, and systemic and local inflammatory diseases.

3. 3. The use according to claim 1 or 2, wherein the antibody has reduced or no effector function.

4. The use according to any one of claims 1 to 3, wherein the antibody does not induce depletion of immune cells.

5. The use according to any one of claims 1 to 4, wherein the antibody does not induce ADCC.

6. The antibody comprises at least the amino acid substitutions L234A and L235A in the human IgG1 Fc region, or S228P and L235E in the human IgG4 Fc region, or a corresponding functional group. The use according to any one of claims 1 to 5, comprising a target mutation.

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