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 specifically, the present invention relates to a method 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 the type 1 interleukin 1 receptor (IL1R1) and mediates IL-1 signaling. 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 act as the IL-1β / IL-1R1 / IL-1RAcP complex to regulate NFκB. 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. It forms a complex (IL-36 / IL-36R / IL-1RAcP) that 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 heavily truncated cytoplasmic domain and does not signal the cell upon binding to IL-1, but instead is involved in regulating IL-1-mediated responses by acting as a decoy receptor.

[0005] The production of IL-1 is triggered by infections, microbial toxins, inflammatory agents, and allergic responses. The overall main 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. If excess IL-1 is produced or expression of IL-1 is not properly regulated, disease states can develop. Thus, IL-1 is implicated in a variety of inflammatory and immunomodulatory diseases and conditions. It has been proposed that a 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-1 alpha and IL-1 beta 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-1R, have certain drawbacks that prevent their use as therapeutic agents. 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 unnecessary 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 with respect to blocking inflammatory signaling pathways. Summary of the Invention

[0008] Thus, the present invention provides a method for the treatment of 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 description of the drawings]

[0009] [Figure 1-1]Sequences (single letter amino acid code) Complete sequences of the 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 diagram, 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 (single letter amino acid code) Complete sequences of the 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 diagram, 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 (single letter amino acid code) Complete sequences of the 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 diagram, 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 (single letter amino acid code) Complete sequences of the 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 diagram, 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 (single letter amino acid code) Complete sequences of the 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 diagram, 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 (single letter amino acid code) Complete sequences of the 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 diagram, 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 (single letter amino acid code) Complete sequences of the 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 diagram, 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 (single letter amino acid code) Complete sequences of the 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 diagram, 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. [Diagram 2]Human IL-1R3 ELISA 384-microtiter plates were coated with human IL-1R3 protein (0.5 mg / ml, at least 1 h), which represents the human extracellular domain of IL-1R3. After extensive washing steps followed by a blocking step, the antibody was added (12.5 μl per well) and incubated for 1 h at room temperature. Unbound antibody was washed out extensively. The amount of bound antibody was determined by incubating the microtiter plate with a peroxidase-labeled anti-human detection antibody (1 h at room temperature). The peroxidase reaction was started by adding TMB and the absorbance was measured at 450 nm / 620 nm. [Diagram 3] HEK293 reporter assay HEK293T / 17-FR cells were stably transfected with pGL4.32[luc2P / NF-κB-RE / Hygro] vector (Promega) and seeded in 384-well PDL Costar cell culture plates and then incubated with antibodies for 30 min. Cells were then stimulated with IL-1β for 5 h, after which NF-κB activity was measured using the Steady-Glo Luciferase Assay Kit (Promega) according to the manufacturer's protocol. [Figure 4] NFkB Luciferase Reporter Assay Using 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 antibodies for 1 hour, after which cells were stimulated with 10 μl of IL-1β for an additional 5 hours. NFkB modulation was measured by the Steady-Glo™ Luciferase Assay System (Promega) to determine the relative luminescence units of each well relative to unstimulated cells. [Diagram 5]Cell Binding Analysis: Binding to IL-1R3 Expressing Cells Humanized anti-IL-1R3 IgG1-LALA antibodies were tested for binding to cell lines with different IL-1R3 receptor densities using flow cytometry. Humanized anti-IL-1R3 IgG1-LALA antibodies bind to low and high IL-1R3 expressing cell lines. The antibodies do not bind to mouse NIH-3T3 cells. The experiments were performed according to the methods described in Example 4. [Figure 6] Cell Binding Analysis: Cell Binding to Human IL-1R3 Highly Expressing 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. Humanized anti-IL-1R3 IgG1-LALA antibodies MAB-16-0030 and MAB-16-0149 show 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 The binding of humanized anti-IL-1R3 IgG1-LALA antibodies to recombinant human IL-1R3 protein was tested in a biochemical ELISA. The exemplified antibodies show EC50 binding values ​​of 16.3 ng / ml and 29.1 ng / ml, respectively. The experiment was performed according to the method 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 IL-1a and IL-1b, respectively. The humanized anti-IL-1R3 IgG1-LALA antibody shows a better EC50 value than 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 humanized anti-IL-1R3 IgG1-LALA antibody was tested using A-549 cells. EC50 values ​​show that humanized anti-IL-1R3 IgG1-LALA antibody is superior to 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-IL33TM Cells Neutralization of IL-33-mediated cell signaling by humanized anti-IL-1R3 IgG1-LALA antibody was tested using IL-33 stimulated gene reporter HEK-Blue-IL33TM cells (InvivoGen). EC50 values ​​show that humanized anti-IL-1R3 IgG1-LALA antibody is superior to 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. Exemplary humanized anti-IL-1R3 IgG1-LALA antibodies show better EC50 values ​​than 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 antibodies Binding of antibodies to immune cells can result in cell depletion and deleterious effects, for example, by direct induction of apoptotic signaling pathways, stimulation of excessive cytokine release, or antibody-dependent cellular cytotoxicity (ADCC). To exclude that humanized anti-IL-1R3 IgG1-LALA antibodies directly affect PBMC viability, the viability and IL-6 release of PBMCs from three donors were investigated 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 humanized anti-IL-1R3 IgG1-LALA antibodies block IL-1R3 function of immune cells without inducing cell depletion and deleterious effects in a contextual manner. The experiments were performed according to the methods described in Example 11. [Figure 13-2]Viability and IL-6 release of unstimulated PBMCs treated with humanized anti-IL-1R3 IgG1-LALA antibodies Binding of antibodies to immune cells can result in cell depletion and deleterious effects, for example, by direct induction of apoptotic signaling pathways, stimulation of excessive cytokine release, or antibody-dependent cellular cytotoxicity (ADCC). To exclude that humanized anti-IL-1R3 IgG1-LALA antibodies directly affect PBMC viability, the viability and IL-6 release of PBMCs from three donors were investigated 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 humanized anti-IL-1R3 IgG1-LALA antibodies block IL-1R3 function of immune cells without inducing cell depletion and deleterious effects in a contextual manner. The experiments were performed according to the methods 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 by 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 of the invention (MAB-16-0030) 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 by 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 of the invention (MAB-16-0030) 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 Block of Candida albicans-Activated Immune Cells in Whole Blood To test whether the humanized anti-IL-1R3 IgG1-LALA antibody inhibits 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 IFNg, IL-6, TNF-a, 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 IFNg, IL-6, TNF-a, 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 IFNg, IL-6, TNF-a, 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 invention was tested using mouse NIH-3T3 cells based on NFkB luciferase gene reporter assay. A representative antibody according to the 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-mouse IL-1R3 antibodies according to the invention was tested on 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 of applying anti-IL-R3 therapy in 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 and induces an influx of several inflammatory cells (such as monocytes and neutrophils), mimicking the classical inflammatory profile of an acute gouty response. A representative anti-IL1R3 antibody (MAB-16-0531) according to the present invention or IL-1Ra was administered IP 1 hour before injecting 3 mg MSU into the peritoneum. Measurement of cells in the peritoneal cavity shows that anti-IL-1R3 antibody treatment significantly inhibits MSU-induced infiltration of lymphocytes, monocytes and granulocytes. The experiment was carried out 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 groups compared to MSU-stimulated groups. *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 cell influx in vivo. (a) Total WBC from BAL fluid. (b) Flow-defined cells different from (a). (c) Histology of mice with thick dots marked group mean (WBC) using H&E and PAS stained lung tissue. Compared to 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 cell influx in vivo. (a) Total WBC from BAL fluid. (b) Flow-defined cells different from (a). (c) Histology of mice with thick dots marked group mean (WBC) using H&E and PAS stained lung tissue. Compared to 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 applied. (c) Blind visual scoring of n=6 at end of 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 applied. (c) Blind visual scoring of n=6 at end of 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 an in vivo model: IL36α / β / γ, IL17F and TNFα mRNA levels in skin biopsies of imiquimod-treated mice. [Figure 23-2] Imiquimod psoriasis in an 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. Anti-IL1R3 antibody MAB-16-0030, containing either IgG1-LALA or IgG1 Fc portions, was tested for activity of Fc receptor-mediated NF-kB signaling in effector cells. Binding of increasing concentrations of IgG1 antibody to hIL1R3-expressing SK-MEL-30 cells induces Fc receptor-mediated signaling in Jurkat gene reporter effector cells, in contrast to IgG1-LALA antibody. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The present invention relates to IL-1α, IL-1β, IL-33, IL-36, IL1RA and / or IL1R3, as well as their barriers. The disease is 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 indicated.

[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, as well as 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. By it is meant animals, which includes the families (hares and rabbits) and the family 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 an antibody that is further modified, preferably a humanized antibody, a chimeric antibody, a fragment thereof, or an antibody that is further genetically engineered and recombinantly produced, 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] A "natural antibody" is a glycoprotein that is usually heterotetrameric, 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. It is believed that certain amino acid residues 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 means 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 does not take into account any conservative substitutions as part of the sequence identity. 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" has the same meaning as 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 IgG antibodies and is a class of receptors that are classified into three subclasses: FcγRI, FcγRII, and FcγRIII. Includes receptors including gene variants and alternative splice forms. FcγRII receptor The body contains FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. 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 (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 may result, for example, from the binding of an Fc effector domain to an Fc receptor on a phagocyte or an immune cell with lytic activity, or from the binding of an Fc effector domain 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 antigen to which the intact antibody binds. 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 competitive assay, or conversely, blocks the binding of the antibody to its antigen by 50% or more in a competitive assay. Exemplary competitive assays are provided herein.

[0024] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" are non-specific cell killing that expresses FcR. It 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 only FcγRIII, 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 forms the complex C1, the first component of the complement-dependent cytotoxicity (CDC) pathway, together with two serine proteases, C1r and C1s. 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 region carried by the heavy chain. There are five major antibody classes: IgA, IgD, IgE, IgG, and IgM, of which several The subclass (isotype) of the antibody, e.g., IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgA 1 and IgA 2 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 the desired therapeutic or prophylactic result.

[0029] The term "Fc region" refers to the C-terminal end of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term Fc 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" comprises at least one "amino acid modification" as defined herein. The term "Fc variant" as used herein refers to a polypeptide comprising a modification in the Fc domain. The Fc variants of the present invention are defined by the amino acid modifications that compose them. Thus, for example, P329G is an Fc variant with a substitution of proline with glycine at position 329 compared 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 EU antibody numbering. (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 additions, deletions or substitutions that are not naturally occurring. Variants may include naturally occurring and non-naturally occurring amino acids. Variants may include non-naturally occurring amino acids. Examples include U.S. Pat. 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, all of which are incorporated by reference.

[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. 111-234.) 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 entirely). (Incorporated by reference herein). 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 that binds to the Fc region of the host to form an Fc / Fc ligand complex, preferably Or it means a polypeptide.

[0034] As used herein, "Fc gamma receptor," "FcγR," or "Fc gamma R" refers to an IgG antibody Fc domain. 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 FcγR includes, but is not limited to, FcγRIII (CD16), including FcγRIIB-NA1 and FcγRIIB-NA2 (Jefferis et al., Immunol Lett 82 (2002) 57-65, entirely incorporated by reference), as well as any human FcγR or FcγR isoform or allotype yet to be discovered. The mouse FcγR may be derived from any organism, including, but not limited to, human, mouse, rat, rabbit, and monkey. Mouse FcγR includes, but is 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 that is at least partially encoded by a gene. FcRn can be from any organism, including but not limited to human, mouse, rat, rabbit, and monkey. As known in the art, a functional FcRn protein contains two polypeptides, often referred to as heavy and light chains. 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 the complex of beta-2-microglobulin and FcRn heavy chain.

[0036] By "immunoconjugate" is meant 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 portion 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 an antibody 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 of the modified strand is that which, 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 per se.

[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 the respective antigen, adding the antibody under conditions allowing the formation of an immune complex with the respective antigen or protein, and detecting the immune complex by measuring the optical density value (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 that contains 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, whereas for detection / assay 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] An "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 invention, for example, do not significantly cross-react with other epitopes on 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 herein refer to human proteins. Where a protein from another species is meant, this is explicitly 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 promotes thymic regulation by inducing IL-2 release, B cell maturation and proliferation, and fibroblast growth factor activity. Stimulates cell proliferation. The IL-1 protein is involved in inflammatory responses 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 that, sequentially activates the NF-kappaB 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 IL-36 receptor that binds to and, through it, mediates the expression of IL1RL2 / IL-36R in target cells involved in the proinflammatory response. The 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 kinase. It consists of a p100 subunit (P19838) and a p100 subunit (Q00653).

[0050] "Inhibition of NFkB" is measured by the present invention as 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, it is explicitly mentioned.

[0051] As used herein, the term "variable region (or domain) of an antibody according to the invention" (light chain variable region (VL), 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 the antibody to an antigen. The variable light and heavy chain regions have the same overall structure, and each region consists of four framework domains whose sequences are largely conserved and which are connected by three complementarity determining regions, CDRs. The antibodies according to the invention comprise a VH region and a VL region or portions thereof, which together are sufficient for specific binding to a respective antigen.

[0052] The term "antigen-binding portion of an antibody" as used herein refers to the amino acid residues of an antibody that are involved in antigen binding. An 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 set forth in the following IEEE International Conference on Molecular Biology, 1999, pp. 111-114, 2003. Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence may be determined by truncating or subtracting the FR or CDR of the variable region. 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 a "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, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, 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 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, neurinoma, upper lip ... lower lipoma, upper lipoma, lower lipoma, lower lipoma, upper lipoma, lower lipoma, lower lipoma, lower lipoma, lower lipoma, lower lipoma, lower lipoma, lower lipoma, lower lipoma, lower lipoma, lower lipoma, lower lipoma, lower lipoma, lower lipoma, lower lipoma, lower lipoma, lower lipoma, lower lipoma, lower lip The cancer may be idiopathic, including idiopathic leukemia, 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 to providing an adequate method for treating inflammatory conditions 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 localized inflammatory diseases.

[0056] The present invention relates to a method comprising administering to a subject a therapeutically effective amount of an antagonist IL1R3 antibody. and a method for treating, inhibiting or ameliorating an inflammatory condition and / or disorder in a subject in need thereof. Regarding.

[0057] In particular, the present invention encompasses anti-IL1R3 antibodies for use in treating an IL1R3-mediated inflammatory condition and / or disorder in a subject. Such inflammatory conditions and / or disorders include inflammatory conditions, immune disorders, fibrotic disorders, eosinophilic disorders, and the like. The inflammation 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, including Schnitzler's syndrome, Behcet'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 pancreatic fibrosis, and inflammatory bowel disease. Histiocytosis, Erdheim-Chester / Histiocytosis, SAPHO, PFAPA, Multicentric Castleman The primary inflammatory disease may be systemic and local, such as chronic granulomatous disease, Jessner-Kanoff disease, primary Sjögren's fatigue syndrome, Kawasaki disease, chronic granulomatous colitis, sweat gland abscess, autoimmune inner ear disease, major traumatic brain injury), or genetic systemic inflammatory diseases such as Familial Mediterranean Fever (FMF), CAPS, TRAPSa, HIDS, PAPA, PASH, DIRA, Blau syndrome / granulomatous arthritis, mevalonate kinase deficiency, Majeed syndrome, 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. The metabolic rheumatic disorder may be selected from the group of gout, pseudogout, drug-induced gout and chronic active (refractory) gout.

[0061] For the treatment of said 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. The therapeutic agents 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 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 an anti-IL1R3 antibody for the treatment of an inflammatory condition and / or disorder, wherein the inflammatory condition is a 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 invention is used to treat pancreatic cancer, liver cancer, lung cancer (associated with inflammation due to asbestos, infections, smoking, silica), non-small cell lung cancer, rectal cancer / colon-related cancer (associated with inflammation). The present invention is for use in the treatment of chronic inflammation associated with cancer, in cancers 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 tumor microenvironment cells that secrete inflammatory cytokines such as IL-1α, IL-1β, IL-33, IL-36, etc. In some instances, 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 a method of treating a subject, wherein the subject is suffering from one or more cytotoxic These tumors are characterized by resistance or inadequate response to treatment with cytostatic, cytostatic, or targeted anticancer agents.

[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 anticancer drugs.

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

[0074] In the latter case, the antibody may be coupled to one or more cytotoxic, cytostatic or targeted anti-cancer agents. It is preferably 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 analogues, peptide antibiotics, and platinum-based agents.

[0076] Preferably, the targeted anti-cancer drug 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] It is further preferred that 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 therapeutic candidates may be, but are not limited to, anti-PD-L1 compounds such as rifampicin, anti-PDL1 compounds such as 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 is done by some compounds of the prior art), but through inhibition of cancer-associated inflammation by modulating the IL1R3 signaling pathway.

[0079] The antibodies of the invention offer a highly advantageous activity profile, since they allow for effective inhibition of cancer-associated chronic inflammation, and at the same time do not affect the viability of target cells expressing IL-1R3, thus 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 and very frequent (daily) treatment intervals. Antibodies targeting a single cytokine (such as Ilaris) result in unnecessary signaling / activation of other IL-1 family cytokines. Furthermore, the IL-1 family These cytokines are known to induce both proinflammatory 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 proinflammatory signaling pathways. The antibodies described herein are preferably monoclonal antibodies with high affinity, high specificity, and potent neutralizing activity for IL-1R3. Thus, the present invention also encompasses IL-1R3 antibodies with high affinity and specificity for IL-1R3, with 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 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 the corresponding substitutions in another organism. The aim is to include functional mutations that

[0083] In one embodiment of the invention, the anti-IL1R3 antibody comprises a) a heavy chain variable region (VH) comprising the 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 the 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. M, 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 comprises a) a heavy chain variable region (VH) comprising the 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 a complementarity determining region 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 is 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 the 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 the 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 is selected from the group consisting of the VH regions 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 the VL regions 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.

[0089] In another embodiment, the subject is a mouse and the antibody has the heavy chain variable (VH) 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 that comprises 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 VH region 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, and more preferably at least 90% identical to a VH region of the VH region of the

[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 sequence identity to:

[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 which is an antibody of the present invention which 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, and which comprises a light chain variable (VL) region which 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: 35-68, 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. The VL may include

[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 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 in the CDR The present invention also includes affinity matured antibodies, which may be produced by methods known to those skilled in the art. Marks et al., Bio / Technology 10:779-783 (1992) describes describe affinity maturation by VH and VL domain shuffling. or random mutagenesis of framework residues as 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 a specific embodiment, a total of 1 to 10 amino acids are substituted 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 a serine. This substitution is preferably at position 90 in the light chain variable region (VL). In a preferred embodiment, the cysteine ​​at position 90 of SEQ ID NO:62 is replaced with a serine. However, the antibodies of the invention may also include a substitution at position 90 of the amino acid at position 90 of SEQ ID NO:62. 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. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside 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 said antibody comprising at least a portion of said antibody sufficient to confer specific binding to IL-1R3, 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) is also included.

[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, 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 consisting 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 that comprises at least a portion of said antibody sufficient to confer specific binding to IL-1R3, is also disclosed herein as 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, the 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. The antibody according to the invention also comprises SEQ ID NO: 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 the sequence 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 NO: 11 and 45, or SEQ ID NO: 12 and 46. Alternatively, the antibody according to the invention comprises SEQ ID NO: 13 and 47, or SEQ ID NO: 14 and 48, or SEQ ID NO: 15 and 49, or SEQ ID NO: 16 and 50, SEQ ID NO: 17 and 51, or SEQ ID NO: 18 and 52, or SEQ ID NO: 19 and 53, or SEQ ID NO: 20 and 54, or SEQ ID NO: 21 and 55, or SEQ ID NO: 22 and 56, or SEQ ID NO: 23 and 57, or SEQ ID NO: 24 and 58, or SEQ ID NO: 25 and 59, or SEQ ID NO: 26 and 60, or SEQ ID NO: 27 and 61.

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

[0106] Most preferably, the antibody of the invention that specifically binds to IL-1R3, or a fragment or derivative of said antibody comprising at least a portion of said antibody sufficient to confer IL-1R3 specific binding, comprises 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 to 34, 173, and 176, and a VL region selected from the group of SEQ ID NOs: 35 to 68, 174, and 177. Includes the area.

[0107] Antibodies of the invention that specifically bind to IL-1R3, or fragments or derivatives of said 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 the fragments of 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, 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 a preferred therapeutic application of the antibodies of the invention, the effector function (such as ADCC) of the antibodies of the invention is 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 the undesired depletion of immune cells.

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

[0110] Therefore, the present invention also relates to a human IgG1 Fc region having at least L234A and L235A, or Contains the amino acid substitutions S228P and L235E of the human IgG4 Fc region, or functionally equivalent mutations Concerning said 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 that comprises at least a portion of said antibody sufficient to confer IL-1R3 specific binding, is any of the 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 invention have the advantage of being very potent with respect to binding to their target. They show a strong binding capacity to the antigen IL1R3, but not to other receptors. This is not the case. The binding characteristics of the 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 show a half maximal effective concentration (EC50) of less than 30 ng / ml, preferably less than 20 ng / ml. In other embodiments, they show an EC50 of less than 15 ng / ml, 10 ng / ml or less than 5 ng / ml. Preferred antibodies according to the invention show an EC50 of 16.3 ng / ml in a biochemical ELISA experiment (see FIG. 7).

[0116] The antibodies according to the invention also showed that human IL1R3 binds to the IL1R3 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 FIG. 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 IL-1 alpha and / or IL-1 beta stimulated NFkB activity. 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, for example, Figure 3.)

[0122] The antibodies of the 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 due to its action on the NFkB receptor (see, for example, FIG. 8).

[0123] The antibodies of the 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, FIG. 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 includes the above-mentioned antibody which inhibits NFkB activity.

[0125] Furthermore, in A549-NFkB-RE-Luc cell lysates, 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 (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 said antibody according to the invention (Steady-Glo™ Luciferase Assay System; Promega; Kaspersky Lab). Catalog number E2510).

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

[0127] Preferably, said IL-1 alpha 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, said IL-1 alpha stimulated luciferase activity is inhibited by 95%.

[0128] Preferably, said IL-1 beta 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, said IL-1 beta 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%.

[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 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 illustrated in FIG. 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-a 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-b 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 the antibodies selected in 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 20000 ng / ml, preferably less than 20000 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 inventors have found that the antibodies according to the 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 of 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 action 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. Thus, simultaneous inhibition of signaling mediated by multiple cytokines exerts effective control of the inflammatory process. Inhibiting multi-cytokine signaling via blocking the IL1R3 receptor This is an important aspect of the antibodies of the present invention.

[0135] Binding of antibodies to immune cells can result in cellular depletion and deleterious effects, for example, by direct induction of apoptotic signaling pathways, stimulation of excessive cytokine release, or by 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 they do not induce the release of IL-6 in PBMCs (see FIG. 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 a donor. The antibodies inhibit cytokine release mediated by any specific or combination of 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] Also, in one embodiment, the anti-IL-1R3 IgG1-LALA antibody according to the invention can inhibit the release of IFNg, IL-6, TNF-a, IL-13, IL-17 and IL-10 in a mixed lymphocyte reaction (see FIG. 16).

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

[0140] As used herein, "pharmaceutical carriers" include 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 appreciated by those of skill 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, it may be necessary to coat the compounds with or co-administer them with a material to prevent inactivation. For example, the compounds may be administered to a subject in a suitable carrier, such as liposomes or a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Pharmaceutical carriers include sterile, injectable, These media and agents include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of the ready-to-use solutions or dispersions. The use of such media and agents for pharma- ceutical 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, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, 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 in the compositions, for example, sugars, sodium chloride, and the like. 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 of the present invention and / or pharmaceutical compositions of the present invention, which may be used in a suitable hydrated form, are formulated into pharma-ceutically 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 the 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 of specific binding 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 of specific binding 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, the antibodies according to the invention exhibit reduced or no Fcγ receptor signaling. It is further preferred that they do not induce ADCC.

[0151] In one embodiment, the antibody of the invention, or a fragment or derivative of said antibody, which 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 invention relates to an antibody of the invention, or a fragment or derivative of said antibody, 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, the serum Transition-induced rheumatoid arthritis, collagen-induced arthritis, antibody-induced arthritis, collagen antibody-induced arthritis, K / BxN antibody transitional 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 COPD, tracheal instillation of elastase COPD, fluorescein isothiocyanate (FITC)-induced lung injury / fibrosis, radiation-induced fibrosis Fibrosis, silica-induced pulmonary fibrosis, asbestos fiber-induced pulmonary fibrosis, DSS-induced colitis, nitrobenzenesulfonic acid-induced colitis, oxazolone-induced colitis, adoptive transfer colitis, house dust mite (HDM), cockroach, or Alternaria alternata-induced asthma, egg white albuginea amine-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 mouse models, 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. EXAMPLES

[0154] The following examples are used in conjunction with the figures and tables to illustrate the 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 by a POD-labeled antibody. Samples are tested at a 1:2 dilution. material: Plates: 384-well NUNC Maxisorp plates; Catalog number 464718 Protein: Fc-tagged human IL-1R3 (concentration 1.5mg / ml; assay concentration 0.5μg / ml) Standard Ab: P013-02 (concentration 1mg / ml; starting assay concentration 2μg / ml) Detection Ab: Anti-rabbit IgG, peroxidase-conjugated species-specific whole antibody (from donkey) (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 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 number SB02 HCl: 1M Titripur Hydrochloric Acid; Merck; Catalog Number 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 wash buffer. 5. Add 12.5 μL of Elisa Buffer containing antibody and incubate for 1 hour at room temperature. 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. 8. Wash six times with wash buffer. 9. Add 15 μL TMB. 10. After sufficient progress is made, add 15 μL HCl. 11. Read absorbance at 450nm / 620nm.

[0156] Example 2: h-IL-1R3 specific inhibitors of the hIL-1R3 receptor by luciferase reporter experiments Characterization of heterologous 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; Cat. No. 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 0.2mg / ml; start working concentration 6μg / ml) Kit: Steady-Glo Luciferase Assay System; Promega; Catalog number E2510 Cell culture medium: DMEM medium; PAN Biotech; Catalog number P04-04510 FCS: Fetal calf serum, HyClone; Thermo; Catalog number St30070.03 293T / 17-FR medium: DMEM medium, 10%FCS, (+20μg / ml hygromycin-B) procedure: 1. Cell culture procedure: Use trypsin / EDTA (incubate at room temperature for 30 seconds only) to culture the cells every Monday (seeding: 5x10 6 cells / T175 flask) and Friday (seeding: 3x10 6 Cells / T175 flask) at confluency Split 293T / 17-FR cells. 2. Add cells (0.25x10) in 25μl DMEM+10% FCS to a 384-well PDL plate (Corning cat#3844). 6 Cells / ml) were seeded and incubated at 37°C, 5% CO 2 Incubate overnight at 4°C. 3. Aspirate the medium and add 12.5 μL of conditioned medium containing antibody or simply conditioned medium. Incubate at 37°C, 5% CO 2 Incubate for 30 min at 4°C (program: 3 aspirations and sample transfers) 4. Add 12.5 μl of DMEM + 10% FCS containing IL-1b and incubate at 37°C, 5% CO 2 5. Equilibrate the cultures 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-NFkB-RE-Luc stable transfected cells (Signosis) are pipetted into 384-well plates and incubated overnight. On the second day, anti-IL1R3 antibody is bound to the A549-NFkB-RE-Luc stable transfected cells and stimulated by addition of IL-1 (α or β). This results in transcription of the luciferase gene by activation of the NFkB signaling pathway and cell lysis. It can be measured by adding luciferin to the solution. It is tested whether the antibodies are able to inhibit activation of the NFkB pathway and, therefore, reduce the luminescent signal. material: Plates: 384-well low flange white flat bottom polystyrene TC treated microplates, sterile ;Corning;Cat. No. 3570 Protein: IL-1α (P026_09); recombinant human IL-1 alpha / IL-1F1; 10 μg / mL; R&D Systems; Catalog number 200-LA-002 IL-1β (P026_10); recombinant human IL-1 beta / IL-1F2; 25 μg / mL; R&D Systems; Catalog number 200-LB-005 Standard Ab: MAB-15-0115; MAB Discovery GmbH; 2.51mg / ml; working concentration 10μg / ml Cells: A549-NFκB-RE-Luc stably transfected cells; Signosis; Catalog number SL-0014 Culture medium: DMEM; PAN; Catalog number P04-04510 FCS: Fetal Calf Serum South Africa Low IgG; PAN; Catalog Number 1552-P120909 Penicillin / Streptomycin: 10,000 U penicillin / ml; 10mg streptomycin / ml; PAN Biotech; Catalog number P06-07100 Detachment agent: Trypsin-EDTA 1x; PAN; Catalog number P10-023100 (4mL for T175 / 2mL 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 number E2510 procedure: 1. Culture A549-NFκB-RE-Luc stable transfected cells in cell medium (1.7E+04 cells / cm for 3 days) 2 ; 2.28E+04 cells / cm in 2 days 2 ) for no more than 10 passages. 2. Plate 40,000 A549-NFκB-RE-Luc stable transfected cells in 25 µL of medium per well (concentration = 1.6x10 6 Plate out 100 μl of 1000 cells / mL into white cell culture flat-bottom 384-well plates. 37℃ / 5%CO 2 Incubate overnight at 4°C. 3. Aspirate the medium from the plate and use the CyBio pipetting robot to pipette 10 µL of the sample into the wells. Add the pull or standard in medium to the plate (program: "Medium removal and sample transfer" in folder P026 / NFκB). Incubate at 37 °C / 5% CO 2 Incubate at 4 °C for 1 hr. 4. Using the CyBio pipetting robot, dispense 10 μL of medium containing IL-1 (α or β) into the plate. The cells were incubated at 37 °C / 5% CO for 1 h (program: "Transfer from reservoir" in folder P026 / NFκB) (working concentration: 0.2 ng / mL; assay concentration: 0.1 ng / mL). 2 Incubate at 4°C for 5 hours. Prior to performing step 4, dissolve Steady-Glo Substrate in Steady-Glo Buffer according to the Steady-Glo protocol and equilibrate this solution and the assay plate 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 concentration of each well. 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 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. The antibody was incubated with cells in staining buffer at a concentration of 10 μg / ml for 30 min at 4° C. For EC50 SK-MEL-30 cell binding assay, cells were stained with ink in a 1:2 dilution series starting at 20 μg / ml. Cells were washed with staining buffer and incubated with Alexa-488-conjugated goat anti-human secondary antibody (Dianova) for 30 min at 4°C. Cells were washed with staining buffer and resuspended in buffer containing 1:100 diluted DRAQ7 (Abcam) dead cell stain. 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 0.25 μg / ml of 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 washing 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 min. 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. Plates were washed six times with wash buffer before adding TMB substrate solution (Invitrogen, 15 μl / well). After 5 min incubation, stop solution (1M HCl, 15 μl / well) was added and the plates were read using a Tecan M1000 plate reader. The absorbance (450 nm / 620 nm) was measured. Fit curves and EC50 calculations were performed using Excel (Microsoft ) and XLfit (IDBS).

[0160] Example 6: IL-1α and IL-1β functional neutralization assays A-549-NFκB-RE-Luc (Signosis) was cultured in DMEM, 10% FCS, 1% Pen / Strep for 5 days, and then 25 μL was added to a 384-well white flat-bottom polystyrene tissue culture-treated microplate (Corning). Cells were seeded at a cell density of 40,000 cells / well in culture medium. Cells were incubated at 37°C / 5% CO 2 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 in 10 μL of medium and incubated at 37°C / 5% CO 2 Recombinant human IL-1α or IL-1β (R&D Systems) protein was added to 10 μl of medium at a final concentration of 0.1 ng / ml and the plates were incubated at 37°C / 5% CO 2 The mixture was incubated for 5 hours in 20 μl Steady-Glo TM (Promega) solution was added to each well, mixed thoroughly, and The plates were incubated at room temperature for 10 min and then developed using a Tecan M1000 plate reader. The light was measured. The fitting curves and EC50 calculations were performed using Excel (Microsoft) and XLfit (IDBS). I used it.

[0161] Example 7: IL-1α and IL-1β functional neutralization assays - 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 medium of DMEM, 10% FCS, 1% Pen / Strep. Cells were incubated at 37°C / 5% CO 2 Overnight 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 in 12.5 μl of medium and incubated at 37°C / 5% CO 2 Recombinant human IL-1α or IL-1β (R&D Systems) protein was added to 12.5 μl of medium at a final concentration of 0.1 ng / ml and the plates were incubated at 37°C / 5% CO 2 The secreted human IL-6 levels in the cell supernatant were assayed according to the manufacturer's instructions. The IL-6 concentration was measured using the DuoSet Human IL-6 ELISA Kit (R&D Systems, Cat. No. DY206-05) by 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 resuspended in 15 μL of medium. 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 for 1 h. Varying concentrations of monoclonal or polyclonal (goat anti-human leukocyte antigen) were used to culture the cells. Add 10 μL of IL-1R3 (AF676, R&D Systems) antibody to a 5 μL medium volume and incubate the plate at 37 °C / 5% CO 2 Recombinant human IL-33 (R&D Systems) protein was added to 5 μL of medium. to a final concentration of 5ng / ml and the plate was incubated at 37°C / 5% CO 2 The mixture was incubated overnight at 4°C for 10 min. The cell supernatant was transferred to a clear flat-bottom polystyrene tube containing 20 μL of 2xQUANTI-Blue reagent (InvivoGen). NBS TM The plates were incubated at 37° C. for 45 min 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, 20 μg / ml hygromycin After 5 days of incubation in 20 μL of medium, cells were seeded into 384-well white flat-bottom, cell culture-treated plates (Corning) at a cell density of 30,000 cells / well. Cells were incubated at 37°C / 5% CO 2 The plates were incubated overnight at 37°C / 5% CO. 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 at 37°C / 5% CO 2 Recombinant human IL-36g (R&D Systems) protein was incubated for 60 min. Proteins were added to 10 μl of medium to a final concentration of 15 ng / ml and plates were incubated at 37°C / 5% CO 2 The plates were incubated for 5 hours in 20 μL of Steady-Glo TM Add (Promega) solution to each well and mix thoroughly. The plate was then incubated at room temperature for 10 minutes and then read using a Tecan M1000 plate reader. Luminescence was read using a 3D CT scanner. 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 expressing either IL-1α, IL-33, or IL-36α. The study was performed in a 5-cell line to determine effects on signaling pathways involving the 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 complete F-12K medium (10% FCS, 1% Pen / Strep) in a T75 flask (37 °C). , 5% CO 2) and split on average twice / week, not exceeding 15 passages before assay. A549 cells were seeded (50.000 / well) in 96-well flat-bottom plates, rested for 3 h, and then pre-incubated with MAB-16-0030 (20 μg / mL-1 μg / mL) or IL-1Ra (10 μg / mL) for 1 h. Cells were then stimulated with recombinant human IL-1α (50 pg / mL, Peprotech) for 24 h, after which supernatants were harvested 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. The cell line was cultured in T75 flasks (37°C, 5% CO 2 ) in complete Iscove's modified Dulbeccos medium (IMDM, 10% FCS, 1% Pen / Strep), split an average of three times a week, and cultured until the cell density reached 2 * 10 6 The concentration of HMC-1 cells was not to exceed 1 / mL and not to exceed 15 passages before assay. HMC-1 cells were seeded (30.000 / well) in 96-well flat-bottom plates, incubated for 3 h, and then pre-incubated with MAB-16-0030 (20 μg / mL-1 μg / mL) or IL-1Ra (10 μg / mL) for 1 h. Cells were then stimulated with recombinant human IL-33 (20 ng / mL, RnD Systems) for 24 h, after which supernatants were harvested 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 T75 flasks (37°C, 5% CO 2 ) and cultured at 4 °C. HaCaT cells were split on average 3 times per week, not exceeding 15 passages before saturation. After 3 h, cells were preincubated with MAB-16-0030 (20 μg / mL-1 μg / mL) or IL-1Ra (10 μg / mL) for 1 h. Cells were then stimulated with recombinant human IL-36α (50 ng / mL, RnD Systems) for 24 h, 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 with MAB-16-0030 (20 μg / mL). After 10 min, 3 days, and 5 days, PBMCs were incubated with MTT (20 μL) for 2 h, after which the absorbance at 570 nM was measured in an ELISA reader. Using the known linearity between absorbance and viable cell converting MTT, viable cell counts were calculated using media alone as a control set at 100%. 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 ), to assess any possible stimulatory effects of MAB-16-0030 alone.

[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 h before stimulation. The mice were incubated with either MAB-16-0030 (20-0.1 μg / mL) or IL-1Ra (10 μg / mL). The following stimuli were used: LPS (10 ng / mL, 24 h, RPMI without FCS), anti-human CD3 / CD28 (1.25 μg / mL; 0.5 μg / mL (eBioscience) for 3 days, RPMI 10% FCS), IL-12 / -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 for 1 h with medium alone (RPMI, no FCS), MAB-16-0030 (20-0.1 μg / mL), or IL-1Ra (10 μg / mL), and then incubated with Candida albicans (0.5 * 10 6 / mL), final Stimulation was performed in a volume of 1 mL. Incubation was continued for 24 hours (37°C, 5% CO 2 ), supernatants were harvested 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 for 5 days (RPMI, 10% FCS) with either medium alone, MAB-16-0030 (20-1 μg / mL) or IL-1Ra (10 μg / mL). 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 cultured 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 The cells were incubated at 37°C / 5% CO 2 The cells were incubated overnight at 37°C / 5% CO. The medium was aspirated and 12.5 μL of antibody solution containing MAB-16-0531 was added to the cells at various concentrations. 2 After incubation at 4°C for 1 h, 12.5 μL of mouse IL-1β was added to the medium to give a final concentration of The concentration was adjusted to 50 pg / ml. The cells were incubated at 37°C / 5% CO 2 The plates were incubated for 5 hours in 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 luminescence was measured 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. 6 The cells were incubated at 37°C / 5% CO 2The cells were incubated at 37°C / 5% CO for 2 h. 10 μL of antibody MAB-16-0531 was added to the cells at various concentrations in the medium. 2 After incubation at 4°C for 1.5 hours, 25 μL of human IL-1β was added to the medium. , to a final concentration of 50 pg / ml. Cells were incubated at 37°C / 5% CO 2 Secreted mouse IL6 in the culture supernatant was analyzed by ELISA (DuoSet ELISA; R&D Systems; Catalog number 840171. Fit curves and EC50 calculations were 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 used per mouse by intraperitoneal injection (IP) as a stimulant, and anti-IL-1R3 (MAB-16-0531, 500 μg / mouse) or IL-1Ra (10 mg / kg) was used for inhibition. Saline was used as both a stimulation and inhibition control. Four mice were used as control mice (saline (inhibition) + saline (stimulation)), MSU These included 8 mice with only MAB (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 h prior to MSU challenge. Mice were placed in a comfortable restroom 6 h 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 activity is associated with elastase production. The IP fluid and IP cell lysates in Example 17 were analyzed for the levels of neutrophil elastase marker and MPO levels. IP lysates were prepared in TritonX 0.5%. Cytokines were measured in both IP fluid and IP cell lysates. If the amount of cytokines in the IP fluid was low, 8 mL of the fluid was concentrated (final range: 4.7-10.9x 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. 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 standard blood phosphatase assay kits (BioRad) and BSA as a standard. As shown in Fig. 20a-c, treatment with the a-mIL1R3 antibody MAB-16-0531 significantly reduces MSU-induced intracellular elastase and elastase in the IP fluid. Levels of IL-6, G-CSF, KC, and CCL-2 are strongly reduced in the IP fluid of MAB-16-0531 mice. Systemically, MPO and KC are also reduced in spleen lysates, and IL-6 and G-CSF are reduced in plasma (Fig. 20d). ).

[0173] Example 19: In vivo model of OVA-induced allergic asthma 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 on days 1, 14, and 21. Mice were IP sensitized with MAB on days 25–28. Mice were IP-injected with 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 min 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. This was performed by lavaging the airways. Cell phenotyping using flow cytometry was then performed. The following monoclonal antibodies (mAbs) against mouse targets were used for flow cytometry following mouse Fc block (BD Biosciences): 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) (10 mg / ml BSA, 0.1 mg / ml NaN 3 FlowJo software (Treestar) was used to perform staining 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 embedded in paraffin, sectioned, and stained with H&E and PAS. Histology was evaluated using a Leica DM2000 LED microscope and LAS V4.12 software. Figure 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 useful in preventing imiquimod-induced An in vivo model of psoriasis was used for testing. The backs of WT12-week-old C57BL / 6 mice (Jackson Laboratories) were shaved and treated with a depilatory cream (Nair). Application of 75 mg IMQ (Aldara 5% IMQ) or control cream (Vaseline cream) was administered to the backskin (IMQ; IMQ (n=10) and a-mIL1R3 (n=9) groups, control cream; vehicle group (n=3)) and to the skin for 1 day. IP injections were given every other day (days 1-5) on alternate days (days 1, 3, and 5). Vehicle and IMQ groups received mouse IgG control (20 mg / kg, MAB Discovery), and treatment groups received MAB-16-0531 (20 mg / kg, MAB Discovery). Ears were treated with a control cream (all Mice were given the appropriate irritant cream (vehicle; 5 mg Vaseline cream) on the right ear. a-mIL1R3 (5 mg IMQ cream) was 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 (for erythema and scaling, The scores ranged from 0 (no response) to 4 (maximally affected). RNA was isolated from skin needle 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 50 ng cDNA in a 100% PBS-derived 100-kDa 100-mL ... cytokine-specific primers (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 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 reduced dermal 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 IgG-1 and -IL1R2 in inducing Fc-mediated effector cell functions such as ADCC To test the activity of the IgG1-LALA version, MAB-16-0030 was injected into 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 Afterwards, 4000 effector cells / well (ADCC Bioassay effector cells, Jurkat, Promega Cat.#G701A) were added in RPMI medium containing 4% low IgG-FCS. Antibodies were then added to final concentrations ranging from 10000 to 0.002 ng / ml, and the plate was incubated at 37°C, 5% CO 2 The activity of NF-kB signaling in luciferase gene reporter Jurkat cells was measured using the prepared 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). As shown in Fig. 1, only the IgG1 version of MAB-16-0030 inhibited effector Jurkat reporter cells induced NF-kB signaling, whereas effector cell activation was abolished with the IgG1-LALA version.

Claims

1. An anti-IL1R3 antibody for use in treating an IL1R3-mediated inflammatory condition and / or disorder in a subject.

2. The inflammatory condition and / or disorder may be an inflammatory condition, an immune disorder, a fibrotic disorder, an eosinophilic disorder, a sensitizing disorder, 2. The anti-IL1R3 antibody of claim 1, wherein the antibody is selected from the group consisting of infectious diseases, pain, central nervous system disorders, ophthalmologic disorders, genetic systemic inflammatory diseases, and systemic and local inflammatory diseases.

3. The anti-IL1R3 antibody of claim 2, wherein the inflammatory condition is a metabolic rheumatic disorder associated with hyperuricemia.

4. The anti-IL1R3 antibody of claim 3, wherein the metabolic rheumatic disorder is selected from the group consisting of gout, pseudogout, drug-induced gout and chronic active (refractory) gout.

5. The IL1R3 antagonist antibody is administered in combination with a therapeutic agent for the treatment of gout. The anti-IL1R3 antibody of claim 4.

6. The anti-IL1R3 antibody of claim 2 , wherein the inflammatory condition is cancer-associated chronic inflammation.

7. The IL1R3 antagonist antibody is a cytotoxic anticancer agent, a cytostatic anticancer agent, or a cytostatic anticancer agent. The anti-IL1R3 antibody of claim 6 , administered in combination with a cancer drug and / or a targeted anti-cancer drug.

8. The subject is administered one or more cytotoxic anti-cancer agents, cytostatic anti-cancer agents, and / or targeted anti-cancer agents. The anti-IL1R3 antibody according to claims 6 to 7, which is resistant to treatment with an anticancer drug. 。

9. The anti-IL1R3 antibody of any one of claims 1 to 8, wherein the antibody has reduced or absent effector function.

10. The anti-IL1R3 antibody of any one of claims 1 to 9, wherein the antibody does not induce immune cell depletion.

11. The anti-IL1R3 antibody of claim 9 or 10, wherein the antibody does not induce ADCC.

12. The antibody comprises at least the amino acid substitutions L234A and L235A in the human IgG1 Fc region, or 12. Any one of claims 9 to 11, comprising S228P and L235E of the human IgG4 Fc region, or corresponding functional mutations. The anti-IL1R3 antibody according to any one of claims 1 to 4.

13. The subject is a human subject, and the antibody is a) a heavy chain variable region (VH) comprising the 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 of SEQ ID NOs: 69-85; the CDR-H2 region comprises an amino acid sequence selected from the group of SEQ ID NOs: 86-102; and the CDR-H3 region comprises an amino acid sequence selected from the group of SEQ ID NOs: 103-119; and b) a light chain variable region (VL) comprising the 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 of SEQ ID NOs: 120 to 136; the CDR-L2 region comprises an amino acid sequence selected from the group of SEQ ID NOs: 137-153; and The CDR-L3 region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 154 to 170 and SEQ ID NO:

175. Contains arrays, The anti-IL1R3 antibody of any one of claims 1 to 12, comprising:

14. the subject is a human subject, and the antibody comprises a heavy chain of SEQ ID NOs: 1-34, and 173. and a light chain variable (VL) region selected from the group consisting of SEQ ID NOs: 35-68, and 174. The anti-IL1R3 antibody of any one of claims 1 to 13, comprising a VL region that is at least 90% identical to the VL region of

15. The subject is a mouse, and the antibody is a) a heavy chain variable region (VH) comprising the 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 the 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; The CDR-L3 region comprises the amino acid sequence of SEQ ID NO: 183; The anti-IL1R3 antibody of any one of claims 1 to 12, comprising:

16. The subject is a mouse, and the antibody has a heavy chain variable (VH) region of SEQ ID NO:

176. and a VH region that is at least 90% identical to the light chain variable (VL) region of SEQ ID NO:

177. The anti-IL1R3 of any one of claims 1 to 12, comprising a VL region that is at least 90% identical. antibody.

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