Anti-IL1RAP Antibodies

Abstract

The present invention relates to an antibody or antigen-binding fragment thereof having binding specificity for interleukin-1 receptor accessory protein (IL1RAP), a polynucleotide encoding the antibody, a vector comprising the polynucleotide, and a recombinant host cell comprising the polynucleotide or vector, a method for producing the antibody or antigen-binding fragment, a composition comprising the antibody or antigen-binding fragment, the polynucleotide, the vector, and / or the host cell, and an antibody or antigen-binding fragment, polynucleotide, vector, host cell, and / or composition for use in medicine and / or for use in the prevention and / or treatment and / or amelioration and / or detection and / or diagnosis of a disease or disorder susceptible to treatment with an inhibitor of IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and / or IL-36γ signaling, and / or wherein the disease or disorder is associated with cells expressing IL1RAP.

Description

[Technical field]

[0001] The present invention relates to anti-IL1RAP antibodies or fragments thereof having anti-inflammatory, anti-fibrotic and / or anti-tumor properties and their use in the prevention, treatment, mitigation, detection and / or diagnosis of diseases or disorders associated with IL-1α, IL-1β, IL-33, IL-36α, IL-36β and / or IL-36γ signaling. [Background technology]

[0002] The interleukin-1 (IL-1) family of cytokine ligands and their receptors are involved in inflammation, autoimmunity, immunoregulation, fibrosis, cell proliferation, tumor growth, tumor metastasis, and host defense. They contribute to the pathology of inflammatory, autoimmune, immunoregulatory, fibrotic, and degenerative diseases and disorders, as well as cell proliferative and neoplastic diseases and disorders, including cancer (Garlanda C, The interleukin-1 family: back to the future. Immunity, 39: 1003-1018 (2013)).

[0003] Both of these diseases impose a significant burden on individuals and society, and there remains a need for therapies to treat, ameliorate, prevent, diagnose, or detect inflammatory, autoimmune, immunoregulatory, fibrotic, degenerative, and cell proliferative diseases or disorders associated with the IL-1 family of cytokine ligands and receptors.

[0004] The IL-1 family includes a number of agonistic cytokines, including IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and IL-36γ, each of which binds to a unique IL-1 family cell membrane receptor. When a cytokine binds to its cognate receptor, a co-receptor called the IL-1 receptor accessory protein (IL1RAP) forms a receptor complex, which triggers intracellular signal transduction and activation of transcription factors, including NF-κB, resulting in an inflammatory response. IL1RAP is a coreceptor for IL-1 receptor I (IL1R1; binds IL-1α and IL-1β), IL-33 receptor (ST2, IL1RL1; binds IL-33), and IL-36 receptor (IL1RL2; binds IL-36α, IL-36β, and IL-36γ) and is required for downstream signaling of the cytokines IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and IL-36γ.

[0005] Antibodies that bind to IL1RAP can block cytokine signals downstream of IL1RAP-dependent receptors. Interestingly, apart from this blocking function, antibodies that bind to IL1RAP can also be used for other functions, such as inducing antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cellular phagocytosis (ADCP), thereby leading to the killing of target cells, such as IL1RAP-expressing tumor cells.

[0006] Previously, antibodies capable of reducing, inhibiting, and / or blocking the signaling pathway of IL-1 family cytokine ligands have been produced according to the characteristics of each antibody. For example, WO2015 / 132602 discloses an anti-IL1RAP antibody that inhibits, to varying degrees, the signaling of IL-1α, IL-1β, and IL-33.

[0007] Blocking all six cytokines (IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and IL-36γ) is expected to be beneficial for therapeutic approaches targeting IL-1 family cytokine ligands and receptors. IL-1α and IL-1β are potent proinflammatory cytokines and their involvement in acute inflammation has been widely described. IL-33 has traditionally been considered a Th2 cytokine that induces the production of type 2 cytokines such as IL-5 and IL-13. Less is known about the function of IL-36, but several studies suggest that it is involved in immune cell activation and induces the release of inflammatory cytokines. Targeting all of these cytokine pathways could more completely downregulate disease-related processes, e.g., inflammatory processes, improving the treatment outcomes of patients with inflammatory diseases or disorders.

[0008] To date, there is a lack of therapeutic options that address various aspects of diseases or disorders associated with interleukin-1 (IL-1) family cytokine ligand and receptor (IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and / or IL-36γ) signaling. Current therapies only target separate pathways associated with IL-1 family cytokine ligands, and simultaneously downregulating multiple pathways associated with IL-1 family cytokine ligands, e.g., in the context of inflammation and fibrosis, remains a clinical challenge. As a result, there remains an urgent need for improved therapies that target the multifaceted aspects associated with IL-1 family cytokine ligands and receptors. Summary of the Invention [Means for solving the problem]

[0009] The inventors of the present invention have generated antibodies and variants thereof that bind to IL-1RAP domain 2 and have the ability to inhibit downstream signaling of six IL1RAP-dependent ligands: IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and / or IL-36γ. Due to this excellent property, the antibodies can be used for the prevention, treatment, mitigation, detection, and / or diagnosis of diseases and disorders associated with IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and / or IL-36γ signaling, thereby simultaneously targeting several downstream pathways associated with these cytokines. As a result, the antibodies can be used to target several aspects and processes of diseases and disorders related to IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and / or IL-36γ signaling, such as inflammatory and fibrotic aspects in inflammatory or fibrotic diseases or disorders, or inflammatory, fibrotic, or proliferative aspects in neoplastic diseases or disorders. The antibodies described herein can simultaneously target multiple cytokine pathways, such as IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and IL-36γ pathways, which is clinically advantageous in treating diseases or disorders where blocking only one or a few cytokines would not be sufficiently effective in treating or preventing the disease or disorder. It is further disclosed that the antibodies can be humanized and deimmunized to result in antibody variants with optimized and fine-tuned properties.

[0010] A first aspect of the present invention relates to an antibody, or antigen-binding fragment thereof, having binding specificity for interleukin-1 receptor accessory protein (IL1RAP), the antibody or antigen-binding fragment being A light chain variable region, a) a CDR-L1 comprising or consisting of an amino acid sequence selected from the group consisting of ESISTA (SEQ ID NO: 1), QASESISTALA (SEQ ID NO: 7), and QASESISTALA (SEQ ID NO: 13); b) a CDR-L2 comprising or consisting of an amino acid sequence selected from the group consisting of KAS, KASTLPS (SEQ ID NO: 8) and KASTLPS (SEQ ID NO: 14); and and c) a CDR-L3 comprising or consisting of an amino acid sequence selected from the group consisting of QQGFSSGNVHNA (SEQ ID NO: 3), QQGFSSGNVHNA (SEQ ID NO: 9), and QQGFSSGNVHNA (SEQ ID NO: 15). and / or A heavy chain variable region, d) a CDR-H1 comprising or consisting of an amino acid sequence selected from the group consisting of GPSLSHFD (SEQ ID NO: 4), HFDIT (SEQ ID NO: 10), and GPSLSHFDIT (SEQ ID NO: 16); e) a CDR-H2 comprising or consisting of an amino acid sequence selected from the group consisting of ISPGVST (SEQ ID NO: 5), TISPGVSTYYASWAKS (SEQ ID NO: 11), and TISPGVSTYYASWAKS (SEQ ID NO: 17); and f) a CDR-H3 comprising or consisting of an amino acid sequence selected from the group consisting of ARGGVGSSWKAFDL (SEQ ID NO: 6), GGVGSSWKAFDL (SEQ ID NO: 12), and ARGGVGSSWKAFDL (SEQ ID NO: 18).

[0011] A second aspect of the invention relates to a polynucleotide encoding an antibody or antigen-binding fragment of the first aspect of the invention, or its component polypeptide chains.

[0012] A third aspect of the present invention relates to a vector comprising a polynucleotide according to the second aspect of the present invention.

[0013] A fourth aspect of the invention relates to a recombinant host cell comprising a polynucleotide according to the second aspect of the invention or a vector according to the third aspect of the invention.

[0014] A fifth aspect of the invention relates to a method of producing an antibody or antigen-binding fragment thereof according to the first aspect of the invention, said method comprising culturing a host cell according to the fourth aspect of the invention comprising a polynucleotide according to the second aspect of the invention or a vector according to the third aspect of the invention under conditions allowing expression of the encoded antibody or antigen-binding fragment thereof.

[0015] A sixth aspect of the invention provides an antibody or antigen-binding fragment of the first aspect of the invention, A polynucleotide according to the second aspect of the present invention, A vector according to the third aspect of the invention, and / or A host cell according to the fourth aspect of the invention, in a pharmaceutical composition, wherein said composition further comprises a pharma- ceutically acceptable diluent, carrier, or excipient.

[0016] A seventh aspect of the present invention is a method for producing a composition comprising the steps of: For medical use, An antibody or antigen-binding fragment according to the first aspect of the invention, a polynucleotide according to the second aspect of the invention, A vector according to the third aspect of the present invention, A host cell according to the fourth aspect of the invention, and / or The sixth aspect of the present invention relates to a composition.

[0017] An eighth aspect of the present invention is a method for producing a composition comprising the steps of: An antibody or antigen-binding fragment according to the first aspect of the invention, A polynucleotide according to the second aspect of the present invention, A vector according to the third aspect of the present invention, A fourth host cell of the invention, and / or A sixth aspect of the present invention relates to a composition for use in the prevention and / or treatment and / or alleviation and / or detection and / or diagnosis of a disease or disorder susceptible to treatment with an inhibitor of IL-1α, IL-1β, IL-33, IL-36α, IL-36β and / or IL-36γ signaling, and / or wherein said disease or disorder is associated with cells expressing IL1RAP.

[0018] A ninth aspect of the present invention is a method for producing a composition comprising the steps of: An antibody or antigen-binding fragment according to the first aspect of the invention, A polynucleotide according to the second aspect of the present invention, A vector according to the third aspect of the present invention, A host cell according to the fourth aspect of the invention, and / or The composition according to the sixth aspect of the present invention comprises For use in inducing cell death and / or inhibiting the growth and / or proliferation of pathological cells associated with a neoplastic disease of a subject, or their stem or progenitor cells, wherein said cells express IL1RAP.

[0019] A tenth aspect of the present invention is a method for producing a composition comprising the steps of: An antibody or antigen-binding fragment according to the first aspect of the invention, A polynucleotide according to the second aspect of the present invention, A vector according to the third aspect of the present invention, A host cell according to the fourth aspect of the invention, and / or The use of the composition according to the sixth aspect of the present invention, in the preparation of a medicament for the prevention, treatment, mitigation, detection, and / or diagnosis of a disease or disorder susceptible to treatment with an inhibitor of IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and / or IL-36γ signaling, and / or said disease or disorder is associated with cells expressing IL1RAP.

[0020] An eleventh aspect of the present invention is a method for producing a composition comprising the steps of: An antibody or antigen-binding fragment according to the first aspect of the invention, A polynucleotide according to the second aspect of the present invention, A vector according to the third aspect of the present invention, A host cell according to the fourth aspect of the invention, and / or The use of the composition according to the sixth aspect of the present invention, For use in the preparation of a medicament for the detection and / or diagnosis of a disease or disorder associated with cells expressing IL1RAP.

[0021] A twelfth aspect of the present invention relates to a method for the prevention and / or treatment and / or alleviation and / or detection and / or diagnosis of a disease or disorder susceptible to treatment with an inhibitor of IL-1α, IL-1β, IL-33, IL-36α, IL-36β and / or IL-36γ signaling, and / or said disease or disorder relates to IL1RAP expressing cells in a subject, an antibody or antigen-binding fragment according to the first aspect of the invention, a polynucleotide according to the second aspect of the invention, - a vector according to the third aspect of the invention, a host cell according to the fourth aspect of the invention, and / or - administering an effective amount of a composition according to the sixth aspect of the invention.

[0022] A thirteenth aspect of the present invention relates to an in vitro method for detecting cells expressing IL1RAP in a subject, said method comprising: (a) providing a sample of cells from a subject to be tested, such as a tissue biopsy or blood sample; (b) optionally extracting and / or purifying cells present in the sample; (c) contacting the antibody or antigen-binding fragment of the first aspect of the invention with cells present in said sample; (d) determining whether the antibody or antigen-binding fragment thereof binds to the cell; wherein said binding of said antibody or antigen-binding fragment thereof to cells indicates the presence of a disease or disorder associated with cells expressing IL1RAP in said tissue of the subject.

[0023] A fourteenth aspect of the invention relates to an in vitro method for identifying a patient having a disease or disorder associated with cells expressing IL1RAP, who would benefit from treatment with an antibody or antigen-binding fragment of the first aspect of the invention, comprising: (a) providing a cell sample, such as a tissue biopsy or blood sample, from a patient to be tested; (b) optionally extracting and / or purifying cells present in the sample; (c) contacting the antibody or antigen-binding fragment of the first aspect of the invention with cells present in said sample; (d) determining whether the antibody or antigen-binding fragment thereof binds to the cell; Here, binding of the antibody or antigen-binding fragment thereof to cells expressing IL1RAP is indicative of a patient that would benefit from treatment with the antibody or antigen-binding fragment of the first aspect of the invention.

[0024] A fifteenth aspect of the invention relates to a method for treating a patient having a disease or disorder associated with expression of IL1RAP in a cell, said method comprising: a) selecting a patient identified as having a disease or disorder associated with cells expressing IL1RAP using a method according to the fourteenth aspect of the invention; and b) administering to said patient a therapeutic agent effective to treat said disease or disorder.

[0025] A sixteenth aspect of the present invention relates to a method for detecting cells expressing IL1RAP, said method comprising: (a) contacting an antibody or antigen-binding fragment thereof according to the first aspect with a cell to be analyzed for expression of IL1RAP; (b) determining whether the antibody or antigen-binding fragment thereof binds to the cell; wherein said binding of said antibody or antigen-binding fragment thereof to cells indicates the presence of a disease or disorder associated with cells expressing IL1RAP in said tissue of the subject.

[0026] A seventeenth aspect of the present invention relates to a method for reducing inflammation in a subject having peritonitis, said method comprising: an antibody or antigen-binding fragment according to the first aspect of the invention, a polynucleotide according to the second aspect of the invention, - a vector according to the third aspect of the invention, a host cell according to the fourth aspect of the invention, and / or - administering an effective amount of a composition according to the sixth aspect of the invention.

[0027] An eighteenth aspect of the invention relates to a method for reducing disease severity in a subject having psoriasis or psoriatic arthritis, said method comprising: an antibody or antigen-binding fragment according to the first aspect of the invention, a polynucleotide according to the second aspect of the invention, - a vector according to the third aspect of the invention, a host cell according to the fourth aspect of the invention, and / or - administering an effective amount of a composition according to the sixth aspect of the invention.

[0028] A nineteenth aspect of the present invention relates to a method of reducing inflammation of atherosclerotic plaque in a subject having atherosclerosis, said method comprising: an antibody or antigen-binding fragment according to the first aspect of the invention, a polynucleotide according to the second aspect of the invention, - a vector according to the third aspect of the invention, a host cell according to the fourth aspect of the invention, and / or - administering an effective amount of a composition according to the sixth aspect of the invention.

[0029] A twentieth aspect of the present invention relates to a method of reducing atherosclerotic plaque volume and / or atherosclerotic plaque size in a subject having atherosclerosis, said method comprising: an antibody or antigen-binding fragment according to the first aspect of the invention, a polynucleotide according to the second aspect of the invention, - a vector according to the third aspect of the invention, a host cell according to the fourth aspect of the invention, and / or - administering an effective amount of a composition according to the sixth aspect of the invention.

[0030] A twenty-first aspect of the present invention relates to a method for reducing inflammation and / or fibrosis in a subject having myocarditis, said method comprising administering to said subject: an antibody or antigen-binding fragment according to the first aspect of the invention, a polynucleotide according to the second aspect of the invention, - a vector according to the third aspect of the invention, a host cell according to the fourth aspect of the invention, and / or - administering an effective amount of a composition according to the sixth aspect of the invention.

[0031] A twenty-second aspect of the present invention relates to a method for counteracting deterioration of cardiac function in a subject having myocarditis or autoimmune myocarditis, said method comprising administering to said subject: an antibody or antigen-binding fragment according to the first aspect of the invention, a polynucleotide according to the second aspect of the invention, - a vector according to the third aspect of the invention, a host cell according to the fourth aspect of the invention, and / or - administering an effective amount of a composition according to the sixth aspect of the invention.

[0032] A twenty-third aspect of the invention relates to a method for reducing skin fibrosis in a subject having systemic sclerosis, said method comprising administering to said subject: an antibody or antigen-binding fragment according to the first aspect of the invention, a polynucleotide according to the second aspect of the invention, - a vector according to the third aspect of the invention, a host cell according to the fourth aspect of the invention, and / or - administering an effective amount of a composition according to the sixth aspect of the invention.

[0033] A twenty-fourth aspect of the present invention relates to a method of reducing pulmonary fibrosis in a subject with systemic sclerosis, said method comprising: an antibody or antigen-binding fragment according to the first aspect of the invention, a polynucleotide according to the second aspect of the invention, - a vector according to the third aspect of the invention, a host cell according to the fourth aspect of the invention, and / or - administering an effective amount of a composition according to the sixth aspect of the invention. [Brief description of the drawings]

[0034] [Figure 1] Figure 1: Anti-IL1RAP antibody mCAN10 suppresses inflammation in an acute peritonitis model. WT or KO mice were immunized with MSU crystals and peritoneal lavage fluid was subjected to flow cytometry for quantification of infiltrating cells 6 hours after immunization (A). WT mice were treated with mCAN10, IL1RA, isotype control antibody (isotype), or PBS only 1 hour before immunization with MSU crystals. Six hours after immunization, peritoneal lavage fluid was collected for quantification of infiltrating cells by flow cytometry (B) or for quantification of cytokines and chemokines by Luminex assay (C). [Diagram 2] Figure 2: Anti-IL1RAP antibody mCAN10 suppresses disease severity in psoriasis and psoriatic arthritis models. Psoriasis was induced in BALB / c mice by administering imiquimod to the shaved backs for 7 days. During this time, mice were treated three times with mCAN10, anti-IL-1β antibody (anti-IL-1β), isotype control antibody (isotype anti-IL-1β, isotype mCAN10), dexamethasone, or PBS alone (vehicle). Disease severity was assessed by scoring inflammation and erythema in the dorsal area over a 7-day period (A and B). Psoriatic arthritis was induced in B6N.Q.NCF1 mice by administering mannan derived from Saccharomyces cerevisiae. Mice were treated as in A and B, and disease severity was assessed by scoring inflammation in all front paw joints over a 7-day period (C and D). On day 7, mice were sacrificed, blood was collected, and plasma IL-17 concentrations were analyzed (E). [Diagram 3]Figure 3: mCAN10 suppresses inflammation in aortic plaques in an atherosclerosis model. The development of atherosclerotic lesions was induced by feeding apolipoprotein E (apoE) KO mice a high cholesterol diet (HCD). Four weeks after HCD administration, the mice were administered mCAN10 or an isotype control antibody (Iso) every other week (twice a week) for 6 weeks. During this time, the mice were fed the HCD. After the experiment was completed, the mice were sacrificed and the aortas were harvested for flow cytometry analysis of myeloid cells such as CD45+ cells (A), CD11b+ cells, and Ly6G+ neutrophils (B and C), and T lymphocytes such as TCR-β+ cells, CD4+ cells, and CD8+ cells (DF). [Figure 4] Figure 4: mCAN10 reduces aortic plaque size in an arteriosclerosis model. Mice were treated as described for Figure 3. After completion of the experiment, mice were sacrificed, hearts were harvested, and sections of the aortic root were stained for lipid accumulation with Oil Red O to compare both plaque volume (A) and area (B). [Diagram 5] Figure 5: Anti-IL1RAP antibody mCAN10 suppresses inflammation and fibrosis in experimental autoimmune myocarditis (EAM). EAM was induced in BALB / c mice by immunizing them twice with α-myosin heavy chain peptide emulsified in complete Freund's adjuvant. Starting on day 7 after the final immunization, mice were administered mCAN10, isotype control antibody (isotype), or PBS alone every other week for 4 weeks. After the experiment was completed, mice were sacrificed and hearts were harvested, and heart sections were stained with H&E to evaluate the degree of inflammation (A and B) or stained with Masson's trichrome to evaluate the degree of fibrosis (C and D). [Figure 6]Figure 6: Anti-IL1RAP antibody mCAN10 corresponds to cardiac dysfunction in experimental autoimmune myocarditis. EAM was induced in BALB / c mice by immunizing them twice with α-myosin heavy chain peptide emulsified in complete Freund's adjuvant. Starting on the day of the final immunization (day 7), mice were administered mCAN10, anti-IL-1β antibody (anti-IL-1β), or isotype control antibody (isotype anti-IL-1β, isotype mCAN10) every other week for 5 weeks (A). Alternatively, mice were treated daily with mCAN10, isotype control antibody (isotype mCAN10), IL1RA, prednisone, or vehicle control (vehicle IL1RA, vehicle prednisone) for 5 weeks (B). Optionally, mice received mCAN10, isotype control antibody (isotype), or vehicle control (PBS) as in A, but treatment began 7 days after the last immunization (day 14) (C). Cardiac function was assessed by transthoracic echocardiography at the start of the study and on days 28 and 42. [Figure 7] Figure 7: mCAN10 ameliorates skin fibrosis in scleroderma chronic graft-versus-host disease (scl-cGvHD) mouse model. Female BALB / c (H-2d) recipient mice were allogeneically transplanted with bone marrow from male B10.D2 donor mice (H-2d) to create an MHC mismatch model that develops scl-cGvHD. As a control, the same recipient mice were syngeneically transplanted with bone marrow from female BALB / c (H-2d) donor mice, and no disease developed. On day 21 after transplantation, mCAN10 (initial dose 20 mg / kg, followed by 10 mg / kg) or the same amount of isotype control antibody (Iso) was administered every other week for 4 weeks. Alternatively, these mice were treated with 50 mg / kg nintedanib orally daily for 4 weeks. Mice that received syngeneic transplants were treated with isotype control antibody (Iso) alone. Mice were sacrificed on day 49 and skin samples from the upper back were taken for histological evaluation for quantification of skin thickness (A), fibroblast number (B), or hydroxyproline content (C). [Figure 8]Figure 8: mCAN10 ameliorates pulmonary fibrosis in a mouse model of scl cGvHD. Mice were treated as described for Figure 7. After sacrificing the mice 49 days after transplantation, lungs were harvested and subjected to histological evaluation for determination of Ashcroft score (A) and area stained with Sirius Red (B) or quantification of hydroxyproline content (C). [Figure 9] Figure 9: mCAN10 ameliorates weight loss in a mouse model of scl cGvHD. Mice were treated as described for Figure 7. After transplantation, mice were continuously weighed for the duration of the study, with a final assessment made 49 days after transplantation. [Figure 10] Figure 10: mCAN10 alters the expression profile of IL-1 family genes in scl cGvHD model mice. Mice were treated as described for Figure 7. After sacrificing mice 49 days after transplantation, skin samples from the upper back were used for RNA sequencing to generate heat maps visualizing the changes in gene expression levels of the indicated IL-1 family members. Results are shown for four samples from each group of allografted mice (Allo), allografted mice (Syn), and allografted mice treated with mCAN10 (Treat). [Figure 11] Figure 11: mCAN10 alters the expression of genes in a mouse model of scl cGvHD that are also differentially expressed in systemic sclerosis patients. Mice were treated as described for Figure 7. After sacrificing the mice 49 days after transplantation, skin samples from the upper back were used for RNA-seq analysis. Transcriptome profiles of systemic sclerosis (SSc) patients were retrieved from the patient cohort NCBI / GEO / GSE130955, which includes 143 patients and 22 healthy controls. The number of overlapping genes was visualized with a Venn diagram. [Figure 12]Figure 12: Dose-dependent binding of chimeric 48D2 to cell membrane IL1RAP. Chimeric 48D2 or hIgG1 (hIg = human immunoglobulin) isotype control antibody was added at increasing concentrations to SKMEL-5 cells and extracellular binding was analyzed by flow cytometry. Chimeric 48D2 specifically bound to IL1RAP on the cell membrane in a dose-dependent manner with higher mean fluorescence intensity (MFI) compared to the hIgG1 isotype control antibody. [Figure 13] Figure 13: Inhibition of interleukin signaling by chimeric 48D2. The ability of chimeric 48D2 to block IL-1α (A), IL-1β (B), IL-33 (C), IL-36α (D), IL-36β (E), and IL-36γ (F) signaling was examined by HEK-Blue™ assay. hIgG1 isotype antibody was included as a control. Dotted lines indicate positive (cytokine stimulated cells) and negative (cells only) controls and illustrate the window of inhibition. Chimeric 48D2 blocks downstream signals of all six cytokines. [Figure 14] Figure 14: Chimeric 48D2 binds to human, cynomolgus, and porcine IL1RAP. Cross-reactivity of chimeric 48D2 with IL1RAP orthologues was measured by ELISA. Chimeric 48D2 reacts with human (hIL1RAP), cynomolgus (mfIL1RAP), and porcine (ssIL1RAP) IL1RAP, but does not react with mouse (mIL1RAP), rat (rnIL1RAP), rabbit (ocIL1RAP), or dog (clIL1RAP) IL1RAP. [Figure 15]Figure 15: Inhibition of interleukin signaling by humanized 48D2 variants. The ability of combinations of humanized (h)48D2 VH variants VH1, VH2, VH3, VH4, VH5, and VL variants VL1, VL2, VL3, VL4, and VL5 to block IL-1α (AE), IL-1β (FJ), and IL-33 (KO) signaling was investigated in HEK-BLUE™ assays. h48D2 variants with VH5 blocked downstream signaling to the same extent as chimeric 48D2 and were further investigated for inhibition of IL-36α (P), IL-36β (Q), and IL-36γ (R). h48D2 variants with VH1, VH2, VH3, or VH4 inhibited IL-1α, IL-1β, and IL-33 signaling to various degrees. hIgG1 isotype antibody and chimeric 48D2 are included as controls. Dotted lines indicate positive (cytokine stimulated cells) and negative (cells only) controls to illustrate the window of inhibition. Combinations of h48D2 variants carrying VH5 and any VL variant (VL1-VL5) block downstream signaling of six interleukins to the same extent as chimeric 48D2. [Figure 16] Figure 16: Dose-dependent binding of two humanized and non-immunized clones to cell membrane IL1RAP. Two humanized and non-immunized 48D2 clones, h48D2 VH5.GL:VL4 and h48D2 VH5.GL:VL5.GL, were added at increasing concentrations to SKMEL-5 cells and analyzed for extracellular binding by flow cytometry. Chimeric 48D2 and hIgG1 isotype antibodies were included as controls. h48D2 VH5.GL:VL4 and h48D2 VH5.GL:VL5.GL specifically bound to cell membrane IL1RAP in a dose-dependent manner with higher mean fluorescence intensity (MFI) compared to the hIgG1 isotype control antibody and the chimeric 48D2 antibody. [Figure 17]Figure 17: Deimmunized h48D2 clones maintained inhibitory activity on all six interleukin pathways. The inhibitory activity of deimmunized h48D2 clones on IL-1α (A), IL-1β (B), IL-33 (C), IL-36α (D), IL-36β (E), and IL-36γ (F) signaling was evaluated by HEK-BLUE™ assay. hIgG1 isotype antibodies, h48D2 VH5:VL4 and h48D2 VH5:VL5 were included as controls. Dotted lines indicate positive (cytokine stimulated cells) and negative (cells only) controls to illustrate the window of inhibition. All six non-immunized clones had similar inhibitory activity as h48D2 VH5:VL4 and VH5:VL5. Furthermore, the inhibitory activity of the h48D2 variant VH5.GL:VL4 against IL-33 (G), IL-36α (H), IL-36β (I), and IL-36γ (J) was evaluated using HEK-Blue™ cells stably expressing the IL-36 receptor. [Figure 18] Figure 18: Deimmunized h48D2 clones bind human, cynomolgus, and porcine IL1RAP. Cross-reactivity was measured by ELISA. h48D2 VH5.GL:VL4(A) and VH5.GL:VL5.GL(B) cross-react with human (hIL1RAP), cynomolgus (mfIL1RAP), and porcine (ssIL1RAP) IL1RAP, but do not react with mouse (mIL1RAP), rat (rnIL1RAP), rabbit (ocIL1RAP), or dog (clIL1RAP) IL1RAP. [Figure 19]Figure 19: 48D2 in hIgG1 format induces ADCC of IL1RAP expressing SKMEL-5 cells. In vitro ADCC assays were performed using IL1RAP expressing SKMEL-5 cells and human NK cells, and the number of dead SKMEL-5 cells was analyzed by flow cytometry and expressed as % dead (DAPI positive) cells. 48D2 in hIgG1 format can induce NK cells to kill SKMEL-5 cells in a dose-dependent manner. When 48D2 was expressed in hIgG1-LALA, no dose-dependent ADCC was induced. Isotype-hIgG1 and isotype-LALA did not induce ADCC above the background cell death observed in untreated cells. [Figure 20] Figure 20: 48D2 inhibits cytokine-induced IL-6 mRNA expression in human dermal fibroblasts. Human primary dermal fibroblasts were cultured in vitro and stimulated with IL-1α or IL-1β (A) or IL-36α, IL-36β or IL-36γ (B) with or without the addition of chimeric 48D2. Data are presented as fold change (2-ddCT) compared to unstimulated control (0 ng / mL). All cytokines induced IL-6 mRNA expression in a dose-dependent manner, and 48D2 inhibited the increase to levels similar to those of the unstimulated control. [Figure 21] Figure 21: VH5.GL:VL4 inhibits IL-1β-induced cytokine and chemokine release in human whole blood. Blood was drawn from two human donors and incubated with VH5.GL:VL4 for 30 minutes. Blood was then stimulated with either IL-1β or lipopolysaccharide (LPS) for 20 hours. Plates were centrifuged and the plasma layer was collected and analyzed on a Human Cytokine / Chemokine 71-Plex Discovery Assay Array. Levels of G-CSF (A), GROα / CXCL1 (B), IL-17A (C), and TNF-α (D) are shown for one of the two donors. [Figure 22]Figure 22: VH5.GL:VL4 inhibits IL-1β induced cytokine release in a blood loop system Blood was drawn from 10 human donors and transferred into a blood loop system. VH5.GL:VL4 was administered to the blood at 32 μg / ml and 15 minutes later IL-1β was added at 1 ng / ml. The loop was run for 4 hours. Plasma was collected from the blood samples and levels of IL-6 (A) and IL-8 (B) were measured using MSD's MULTI-ARRAY® technology. [Figure 23] Figure 23: VH5.GL:VL4 is internalized in IL1RAP expressing cells. WT and IL1RAP KO SKMEL cells were incubated with fluorescently labeled VH5.GL:VL4 or isotype control (IsoCtrl) for 1, 2 or 4 hours in Ibidi-treat microscope chamber slides. Cell nuclei were stained with DAPI. Optical sections scanned within the cells were interactively analyzed visually and representative images were captured to assess membrane binding and internalization of VH5.GL:VL4 (indicated by arrows). [Figure 24] Figure 24: VH5.GL:VL4 localizes to lysosomes or endosomes upon internalization by IL1RAP-expressing cells. WT SKMEL cells were incubated with fluorescently labeled VH5.GL:VL4 for 1, 2, or 4 hours in Ibidi-treat microscope chamber slides. Additionally, we stained for the markers EEA1 and Lamp1 to detect endosomes and lysosomes, respectively. Cell nuclei were stained with DAPI. Cells were scanned in optical sections and visually analyzed interactively, and representative images were taken to assess the overlap of VH5.GL:VL4 staining with EEA1 and Lamp1 staining. [Diagram 25]Figure 25: VH5.GL:VL4 in hIgG1-LALA format does not induce Fc-mediated cytokine release. Blood was collected from 10 human donors and transferred into a blood loop system. VH5.GL:VL4 was added to the blood at increasing concentrations as indicated. Alternatively, the anti-CD52 antibody alemtuzumab was added. The loop was run for 4 hours. Plasma was collected from the blood samples and levels of IFN-γ (A), IL-6 (B), IL-8 (C) and TNF-α (D) were measured using MSD's MULTI-ARRAY® technology. [Figure 26] Figure 26: VH5.GL:VL4 in hIgG1-LALA format does not induce Fc-mediated complement activation. Blood was collected from 10 human donors and transferred into a blood loop system. VH5.GL:VL4 was added to the blood at increasing concentrations as indicated. Alternatively, the anti-CD52 antibody alemtuzumab was added. The loop was run for 15 minutes. Plasma was collected from the blood samples and complement activation was analyzed by measuring the complement split products C3a (A) and C5a (B) using an ELISA kit from RayBiotech. [Figure 27] Figure 27: Serum concentrations of VH5.GL:VL4 after a single intravenous dose in cynomolgus monkeys. VH5.GL:VL4 was administered intravenously at a single dose of 5, 20, or 50 mg / kg to one male (M) and one female (F) cynomolgus monkey for each dose level. Blood was collected and serum was collected at 0.083, 1, 3, 6, 24, 48, 96, 168, 264, and 336 hours post-dose. Serum samples were transferred to IL1RAP-coated MSD plates, and VH5.GL:VL4 in the samples was detected by adding an anti-human IgG antibody conjugated with an electrochemiluminescence label and measuring the luminescence intensity. [Figure 28]Figure 28: Serum concentrations of VH5.GL:VL4 after a single intravenous or subcutaneous administration to cynomolgus monkeys. VH5.GL:VL4 was administered at a single dose of 10 mg / kg intravenously or subcutaneously to two female cynomolgus monkeys. Blood was collected and serum obtained at 0.083, 0.5, 1, 3, 6, 24, 48, 96, 168, 264, 336, 480, and 672 hours after administration. Serum samples were transferred to an IL1RAP-coated MSD plate, anti-human IgG antibody conjugated with an electrochemiluminescence label was added, and VH5.GL:VL4 in the samples was detected by measuring the luminescence intensity. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to an "antibody" includes a plurality of such antibodies, such as one or more antibodies, at least one antibody, or two or more antibodies. Similarly, reference to an "anti-IL1RAP antibody" can also refer to "anti-IL1RAP antibodies," such as, for example, the antibody variants described in Examples 9-22.

[0036] The term "some embodiments" may include one embodiment or more than one embodiment.

[0037] The use of the words "a" or "an," when used in conjunction with the term "comprising" in the claims and / or specification, may mean "one," but may also be consistent with the meanings of "one or more," "at least one," and "one or more than one."

[0038] IL1RAP antibody A first aspect of the present invention relates to an antibody, or antigen-binding fragment thereof, having binding specificity for interleukin-1 receptor accessory protein (IL1RAP), the antibody or antigen-binding fragment being A light chain variable region, a) a CDR-L1 comprising or consisting of an amino acid sequence selected from the group consisting of ESISTA (SEQ ID NO: 1), QASESISTALA (SEQ ID NO: 7), and QASESISTALA (SEQ ID NO: 13); b) a CDR-L2 comprising or consisting of an amino acid sequence selected from the group consisting of KAS, KASTLPS (SEQ ID NO: 8) and KASTLPS (SEQ ID NO: 14); and and c) a CDR-L3 comprising or consisting of an amino acid sequence selected from the group consisting of QQGFSSGNVHNA (SEQ ID NO: 3), QQGFSSGNVHNA (SEQ ID NO: 9), and QQGFSSGNVHNA (SEQ ID NO: 15). and / or A heavy chain variable region, d) a CDR-H1 comprising or consisting of an amino acid sequence selected from the group consisting of GPSLSHFD (SEQ ID NO: 4), HFDIT (SEQ ID NO: 10), and GPSLSHFDIT (SEQ ID NO: 16); e) a CDR-H2 comprising or consisting of an amino acid sequence selected from the group consisting of ISPGVST (SEQ ID NO: 5), TISPGVSTYYASWAKS (SEQ ID NO: 11), and TISPGVSTYYASWAKS (SEQ ID NO: 17); and f) a CDR-H3 comprising or consisting of an amino acid sequence selected from the group consisting of ARGGVGSSWKAFDL (SEQ ID NO: 6), GGVGSSWKAFDL (SEQ ID NO: 12), and ARGGVGSSWKAFDL (SEQ ID NO: 18).

[0039] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP is: A light chain variable region, a) a CDR-L1 consisting of an amino acid sequence selected from the group consisting of ESISTA (SEQ ID NO: 1), QASESISTALA (SEQ ID NO: 7), and QASESISTALA (SEQ ID NO: 13); b) a CDR-L2 consisting of an amino acid sequence selected from the group consisting of KAS, KASTLPS (SEQ ID NO: 8) and KASTLPS (SEQ ID NO: 14); and c) a CDR-L3 consisting of an amino acid sequence selected from the group consisting of QQGFSSGNVHNA (SEQ ID NO: 3), QQGFSSGNVHNA (SEQ ID NO: 9), and QQGFSSGNVHNA (SEQ ID NO: 15); and / or A heavy chain variable region, d) a CDR-H1 consisting of an amino acid sequence selected from the group consisting of GPSLSHFD (SEQ ID NO: 4), HFDIT (SEQ ID NO: 10), and GPSLSHFDIT (SEQ ID NO: 16); e) a CDR-H2 consisting of an amino acid sequence selected from the group consisting of ISPGVST (SEQ ID NO: 5), TISPGVSTYYASWAKS (SEQ ID NO: 11), and TISPGVSTYYASWAKS (SEQ ID NO: 17); and f) a CDR-H3 consisting of an amino acid sequence selected from the group consisting of ARGGVGSSWKAFDL (SEQ ID NO: 6), GGVGSSWKAFDL (SEQ ID NO: 12), and ARGGVGSSWKAFDL (SEQ ID NO: 18).

[0040] The terms "interleukin-1 receptor accessory protein", "IL1RAP" and "IL1-RAP" as used herein specifically include the human IL1RAP protein as described, for example, in GenBank Accession No. AAB84059, NCBI Reference Sequence: NP_002173.1 and UniProtKB / Swiss-Prot Accession No. Q9NPH3-1. IL1RAP is also known in the scientific literature as IL1R3, C3orf13, FLJ37788, IL-1RAcP, and EG3556.

[0041] As used herein, the term "mCAN10" (short for "murine CAN10") refers to an antibody against mouse IL1RAP. This antibody is also referred to as mouse surrogate anti-IL1RAP antibody. mCAN10 can block signaling by IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and / or IL-36γ, and therefore can be used as a surrogate for anti-human IL1RAP antibodies that may lack cross-reactivity with mouse IL1RAP, have similar functional properties, and can be used to evaluate the therapeutic effect of IL1RAP blockade in mouse disease models.

[0042] The term "48D2" as used herein refers to an antibody against human IL1RAP, even though the antibody may also bind to IL1RAP from other species. For example, it will be understood from Example 10 that 48D2 cross-reacts with IL1RAP from human, cynomolgus monkey, and pig. As will be understood from Examples 9 and 10, the term "48D2" may be used to refer to the chimeric antibody (also referred to as "ch48D2") obtained as described in Example 9 and characterized in Example 10.

[0043] As used herein, the term "h48D2" refers to the humanized antibody 48D2, as generated in Example 11 and further characterized in Example 12. As described in Example 12, several variants of h48D2 were obtained (see, e.g., Table 8). As described in Example 13, several additional variants of h48D2 were obtained by deimmunization.

[0044] Because antibodies and fragments thereof are polypeptides, it will be understood that an "antibody or antigen-binding fragment of the invention" may be referred to as a "polypeptide of the invention" or an "antibody polypeptide, or antigen-binding fragment thereof."

[0045] The antibody or antigen-binding fragment of the present invention has specificity for IL1RAP. By "specificity" is meant that the antibody or antigen-binding fragment is capable of binding to IL1RAP in vivo, i.e. under physiological conditions where IL1RAP is present in the human body. Preferably, the antibody or antigen-binding fragment does not bind to other proteins in vivo. Alternatively, it means that the antibody or antigen-binding fragment is capable of binding to IL1RAP ex vivo or in vitro. Such binding specificity can be determined by methods well known in the art, such as ELISA, immunohistochemistry, immunoprecipitation, Western blot, flow cytometry, etc., using transfected cells expressing IL1RAP. Advantageously, the antibody or antigen-binding fragment is capable of selectively binding to IL1RAP, i.e., binds to IL1RAP at least 10-fold more strongly than to other proteins.

[0046] In some embodiments, the antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP binds to human IL1RAP.

[0047] In some embodiments, the antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP binds to a non-human IL1RAP.

[0048] In some embodiments, the antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP binds to IL1RAP from cynomolgus monkeys.

[0049] In some embodiments, the antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP binds to IL1RAP from porcine.

[0050] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP binds to IL1RAP expressed on the surface of a cell.

[0051] In some embodiments, the antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP binds to an epitope on the extracellular domain of IL1RAP.

[0052] In some embodiments, the antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP binds to soluble IL1RAP.

[0053] In some embodiments, the antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP binds to domain 2 of IL1RAP.

[0054] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP binds IL1RAP at or within amino acids 135-234 of IL1RAP.

[0055] Thus, the antibody or antigen-binding fragment may potentially bind to an epitope in / within domain 2 of IL1RAP (see Wang et al., 2010, Nature Immunology, 11:905-912, the disclosure of which is incorporated herein by reference), i.e., within amino acids 135 to 234 of IL1RAP (see accession number Q9NPH3 in UniProtKB / Swiss-Prot). For example, the epitope to which the antibody or antigen-binding fragment binds may be located between amino acids 135-154, 155-174, 175-194, 195-214, or amino acids 215-234, or between amino acids 174-191 of IL1RAP. However, it will be appreciated that the epitope may be non-linear.

[0056] In further embodiments, as described above, the antibody or antigen-binding fragment of the invention comprises or consists of an antibody mimetic selected from the group comprising or consisting of affibodies, tetranectins (CTLDs), adectins (monobodies), anticalins, DARPins (ancalins), avimers, iMabs, microbodies, peptide aptamers, Kunitz domains, and affilins.

[0057] The term "antibody or antigen-binding fragment thereof" includes substantially intact antibodies as well as antibody fragments and derivatives. An intact antibody can be considered as an antibody comprising a variable light region, a variable heavy region, a constant light region, and a constant heavy region. It also includes chimeric antibodies, humanized antibodies, isolated human antibodies, single chain antibodies, bispecific antibodies, antibody heavy chains, antibody light chains, homodimers and heterodimers of antibody heavy and / or light chains, and antigen-binding fragments and derivatives thereof. Suitable antigen-binding fragments and derivatives include Fv fragments (e.g., single chain Fv and disulfide-linked Fv), Fab-like fragments (e.g., Fab fragments, Fab' fragments, and F(ab)2 fragments), single variable domains (e.g., V H and V L Examples of antibody fragments include, but are not necessarily limited to, domain (Fv) and domain antibodies (dAbs, including single and dual formats [i.e. dAb-linker-dAb]). The potential advantages of using antibody fragments rather than whole antibodies are several fold. The reduced size of the fragments may result in improved pharmacological properties, such as improved penetration into solid tissues. Furthermore, antigen-binding fragments such as Fab, Fv, ScFv and dAb antibody fragments can be expressed in and secreted from E. coli and thus can be readily produced in large quantities.

[0058] The term "antibody or antigen-binding fragment thereof" is also intended to encompass antibody mimetics (e.g., non-antibody scaffold structures that have high stability while allowing for the introduction of variability at specific positions). Those skilled in the art of biochemistry will be familiar with many such molecules, which are described in Gebauer & Skerra, 2009, Curr Opin Chem Biol 13(3):245-255, the disclosure of which is incorporated herein by reference. Exemplary antibody mimetics are: affibodies (also called trinectins; Nygren, 2008, FEBS J, 275, 2668-2676); CTLDs (also called Tetranectins Innovations Pharmac. Technol. (2006), 27-30); adnectins (also called monobodies; Meth. Mol. Biol., 352 (2007), 95-109); anticalins (Drug Discovery Today (2005), 10, 23-33); DARPins (ancalins; Nat. Biotechnol. (2004), 22, 575-582); avimers (Nat. Biotechnol. (2005), 23, 1556-1561); microbodies (FEBS J, (2007), 274, 86-95); peptides These include peptide aptamers (Expert. Opin. Biol. Ther. (2005), 5, 783-797); Kunitz domains (J. Pharmacol. Exp. Ther. (2006) 318, 803-809); affilins (Trends. Biotechnol. (2005), 23, 514-522); affimers (Avacta Life Sciences, Wetherby, UK).

[0059] Also included within the scope of the present invention are chimeric T cell receptors (also known as chimeric T cell receptors, chimeric immune receptors, and chimeric antigen receptors or CARs) (Pule et al., 2003, Cytotherapy 5(3):211-26, the disclosure of which is incorporated herein by reference), which are engineered receptors that graft any specificity onto immune effector cells. Typically, CARs are used to graft the specificity of a monoclonal antibody onto T cells, and the grafting of their coding sequence is facilitated by a retroviral vector. The most common form of such molecules is a fusion that contains a single chain variable fragment (scFv) from a monoclonal antibody fused to the transmembrane and endodomains of CD3-zeta. Upon expressing this fusion molecule, T cells recognize and kill target cells expressing the specificity of the transferred monoclonal antibody.

[0060] Those skilled in the art will further appreciate that the present invention also encompasses modified versions of antibodies and antigen-binding fragments thereof, whether existing now or in the future, which are modified, for example, by the covalent attachment of polyethylene glycol or other suitable polymers (see below).

[0061] Methods for producing antibodies and antibody fragments are well known in the art. For example, antibodies can be produced via any one of several methods using inducing in vivo production of antibody molecules, screening immunoglobulin libraries (Orlandi. et al, 1989. Proc. Natl. Acad. Sci. USA 86:3833-3837; Winter et al., 1991, Nature 349:293-299, the disclosures of which are incorporated herein by reference), or production of monoclonal antibody molecules by cell lines in culture. These include, but are not limited to, hybridoma method, human B cell hybridoma method, and Epstein-Barr virus (EBV) hybridoma method (Kohler et al., 1975. Nature 256:4950497; Kozbor et al., 1985. J. Immunol. Methods 81:31-42; Cote et al., 1983. Proc. Natl. Acad. Sci. USA 80:2026-2030; Cole et al., 1984. Mol. Cell. Biol. 62:109-120, the disclosures of which are incorporated herein by reference).

[0062] Suitable methods for the production of monoclonal antibodies are also disclosed in "Monoclonal Antibodies: A manual of techniques", H Zola (CRC Press, 1988, the disclosures of which are incorporated herein by reference) and in "Monoclonal Hybridoma Antibodies: Techniques and Applications", JGR Hurrell (CRC Press, 1982, the disclosures of which are incorporated herein by reference).

[0063] Similarly, antibody fragments can be obtained using methods well known in the art (see, e.g., Harlow & Lane, 1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory, New York, the disclosures of which are incorporated herein by reference). For example, antibody fragments according to the invention are prepared by proteolytic hydrolysis of the antibody or by expressing DNA encoding the fragment in E. coli or mammalian cells (e.g., Chinese hamster ovary cell culture or other protein expression systems). Antibody fragments can also be obtained by digesting whole antibodies with pepsin or papain using conventional methods.

[0064] As used herein, the term "amino acid" includes the standard twenty genetically encoded amino acids and their corresponding stereoisomers in the "D" form (as compared to the natural "L" form), the omega amino acids and other naturally occurring amino acids, non-conventional amino acids (e.g., α,α-disubstituted amino acids, N-alkyl amino acids, etc.) and chemically derivatized amino acids (see below).

[0065] Specific recitation of an amino acid, such as "alanine," "Ala," or "A," refers to both L-alanine and D-alanine unless expressly stated otherwise. The polypeptides (antibodies or antigen-binding fragments thereof) of the invention may also include other non-conventional amino acids as suitable components, so long as the desired functional property is retained by the antibody or antigen-binding fragment. For the amino acid sequences shown, where appropriate, each coded amino acid residue is represented by a one-letter designation that corresponds to the conventional amino acid trivia name.

[0066] In some embodiments, an antibody or antigen-binding fragment thereof as defined herein of the invention comprises or consists of L-amino acids.

[0067] It will be appreciated by those skilled in the art that for human therapy, human or humanized antibodies are preferably used. Humanized versions of non-human antibodies (e.g. rabbit antibodies as described in Examples 9 and 10) are genetically engineered chimeric antibodies or antibody fragments with preferably minimal portions derived from the non-human antibody. Humanized antibodies include antibodies in which the complementarity determining regions of a human antibody (recipient antibody) are replaced with residues from the complementarity determining regions of a non-human species (donor antibody) such as mouse, rat, or rabbit that have the desired function. In some examples, Fv framework residues of a human antibody are replaced with corresponding non-human residues. Humanized antibodies may also contain residues that are not present in the recipient antibody or in the introduced complementarity determining regions or framework sequences. Generally, humanized antibodies comprise substantially all of at least one, and typically two, variable domains, with all or substantially all of the complementarity determining regions corresponding to those of a non-human antibody and all or substantially all of the framework regions corresponding to those of the relevant human consensus sequence. Humanized antibodies optimally comprise at least a portion of an antibody constant region, such as an Fc region, which is typically derived from a human antibody. As discussed elsewhere herein, humanized antibodies may lack cross-reactivity with mouse IL1RAP. Thus, suitable surrogates (e.g., mCAN10) that exhibit similar functional properties to the humanized antibody can be used in mouse disease models, and the results of the surrogate can be interpreted as reflecting the functionality of the humanized antibody.

[0068] Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues, often referred to as introduced residues, are usually taken from the imported variable domain. Humanization can be essentially performed by replacing human complementarity determining regions with the corresponding rodent complementarity determining regions. Such humanized antibodies are thus chimeric antibodies in which substantially less than an intact human variable domain has been replaced with the corresponding sequence from a non-human species. In practice, humanized antibodies are considered to be typical human antibodies in which some of the complementarity determining region residues and possibly framework residues have been replaced with residues from analogous sites in rodent antibodies.

[0069] Human antibodies can also be identified using various techniques known in the art, such as phage display libraries.

[0070] Optimization of antibodies, e.g. by humanization (see, e.g., Jones et al., 1986, Nature 321:522- 525; Reichmann et al., 1988. Nature 332:323-327; Verhoeyen et al., 1988, Science 239:1534-15361; US ​​4,816,567, the disclosures of which are incorporated herein by reference) and / or deimmunization, e.g., as described in Jones et al. (Methods Mol Biol. 2009; 525:405-23) or in Examples 11 and 13, leads to the generation of antibody variants with fine-tuned properties.

[0071] CDRs The antibodies of the present invention are defined by their characteristic complementarity determining region (CDR) sequences. There are several approaches to define the CDR sequences of an antibody. The CDRs of the antibodies of the present invention have been defined by three different well-known approaches (resulting in three CDR definition categories): 1) the Kabat definition, 2) the IMGT definition, or 3) the combined IMGT and Kabat definition.

[0072] It is important to note that within each CDR definition category (Kabat, IMGT, or a combination of IMGT and Kabat, respectively), the CDR sequences are the same for chimeric antibody 48D2 and all its optimized antibody variants, e.g., humanized antibody variants or humanized / deimmunized antibody variants (see the respective sequences in the "Sequences" section).

[0073] One skilled in the art will appreciate that the set of six CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3) can be defined according to either i) IMGT, or ii) Kabat, or iii) a combination of Kabat and IMGT.

[0074] Furthermore, those skilled in the art will appreciate that the CDRs of the antibodies of the present invention can be defined by other approaches known in the art, such as the CDR definitions according to Al-Lazikani et al., (1997) JMB 273, 927-948), Martin (enhanced Chothia), Gelfand or Honneger. Additionally, approaches such as the AbM definition (a combination of the Kabat and Chothia definitions used in Oxford Molecular's AbM antibody modeling software) or the contact definition (based on analysis of crystal structures) exist and are known in the art. For example, Kabat et al. (Sequences of Proteins of Immunological Interest, 1987 and 1991, NIH, Bethesda, Md.), Lefranc et al. (IMGT unique numbering for immunoglobulin and T cell receptor constant domains and Ig superfamily C-like domains, Dev Comp Immunol. 2005;29(3):185-203), and Dondelinger et al. (Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface / Residue Definition, Front. Immunol., 16 October 2018).

[0075] One of skill in the art, when provided with the IMGT and Kabat CDRs presented herein, can use known information to list other CDR naming conventions or approaches (e.g., Chothia), thus, all CDR naming conventions and approaches are encompassed.

[0076] As shown in the specification, the IMGT and Kabat numbering systems identify slightly different residues as CDRs. In some cases, it may be beneficial to define CDRs according to one numbering system, such as IMGT or Kabat. In many cases, these CDR sequences are short (e.g., shorter than the combined numbering system approach), and therefore provide core sequences important for binding. In other cases, it may be beneficial to use a combination of, for example, IMGT and Kabat CDR sequences.

[0077] In some embodiments, the antibody or antigen-binding fragment thereof that has binding specificity for Interleukin-1 Receptor Accessory Protein (IL1RAP) is A light chain variable region, a) CDR-L1 comprising or consisting of the amino acid sequence of ESISTA (SEQ ID NO: 1); CDR-L2 comprising or consisting of the amino acid sequence of KAS; and CDR-L3 comprising or consisting of the amino acid sequence of QQGFSSGNVHNA (SEQ ID NO: 3); b) CDR-L1 comprising or consisting of the amino acid sequence of QASESISTALA (SEQ ID NO: 7); CDR-L2 comprising or consisting of the amino acid sequence of KASTLPS (SEQ ID NO: 8); and CDR-L3 comprising or consisting of the amino acid sequence of QQGFSSGNVHNA (SEQ ID NO: 9); or c) CDR-L1 comprising or consisting of the amino acid sequence of QASESISTALA (SEQ ID NO: 13); CDR-L2 comprising or consisting of the amino acid sequence of KASTLPS (SEQ ID NO: 14); and CDR-L3 comprising or consisting of the amino acid sequence of QQGFSSGNVHNA (SEQ ID NO: 15); and / or A heavy chain variable region, d) CDR-H1 comprising or consisting of the amino acid sequence of GPSLSHFD (SEQ ID NO: 4); CDR-H2 comprising or consisting of the amino acid sequence of ISPGVST (SEQ ID NO: 5); and CDR-H3 comprising or consisting of the amino acid sequence of ARGGVGSSWKAFDL (SEQ ID NO: 6); or e) CDR-H1 comprising or consisting of the amino acid sequence of HFDIT (SEQ ID NO: 10); CDR-H2 comprising or consisting of the amino acid sequence of TISPGVSTYYASWAKS (SEQ ID NO: 11); and A CDR-H3 comprising or consisting of the amino acid sequence of GGVGSSWKAFDL (SEQ ID NO: 12); or f) CDR-H1 comprising or consisting of the amino acid sequence of GPSLSHFDIT (SEQ ID NO: 16); CDR-H2 comprising or consisting of the amino acid sequence of TISPGVSTYYASWAKS (SEQ ID NO: 17); and CDR-H3 comprising or consisting of the amino acid sequence of ARGGVGSSWKAFDL (SEQ ID NO: 18).

[0078] The relationships between the different defined sets of CDR sequences of antibody 48D2 can be illustrated as follows: - CDR residues highlighted in bold were identified using the IMGT numbering system. - underline The CDR residues highlighted in were identified using the Kabat numbering system. - CDR residues are defined by a combination of the IMGT and Kabat numbering systems (combination of bold and underlined sequences).

[0079] [ka]

[0080] In some embodiments, the CDR sequences of an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP are defined according to IMGT:

[0081] SEQ ID NO:1 Variable light chain complementarity determining region 1 (CDR-L1) ESISTA

[0082] Variable light chain complementarity determining region 2 (CDR-L2) KAS

[0083] SEQ ID NO:3 Variable light chain complementarity determining region 3 (CDR-L3) QQGFSSGNVHNA

[0084] SEQ ID NO:4 Variable heavy chain complementarity determining region 1 (CDR-H1) GPSLSHFD

[0085] SEQ ID NO:5 Variable heavy chain complementarity determining region 2 (CDR-H2) ISPGVST

[0086] SEQ ID NO:6 Variable heavy chain complementarity determining region 3 (CDR-H3) ARGGVGSSWKAFDL

[0087] In some embodiments, the CDR sequences of an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP are defined according to Kabat:

[0088] SEQ ID NO:7 Variable light chain complementarity determining region 1 (CDR-L1) QASESISTALA

[0089] SEQ ID NO:8 Variable light chain complementarity determining region 2 (CDR-L2) KASTLPS

[0090] SEQ ID NO:9 Variable light chain complementarity determining region 3 (CDR-L3) QQGFSSGNVHNA

[0091] SEQ ID NO:10 Variable heavy chain complementarity determining region 1 (CDR-H1) HFDIT

[0092] SEQ ID NO:11 Variable heavy chain complementarity determining region 2 (CDR-H2) TISPGVSTYYASWAKS

[0093] SEQ ID NO:12 Variable heavy chain complementarity determining region 3 (CDR-H3) GGVGSSWKAFDL

[0094] In some embodiments, the CDR sequences of an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP are defined according to a combination of IMGT and Kabat:

[0095] SEQ ID NO:13 Variable light chain complementarity determining region 1 (CDR-L1) QASESISTALA

[0096] SEQ ID NO:14 Variable light chain complementarity determining region 2 (CDR-L2) KASTLPS

[0097] SEQ ID NO:15 Variable light chain complementarity determining region 3 (CDR-L3) QQGFSSGNVHNA

[0098] SEQ ID NO:16 Variable heavy chain complementarity determining region 1 (CDR-H1) GPSLSHFDIT

[0099] SEQ ID NO:17 Variable heavy chain complementarity determining region 2 (CDR-H2) TISPGVSTYYASWAKS

[0100] SEQ ID NO:18 Variable heavy chain complementarity determining region 3 (CDR-H3) ARGGVGSSWKAFDL

[0101] However, one of skill in the art will understand that low levels of variation within the CDR sequences (typically only one or two amino acids) can be tolerated without compromising the specificity of the antibody or antigen-binding fragment for IL1RAP.

[0102] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP comprises a CDR as described above (comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-18), where any one of the amino acids of the CDR is changed to another amino acid, with the proviso that no more than two amino acids, e.g., one amino acid, are so changed.

[0103] Light chain variable region Rabbits were immunized with human and murine IL1RAP as detailed in Example 9. The resulting antibodies were then analyzed for desirable properties, such as binding to IL1RAP and the ability to inhibit IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and / or IL-36γ signaling. 48D2 was identified as an antibody with superior characteristics with respect to these features and has since been refined and optimized with the goal of improving the antibody for clinical and therapeutic applications. This optimization resulted in antibody variants with variable light and heavy chain regions that differ in amino acid sequence. However, the CDRs are identical in all antibody variants. In other words, within each CDR definition category (i) Kabat, ii) IMGT, or iii) a combination of IMGT and Kabat), the CDR sequences (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, or CDR-H3, respectively) are the same in chimeric antibody 48D2 and all of its optimized antibody variants.

[0104] In some embodiments, the antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP comprises or consists of an amino acid sequence selected from the group consisting of ESISTA (SEQ ID NO:1), QASESISTALA (SEQ ID NO:7), and QASESISTALA (SEQ ID NO:13); or consisting of an amino acid sequence selected from the group consisting of KAS, KASTLPS (SEQ ID NO:8) and KASTLPS (SEQ ID NO:14); and / or consisting of an amino acid sequence selected from the group consisting of QQGFSSGNVHNA (SEQ ID NO:3), QQGFSSGNVHNA (SEQ ID NO:9), and QQGFSSGNVHNA (SEQ ID NO:15).

[0105] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP comprises a light chain variable region comprising CDRs, a) CDR-L1 comprising or consisting of the amino acid sequence of ESISTA (SEQ ID NO: 1); CDR-L2 comprising or consisting of the amino acid sequence of KAS; and CDR-L3 comprising or consisting of the amino acid sequence of QQGFSSGNVHNA (SEQ ID NO: 3); b) CDR-L1 comprising or consisting of the amino acid sequence of QASESISTALA (SEQ ID NO: 7); CDR-L2 comprising or consisting of the amino acid sequence of KASTLPS (SEQ ID NO: 8); and CDR-L3 comprising or consisting of the amino acid sequence of QQGFSSGNVHNA (SEQ ID NO: 9); or c) CDR-L1 comprising or consisting of the amino acid sequence of QASESISTALA (SEQ ID NO: 13); CDR-L2 comprising or consisting of the amino acid sequence of KASTLPS (SEQ ID NO: 14); and CDR-L3 comprising or consisting of the amino acid sequence of QQGFSSGNVHNA (SEQ ID NO: 15).

[0106] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP comprises a light chain variable region that comprises, or consists of, the amino acid sequence of SEQ ID NO: 19; or an amino acid sequence having at least 70% sequence identity, e.g., at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 19. The light chain variable region is, for example, part of the chimeric (non-humanized, non-optimized) 48D2 antibody described in Examples 9 and 10.

[0107] In some embodiments, an antibody or antigen-binding fragment thereof with binding specificity for IL1RAP comprises a light chain variable region that comprises, or consists of, an amino acid sequence selected from the group consisting of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25; or comprises an amino acid sequence having at least 70% sequence identity, such as at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25. These light chain variable regions are part of the humanized h48D2 antibody variants described, for example, in Examples 11 and 12.

[0108] In some embodiments, the antibody or antigen-binding fragment thereof with binding specificity for IL1RAP comprises the amino acid sequence of SEQ ID NO:31; or an amino acid sequence having at least 70% sequence identity to SEQ ID NO:31, such as at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity. This light chain variable region is part of the humanized and deimmunized 48D2 antibody variants described, for example, in Examples 13 and 14.

[0109] Percent identity (or sequence identity) can be determined, for example, by the LALIGN program at the Expasy facility site (http: / / www.ch.embnet.org / software / LALIGN_form.html) using as parameters the global alignment option, scoring matrix BLOSUM62, an opening gap penalty of -14, and an extend gap penalty of -4. Alternatively, the percentage sequence identity between two polypeptides, such as portions of an antibody, can be determined using a suitable computer program, for example the GAP program of the University of Wisconsin Genetic Computing Group, it being understood that the percent identity is calculated relative to the polypeptides whose sequences are optimally aligned.

[0110] Alignment may be run using the Clustal W program. The parameters used may be as follows: -Fast pairwise alignment parameters: K-tuple (word) size; 1, window size; 5, gap penalty; 3, number of top diagonals; 5. Scoring method: x%. - Multiple sequence parameters: Gap open penalty; 10, Gap extension penalty; 0.05 -Scoring matrix BLOSUM.

[0111] Alternatively, the BESTFIT program may be used to determine the alignment of local sequences.

[0112] One skilled in the art would consider further modifications of the light chain variable region described above, for example to further optimize the antibody or antigen-binding fragment, For example, one skilled in the art would consider altering amino acids outside of the framework regions, i.e., the epitope-binding CDR regions, thereby leaving the CDR regions unchanged, as is done during humanization and deimmunization procedures.

[0113] In some embodiments, an antibody or antigen-binding fragment thereof with binding specificity for IL1RAP comprises a light chain variable region as described above, wherein any one of the amino acids in the framework regions of the light chain variable region is altered to another amino acid, with the exception that no more than five amino acids, e.g., four amino acids, no more than three amino acids, e.g., two amino acids, or no more than one amino acid are not so altered.

[0114] Heavy chain variable region In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP comprises or consists of an amino acid sequence selected from the group consisting of GPSLSHFD (SEQ ID NO:4), HFDIT (SEQ ID NO:10), and GPSLSHFDIT (SEQ ID NO:16); comprising or consisting of an amino acid sequence selected from the group consisting of ISPGVST (SEQ ID NO:5), TISPGVSTYYASWAKS (SEQ ID NO:11), and TISPGVSTYYASWAKS (SEQ ID NO:17); and The invention comprises a heavy chain variable region comprising CDRs, the heavy chain variable region comprising or consisting of an amino acid sequence selected from the group consisting of ARGGVGSSWKAFDL (SEQ ID NO: 6), GGVGSSWKAFDL (SEQ ID NO: 12), and ARGGVGSSWKAFDL (SEQ ID NO: 18).

[0115] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP comprises: a) CDR-H1 comprising or consisting of the amino acid sequence of GPSLSHFD (SEQ ID NO: 4); CDR-H2 comprising or consisting of the amino acid sequence of ISPGVST (SEQ ID NO: 5); and CDR-H3 comprising or consisting of the amino acid sequence of ARGGVGSSWKAFDL (SEQ ID NO: 6); b) CDR-H1 comprising or consisting of the amino acid sequence of HFDIT (SEQ ID NO: 10); CDR-H2 comprising or consisting of the amino acid sequence of TISPGVSTYYASWAKS (SEQ ID NO: 11); and CDR-H3 comprising or consisting of the amino acid sequence of GGVGSSWKAFDL (SEQ ID NO: 12); or c) CDR-H1 comprising or consisting of the amino acid sequence of GPSLSHFDIT (SEQ ID NO: 16); CDR-H2 comprising or consisting of the amino acid sequence of TISPGVSTYYASWAKS (SEQ ID NO: 17); and CDR-H3 comprising or consisting of the amino acid sequence of ARGGVGSSWKAFDL (SEQ ID NO: 18).

[0116] In some embodiments, the antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP has the amino acid sequence of SEQ ID NO:20; Or, it comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:20, such as at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity. This heavy chain variable region is part of the chimeric (non-humanized, non-optimized) 48D2 antibody described, for example, in Examples 9 and 10.

[0117] In some embodiments, the antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP has an amino acid sequence selected from the group consisting of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30; Or, it comprises an amino acid sequence having at least 70% sequence identity, such as at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30. These heavy chain variable regions are part of the humanized h48D2 antibody variants described, for example, in Examples 11 and 12.

[0118] In some embodiments, the antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP has an amino acid sequence selected from the group consisting of SEQ ID NO:32, SEQ ID NO:33, and SEQ ID NO:34; Or, it comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:32, SEQ ID NO:33, or SEQ ID NO:34, such as at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity. These heavy chain variable regions are part of the humanized and deimmunized 48D2 antibody variants described, for example, in Examples 13 and 14.

[0119] One skilled in the art would consider further modifications of the heavy chain variable regions described above, for example to further optimize the antibody or antigen-binding fragment. For example, one skilled in the art would consider altering amino acids outside of the framework regions, i.e., the epitope-binding CDR regions, thereby leaving the CDR regions unchanged, as is done during humanization and deimmunization procedures.

[0120] In some embodiments, an antibody or antigen-binding fragment thereof with binding specificity for IL1RAP comprises a heavy chain variable region as described above, wherein any one of the amino acids in the framework regions of the heavy chain variable region is altered to another amino acid, with the exception that no more than five amino acids, e.g., four amino acids, no more than three amino acids, e.g., two amino acids, or no more than one amino acid are so altered.

[0121] Variable Light Chain and Variable Heavy Chain Combinations One skilled in the art will appreciate that any of the above-mentioned variants of the light chain variable region can be combined with any of the above-mentioned variants of the heavy chain variable region.

[0122] In some embodiments, an antibody or antigen-binding fragment thereof with binding specificity for IL1RAP comprises a light chain variable region that comprises, or consists of, the amino acid sequence of SEQ ID NO:19 and a heavy chain variable region that comprises, or consists of, the amino acid sequence of SEQ ID NO:20, or an amino acid sequence having at least 70% sequence identity to SEQ ID NO:19 or SEQ ID NO:20, such as at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity. This combination of light and heavy chain variable regions is part of the chimeric (non-humanized, non-optimized) 48D2 antibody described, for example, in Examples 9 and 10.

[0123] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP is: a) a light chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 21 and a heavy chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 26; b) a light chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 21 and a heavy chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 27; c) a light chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 21 and a heavy chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 28; d) a light chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO:21 and a heavy chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO:29; e) a light chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO:21 and a heavy chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO:30; f) a light chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 22 and a heavy chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 26; g) a light chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 22 and a heavy chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 27; h) a light chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 22 and a heavy chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 28; i) a light chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 22 and a heavy chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 29; j) a light chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 22 and a heavy chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 30; k) a light chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 23 and a heavy chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 26; l) a light chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 23 and a heavy chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 27; m) a light chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 23 and a heavy chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 28; n) a light chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 23 and a heavy chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 29; o) a light chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 23 and a heavy chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 30; p) a light chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 24 and a heavy chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 26; q) a light chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 24 and a heavy chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 27; r) a light chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 24 and a heavy chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 28; s) a light chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 24 and a heavy chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 29; t) a light chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 24 and a heavy chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 30; u) a light chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 25 and a heavy chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 26; v) a light chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 25 and a heavy chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 27; w) a light chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 25 and a heavy chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 28; x) a light chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 25 and a heavy chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 29; y) a light chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 25 and a heavy chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 30; or an amino acid sequence having at least 70% sequence identity, such as at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NO:21 to SEQ ID NO:30. These light and heavy chain variable region combinations are part of the humanized h48D2 antibody variants described, for example, in Examples 11 and 12. The following table is an alternative way of depicting exemplary combinations of humanized light and heavy chain variable regions:

[0124] [Table 1]

[0125] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP is: a) a light chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 24 and a heavy chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 30; b) a light chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO:24 and a heavy chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO:32; c) a light chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 24 and a heavy chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 33; d) a light chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 24 and a heavy chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 34; e) a light chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO:25 and a heavy chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO:30; f) a light chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 25 and a heavy chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 32; g) a light chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 25 and a heavy chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 33; h) a light chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 25 and a heavy chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 34; i) a light chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 31 and a heavy chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 30; j) a light chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 31 and a heavy chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 32; k) a light chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 31 and a heavy chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 33; or l) a light chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 31 and a heavy chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 34; or an amino acid sequence having at least 70% sequence identity, such as at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:31, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, or SEQ ID NO:34.

[0126] These light and heavy chain variable region combinations are part of the humanized and / or deimmunized h48D2 antibody variants described, for example, in Examples 13 and 14.

[0127] The following table is an alternative way of depicting exemplary combinations of humanized and / or humanized and deimmunized light and heavy chain variable regions:

[0128] [Table 2]

[0129] It will be appreciated by those skilled in the art that the above defined light chain variable regions, heavy chain variable regions and combinations thereof may further be combined with light / heavy chain constant regions or parts thereof (see below).

[0130] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP comprises a light chain variable region that comprises or consists of SEQ ID NO:24, and a heavy chain variable region that comprises or consists of SEQ ID NO:34.

[0131] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP comprises a light chain variable region that comprises or consists of SEQ ID NO:31, and a heavy chain variable region that comprises or consists of SEQ ID NO:34.

[0132] In some embodiments, an antibody or antigen-binding fragment thereof with binding specificity for IL1RAP comprises a heavy chain variable region as described above, wherein any one of the amino acids in the framework regions of the light chain variable region and / or the heavy chain variable region is altered to another amino acid, with the exception that no more than five amino acids, e.g., four amino acids, no more than three amino acids, e.g., two amino acids, or one amino acid are not so altered.

[0133] Constant region and Fc region One of skill in the art will understand that any light constant region known in the art can be combined with any of the light variable region variants described above, and any heavy constant region known in the art can be combined with any of the heavy variable region variants described above to form a complete antibody.

[0134] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP comprises a light chain constant region, or a portion thereof.

[0135] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP comprises a light chain constant region that is a kappa or lambda light chain.

[0136] In some embodiments, the antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP comprises a κ light chain.

[0137] In some embodiments, the antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP comprises a lambda light chain.

[0138] In some embodiments, an antibody or antigen-binding fragment thereof with binding specificity for IL1RAP comprises a light chain constant region comprising or consisting of the amino acid sequence of SEQ ID NO:35, or an amino acid sequence having at least 70% sequence identity to SEQ ID NO:35, such as at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity.

[0139] In some embodiments, the antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP comprises a heavy chain constant region or a portion thereof.

[0140] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP comprises a heavy chain constant region selected from the group consisting of α, δ, γ, ε, and μ.

[0141] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP comprises a heavy chain constant region that is an immunoglobulin isotype selected from the group consisting of IgA, IgD, IgG, IgE, and IgM.

[0142] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP comprises a heavy chain constant region that is an IgG immunoglobulin isotype.

[0143] In some embodiments, the antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP is an IgG antibody.

[0144] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP comprises a heavy chain constant region of an immunoglobulin subclass selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

[0145] In some embodiments, the antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP is an IgG1 antibody.

[0146] In some embodiments, the antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP is an IgG2 antibody.

[0147] In some embodiments, the antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP is an IgG3 antibody.

[0148] In some embodiments, the antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP is an IgG4 antibody.

[0149] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP comprises, or consists of, a heavy chain constant region comprising, or consisting of, the amino acid sequence of SEQ ID NO:36 or SEQ ID NO:2; or an amino acid sequence having at least 70% sequence identity to SEQ ID NO:36 or SEQ ID NO:2, such as at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity.

[0150] In some embodiments, an antibody or antigen-binding fragment thereof with binding specificity for IL1RAP comprises or consists of a light chain constant region and / or a heavy chain constant region in which any one of the amino acids in said light chain constant region and / or said heavy chain constant region is changed to another amino acid, with the exception that no more than five amino acids, e.g., four amino acids, no more than three amino acids, e.g., two amino acids, or no more than one amino acid are not so changed.

[0151] In some embodiments, an antibody or antigen-binding fragment of the invention comprises the CH1, CH2, and / or CH3 regions of an IgG heavy chain (such as an IgG1, IgG2, IgG3, or IgG4 heavy chain). Thus, the antibody or antigen-binding fragment may comprise part or all of the constant region from an IgG1 heavy chain. For example, the antibody or antigen-binding fragment may be a Fab fragment that is composed of the CH1 and CL constant regions in combination with any of the heavy and light variable regions, respectively, as defined above.

[0152] Similarly, the above-described antibodies or antigen-binding fragments of the invention may further comprise a light chain constant region or a portion thereof. For example, the antibodies or antigen-binding fragments may comprise the CL region from a kappa or lambda light chain.

[0153] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP comprises: - any one of the light chain variable regions listed above; and / or - any one of the heavy chain variable regions listed above; and / or - any one of the light chain constant regions listed above; and / or - comprising, or consisting of, any one of the heavy chain constant regions above.

[0154] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP comprises: a light chain variable region comprising or consisting of an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 19, 21, 22, 23, 24, 25, and 31; and / or - a heavy chain variable region comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 26, 27, 28, 29, 30, 32, 33, and 34; and / or - a light chain constant region comprising or consisting of the amino acid sequence of SEQ ID NO: 35; and / or - a heavy chain constant region comprising or consisting of the amino acid sequence of SEQ ID NO: 36 or 2.

[0155] In some embodiments, the antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP comprises an Fc region.

[0156] The Fc region may also be referred to as the Fc domain.

[0157] In some embodiments, the antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP comprises a naturally occurring Fc region.

[0158] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP comprises a non-naturally occurring Fc region.

[0159] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP comprises a modified, eg, mutated, Fc region with an IgG constant region.

[0160] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP comprises an Fc region, wherein the Fc region comprises one or more of the mutations identified in Table 1.

[0161] The Fc region may be naturally occurring (e.g., part of an endogenously produced antibody) or artificial (e.g., containing one or more point mutations relative to a naturally occurring Fc region).

[0162] As is well known in the art, the Fc region of an antibody mediates its serum half-life and effector functions, such as complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP).

[0163] The Fc region of therapeutic monoclonal antibodies and Fc fusion proteins can be engineered to generate molecules with the desired pharmacological activity (Strohl, 2009, Curr Opin Biotechnol 20(6):685-91, the disclosure of which is incorporated herein by reference).

[0164] (a) Engineered Fc region to extend half-life One approach to improving the efficacy of antibody drugs is to increase the serum persistence of the antibody, allowing for higher circulating levels and less frequent and less expensive administration.

[0165] The half-life of IgG depends on its pH-dependent binding to the neonatal receptor FcRn, which is expressed on the surface of endothelial cells and binds IgG in a pH-dependent manner, protecting it from degradation.

[0166] Some antibodies that selectively bind to FcRn at pH 6.0 but not at pH 7.4 exhibit extended half-lives in various animal models.

[0167] Several mutations located at the interface between the CH2 and CH3 domains, such as T250Q / M428L (Hinton et al., 2004, J Biol Chem. 279(8):6213-6, the disclosure of which is incorporated herein by reference) and M252Y / S254T / T256E + H433K / N434F (Vaccaro et al., 2005, Nat. Biotechnol. 23(10):1283-8, the disclosure of which is incorporated herein by reference), have been shown to increase the binding affinity to FcRn and the half-life of IgG1 in vivo.

[0168] (b) Engineered Fc regions to alter effector function Depending on the application of an antibody drug or Fc fusion protein, it may be desirable to decrease or increase the effector function (such as ADCC). For antibodies that target cell surface molecules, particularly those on immune cells, deprivation of effector function may be required for certain clinical applications.

[0169] Conversely, antibodies for oncology applications (such as treatment of leukemia and solid tumors; see below) can have improved therapeutic activity by enhancing effector function. The four human IgG isotypes bind with different affinities to the activating Fcγ receptors (FcγRI, FcγRIIa, FcγRIIIa), the inhibitory FcγRIIb receptor, and the first component of complement (C1q), resulting in very distinct effector functions (Bruhns et al., 2009, Blood. 113(16):3716-25, the disclosure of which is incorporated herein by reference).

[0170] The binding of IgG to FcγRs or C1q depends on residues located in the hinge region and the CH2 domain. Two regions of the CH2 domain are important for FcγR and C1q binding and have unique sequences in IgG2 and IgG4. Substitution of IgG2 residues at positions 233-236 and IgG4 residues at positions 327, 330 and 331 of human IgG1 has been shown to significantly reduce ADCC and CDC (Armour et al., 1999, Eur J Immunol. 29(8):2613-24; Shields et al., 2001, J Biol Chem. 276(9):6591-604, the disclosures of which are incorporated herein by reference). The "LALA" mutation, L234A / LL235A, has been introduced in several therapeutic IgG1 antibodies to create effector function-silent Fc regions, e.g., Xu et al., 2000, Cell. Immuno. 200:16-26. Furthermore, Idusogie et al. demonstrated that alanine substitutions at different positions, including K322, significantly reduced complement activation (Idusogie et al., 2000, J Immunol. 164(8):4178-84, the disclosure of which is incorporated herein by reference). Similarly, mutations in the CH2 domain of mouse IgG2A were shown to reduce binding to FcγRI and C1q (Steurer. et al., 1995. J Immunol. 155(3):1165-74, the disclosure of which is incorporated herein by reference).

[0171] Numerous mutations have been made in the CH2 domain of human IgG1 and their effects on ADCC and CDC have been tested in vitro (see references cited above). In particular, an alanine substitution at position 333 has been reported to increase both ADCC and CDC (Shields et al., 2001, supra; Steurer et al., 1995, supra). Lazar et al. described a triple mutant (S239D / I332E / A330L) with high affinity for FcγRIIIa and low affinity for FcγRIIb, resulting in enhanced ADCC (Lazar et al., 2006, PNAS 103(11):4005-4010, the disclosure of which is incorporated herein by reference). The same mutations were used to generate antibodies with increased ADCC (Ryan et al., 2007, Mol. Cancer Ther. 6:3009-3018, the disclosure of which is incorporated herein by reference). Richards et al. investigated a slightly different triple mutant (S239D / I332E / G236A) that improved FcγRIIIa affinity and FcγRIIa / FcγRIIb ratio that mediated enhanced phagocytosis of target cells by macrophages (Richards et al., 2008. Mol Cancer Ther. 7(8):2517-27, the disclosure of which is incorporated herein by reference).

[0172] Being devoid of effector functions, IgG4 antibodies display an IgG subclass suitable for receptor blockade (i.e., inhibition of IL-1 signaling) without cell depletion. IgG4 molecules can exchange half molecules in a dynamic process called Fab-arm exchange. This phenomenon can also occur in vivo between therapeutic antibodies and endogenous IgG4.

[0173] The S228P mutation has been shown to block this recombination process, allowing for the design of unpredictable therapeutic IgG4 antibodies (Labrijn et al., 2009, Nat Biotechnol. 27(8):767-71, the disclosure of which is incorporated herein by reference).

[0174] Examples of engineered Fc regions are shown in Table 1 below.

[0175] [Table 3]

[0176] [Table 4]

[0177] *The positions of the Fc amino acid mutations are defined using the EU numbering scheme (see Edelman et al., 1969, Proc. Natl. Acad. Sci. USA, 63:78-85), which may differ from the actual numbering in, e.g., SEQ ID NOs: 36 and 2, but see further detailed explanation regarding mutation numbering below.

[0178] Reference to Table 1 1.Hinton et al 2004 J.Biol.Chem.279(8):6213-6) 2.Vaccaro et al.2005 Nat Biotechnol.23(10):1283-8) 3.Zalevsky et al 2010 Nat.Biotechnology 28(2):157-159 4.Armour KL.et al.,1999.Eur J Immunol.29(8):2613-24 5.Shields RL.et al.,2001.J Biol Chem.276(9):6591-604 6.Masuda et al.2007,Mol Immunol.44(12):3122-31 7. Bushfield et al 2014, Leukemia 28(11):2213-21 8.Okazaki et al.2004, J Mol Biol.;336(5):1239-49 9.Idusogie et al.,2000.J Immunol.164(8):4178-84 10.Datta-Mannan A.et al.,2007.Drug Metab.Dispos.35:86-94 11. Steurer W. et al., 1995. J Immunol. 155(3):1165-74 12.Richards et al.2008 Mol Cancer There.7(8):2517-27 13. US 7,960,512 B2 14.EP 2213683 15.Labrijn AF.et al.,2009.Nat Biotechnol.27(8):767-71

[0179] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP comprises an Fc region, wherein the Fc region comprises one or more mutations selected from the group consisting of L234A, L235A, P329G, G237A, P238S, H269A, A330S, and P331S as defined by the EU index. These mutations may reduce the effector functions of the antibody. The EU index is conventionally used in the art (see generally Kabat et al., 1991).

[0180] Those skilled in the art will understand that the exact positions of the Fc mutations described herein may vary in the antibodies of the present invention, due to species differences in the variable regions resulting in species differences in chimeric antibodies (and humanized and humanized / deimmunized antibodies derived therefrom). Changing the length of the variable heavy region regions leads to shifts in the amino acid positions compared to those of the EU index, but the relative positions of the amino acids in the Fc portion are maintained.

[0181] For example, the LALA mutation is defined by the EU index as occurring at positions L234A, L235A.

[0182] Exemplary antibodies of the invention include, for example: The heavy chain variable region may comprise the variable heavy chain region of SEQ ID NO:20 (non-humanized, non-immunized). QEQLEESGGGLVKPGGSLTLTCTVSGPSLSHFDITWVRQAPGSGLEWIGTISPGVSTYYASWAKSRSTITSNTNLNTVTLKMTSLTAADTATYFCARGGVGSSWKAFDLWGPGTLVTISS And, for example, the constant heavy chain of SEQ ID NO:36 (not including the LALA mutation). Immunoglobulin IgG1 constant heavy chain (heavy chain constant region) (za allotype) [ka] (The bold and underlined amino acid residues indicate the residues that are changed when the LALA mutation is introduced, as shown in SEQ ID NO:2 below).

[0183] Alternatively, exemplary antibodies of the invention can be, for example: It may comprise the variable heavy chain region of SEQ ID NO:20. QEQLEESGGGLVKPGGSLTLTCTVSGPSLSHFDITWVRQAPGSGLEWIGTISPGVSTYYASWAKSRSTITSNTNLNTVTLKMTSLTAADTATYFCARGGVGSSWKAFDLWGPGTLVTISS And, for example, SEQ ID NO:2 is the constant heavy chain (containing the LALA mutation). Immunoglobulin IgG1 constant heavy chain (heavy chain constant region) with "LALA" mutation (za allotype) [ka]

[0184] Amino acid mutations (in the above sequence [ka] ) are at positions 237 and 238 (i.e., L237A, L238A), which correspond to the L234A and L235A mutations according to the EU index in terms of the amino acid sequence of the heavy chain constant region.

[0185] One of skill in the art will appreciate that the same reasoning applies to other named mutations known in the art, such as P329G, G237A, P238S, H269A, A330S, and P331S as defined by the EU index.

[0186] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP comprises an Fc region and any carbohydrate chain attached to the Fc region lacks fucose.

[0187] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP comprises an Fc region that lacks fucose.

[0188] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP comprises an Fc region, and the carbohydrate chains attached to the Fc region are low in fucose.

[0189] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP comprises a fucose-deficient Fc region.

[0190] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP, wherein the antibody or antigen-binding fragment is produced in a FUT8 negative cell line.

[0191] In some embodiments, the antibody is produced in a FUT8 negative cell line. It is possible to generate a FUT8-negative cell line by knocking out the FUT8 gene, which encodes the a-(1,6)-fucosyltransferase enzyme that catalyzes fucose transfer. Antibodies with reduced fucosylation in the Fc region exhibit improved ADCC activity, as described in WO00 / 61739A1. Those skilled in the art will appreciate that a FUT8-negative cell line is useful for producing antibodies with improved ADCC activity.

[0192] In some embodiments, the antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP is an intact antibody or a portion of an intact antibody.

[0193] In some embodiments, antibodies or antigen-binding fragments thereof that have binding specificity for IL1RAP include Fv fragments (e.g., single chain Fvs and disulfide-linked Fvs), Fab-like fragments (e.g., Fab fragments, Fab' fragments, and F(ab)2 fragments), and domain antibodies (e.g., single V H Variable domain or V L The antigen-binding fragment may comprise or consist of an antigen-binding fragment selected from the group consisting of:

[0194] The "Sequences" section below provides sequences of certain exemplary embodiments of the antibodies disclosed and claimed herein.

[0195] Inhibition of signal transduction Interleukins are involved in a variety of diseases and disorders.

[0196] Interleukins, like cytokines, include interleukins of the IL-1 family. Cytokines (e.g., chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors) are important in cell signaling. In other words, cytokines have a signaling function to cells, such as through binding to their respective receptors. For example, signaling of IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and / or IL-36γ includes events that these cytokines trigger on the cell surface or within the cell, as described herein. At the cell surface, these signaling events affect, for example, the identification of receptors, the properties of co-receptors and other molecules, and the binding of molecules outside the cell, on the cell surface, within the cell membrane, or within the cell. These events can be physiological or pathological. These events can be, for example, biological pathways that lead to a reaction within the cell in response to the signaling. This reaction can be physiological or pathological.

[0197] As used herein, "inhibition of signal transduction" is defined as decreasing the activity, confirmation, or production of said signal transduction event or, for example, a receptor or biological pathway or molecular activity. This inhibition is understood to be compared to the situation in the presence of an inhibitor (in the present case, an antibody or antigen-binding fragment thereof having binding specificity for IL1RAP) compared to the situation in the absence of the inhibitor. Those skilled in the art will understand that the degree of inhibition can be determined by methods well known in the art depending on the signal transduction event or pathway or activity to be measured. The method can be, for example, a cellular assay, for the examples as described in Example 10, 12, or 14.

[0198] It should be noted that as used herein, phrases such as "inhibit (for example) IL-1α signaling" are used interchangeably with the phrase "inhibit (for example) IL-1α signaling."

[0199] IL-1 Interleukin-1 (IL-1) is a potent proinflammatory cytokine that can be produced by various types of cells, including mononuclear phagocytes, in response to infection and inflammation. The IL-1 family consists of seven agonists, including IL-1α and IL-1β, and three naturally occurring receptor antagonists, including the IL-1 receptor antagonist (IL-1Ra or IL1RA) (Dinarello, CA, Blood 1996, 87(6):2095-147). Two IL-1 receptors have been identified: IL-1R type I and IL-1R type II. Both receptors can interact with all three forms of IL-1 family molecules. IL-1RI is responsible for mediating cell activation by IL-1. However, the IL-1 / IL-1RI complex cannot signal by itself, but depends on its association with a second receptor chain, the IL-1R accessory protein (IL1RAP) (Dinarello, CA, Blood 1996, 87(6):2095-147). In contrast to IL-1RI, IL-1RII does not induce cellular activation upon binding to IL-1, so IL-1RII functions as a regulatory decoy receptor, resulting in a net reduction in IL-1 that can bind to IL-1RI.

[0200] In addition to IL1 signaling, IL1RAP is important for mediating the effects of IL33 via the ST2 / IL1RAP complex and IL36 via the IL1Rrp2 / IL1RAP complex (Garlanda et al, Immunity. 2013 Dec 12;39(6):1003-18).

[0201] IL-1α can also be released from injured cells and function as an alarmin. IL-1 is a potent proinflammatory cytokine that is induced at sites of local infection or inflammation and participates in the control of various physiological and cellular events (reviewed in Dinarello CA, CHEST, 2000, 118:503-508 and Dinarello, CA, Clin Exp Rheumatol, 2002, 20(5 Suppl 27):S1-13). It can activate several cell types, including leukocytes and endothelial cells. IL-1 promotes the production and expression of inflammatory mediators, such as adhesion molecules, cytokines, chemokines, prostaglandin E2, and nitric oxide (NO), inducing and amplifying immune responses. As a result, local inflammation is amplified and sustained. In addition, the production of inflammatory mediators by IL-1 leads to fever, headache, hypotension, and weight loss. Furthermore, IL-1 is a hematopoietic growth factor and has been shown to reduce leukocyte and platelet nadirs in patients undergoing bone marrow transplantation. IL-1 has also been shown to induce the production of vascular endothelial growth factor to promote angiogenesis, thereby enhancing pannus formation and blood supply in rheumatoid joints. IL-1 has been known to promote bone and cartilage degradation in rheumatic diseases. Finally, IL-1 has been implicated as a key player in the inflammatory response in cardiovascular and fibrotic diseases.

[0202] IL-33 IL-33 is normally released from damaged or necrotic barrier cells (endothelial and epithelial cells) and functions as an endogenous danger signal, an alarmin, to inform the immune system of tissue damage during trauma or infection (Liew FY, Interleukin-33 in health and disease. Nature Reviews Immunology, 16, 676-689 (2016)). IL-33 induces T helper 2 (Th2) cells, mast cells, type 2 innate lymphocytes, eosinophils, and basophils to produce type 2 cytokines (IL-5, IL-13, etc.). IL-33 is also known to target endothelial cells and induce angiogenesis. As a Th2-inducing cytokine, IL-33 has been implicated in asthma, allergic diseases, inflammatory bowel disease, and dermatitis. IL-33 can potently stimulate a variety of cells, and its pleiotropic properties are reflected in its roles in tissue and metabolic homeostasis, infection, inflammation, cancer, and diseases of the central nervous system.

[0203] IL-36 IL-36 cytokines α, β, and γ are expressed in various cell types, including keratinocytes, bronchial epithelium, neurons, dendritic cells, and macrophages. IL-36 is most active in barrier tissues (e.g., skin, lung, and intestine), suggesting that its primary role is to regulate the body's interactions with the environment. IL-36 is known to activate NF-κB and mitogen-activated protein kinase in target cells that express the IL-36 receptor, such as keratinocytes, monocytes, dendritic cells, and CD4 T cells. Emerging evidence indicates that IL-36 signaling is involved in the activation of innate and adaptive immune responses (Ding L,IL-36 cytokines in autoimmunity and inflammatory disease,Oncotarget,Vol.9,(No.2),pp:2895-2901(2018)).

[0204] Aberrant IL-36 plays an important role in inflammatory skin diseases such as psoriasis and atopic dermatitis, and our findings suggest that aberrant IL-36 activity promotes pulmonary, renal, and intestinal inflammatory diseases, highlighting the potential of IL-36 as a therapeutic target for common inflammatory diseases.

[0205] Interestingly, all the cytokines described herein (IL-1α, IL-1β, IL-33, IL-36α, IL-36β, IL-36γ) have been shown to also target stromal cells such as fibroblasts and endothelial cells, thus implicating these cytokines in fibrotic diseases and disorders apart from their well-established effects in inflammatory diseases and disorders.

[0206] The antibody or antigen-binding fragment thereof according to the first aspect of the invention has the ability to inhibit signalling of a cytokine ligand and / or receptor of the interleukin-1 (IL-1) family. A person skilled in the art will appreciate that inhibition occurs through binding of the antibody or antigen-binding fragment to an epitope on IL1RAP.

[0207] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP is capable of inhibiting signal transduction upon binding to IL1RAP.

[0208] One of skill in the art will appreciate that the binding of an antibody or antigen-binding fragment thereof to IL1RAP may affect IL1RAP in different ways, e.g., at the molecular level or at the conformational level, however, it may not be entirely known today how the binding of an antibody or antigen-binding fragment thereof to IL1RAP affects IL1RAP. One possibility is that the binding of the antibody or antigen-binding fragment thereof to IL1RAP affects the association of IL1RAP with the IL-1 receptor, the IL-33 receptor, and / or the IL-36 receptor, respectively. As a result, a proper receptor complex consisting of any one of the IL-1 receptor, the IL-33 receptor, or the IL-36 receptor with IL1RAP as a co-receptor may not form. As a result, when cytokines such as IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and IL-36γ bind to their cognate receptors (IL-1 receptor, IL-33 receptor, or IL-36 receptor, respectively), signaling of these cytokines may be inhibited, e.g., inhibited, e.g., blocked, e.g., essentially completely inhibited, e.g., partially inhibited, etc.

[0209] One skilled in the art will appreciate that not all IL1RAP binding antibodies that bind to any one epitope of IL1RAP are capable of disrupting the association between IL1RAP and the IL-1 receptor, IL-33 receptor, and / or IL-36 receptor, respectively, thereby inhibiting signal transduction. The present invention strives to provide such antibodies.

[0210] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP is capable of inhibiting signaling of interleukin-1 (IL-1) family cytokine ligands and / or receptors.

[0211] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP can inhibit IL1RAP-dependent interleukin-1 (IL-1) family cytokine ligand and / or receptor signaling.

[0212] In some embodiments, the antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP is capable of inhibiting signaling of at least one cytokine selected from the group consisting of IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and IL-36γ, or any combination thereof.

[0213] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP can inhibit signaling of IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and IL-36γ.

[0214] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP is capable of inhibiting IL-1α signaling.

[0215] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP is capable of inhibiting IL-1β signaling.

[0216] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP is capable of inhibiting IL-33 signaling.

[0217] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP is capable of inhibiting IL-36α signaling.

[0218] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP is capable of inhibiting IL-36β signaling.

[0219] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP is capable of inhibiting IL-36γ signaling.

[0220] Those skilled in the art will appreciate that inhibition of signal transduction can be to various degrees. Inhibition of signal transduction can be complete, such as blocking signal transduction, or substantially complete. Inhibition of signal transduction can be partial, such as non-complete, such as reducing signal transduction. Given the uncertainty in assessing completeness associated with the methodology used to measure inhibition of signal transduction, "substantially complete" shall be understood as "complete."

[0221] For example, signaling can be inhibited by at least 10%, 20%, 30%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more relative to signaling in the absence of the antibody or antigen-binding fragment of the invention.

[0222] In some embodiments, inhibition of signal transduction is between 10-100% compared to signal transduction in the absence of the antibody or antigen-binding fragment of the present invention. More preferably, inhibition of signal transduction is between 25-100%. Even more preferably, inhibition of signal transduction is between 50-100%.

[0223] Signaling can be inhibited 100% compared to signaling in the absence of the antibody or antigen-binding fragment of the invention.

[0224] The degree of inhibition of IL-1, IL-33, and / or IL-36 signaling by the antibodies or antigen-binding fragments of the present invention can be determined by methods well known in the art, for example, the methods used in Examples 10, 12, and 14.

[0225] In a preferred embodiment, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP is capable of substantially completely inhibiting signal transduction.

[0226] In a preferred embodiment, inhibiting signal transduction is substantially complete inhibition of signal transduction.

[0227] In a preferred embodiment, the antibody or antigen-binding fragment thereof having binding specificity for IL1RAP can substantially completely inhibit the signaling of at least one cytokine selected from the group consisting of IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and IL-36γ, or any combination thereof.

[0228] In a preferred embodiment, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP is capable of substantially completely inhibiting IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and IL-36γ signaling.

[0229] The antibody may further inhibit signal transduction to some extent, i.e. partially rather than substantially completely. As a result, inhibition of signal transduction may mean partial inhibition rather than substantially complete inhibition.

[0230] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP can partially inhibit signal transduction.

[0231] In some embodiments, inhibiting signaling is partial inhibition of signaling.

[0232] In some embodiments, the antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP can partially inhibit signaling of at least one cytokine selected from the group consisting of IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and IL-36γ, or any combination thereof.

[0233] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP can partially inhibit signaling of IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and IL-36γ.

[0234] In some embodiments, an antibody or antigen-binding fragment thereof with binding specificity for IL1RAP can inhibit IL-1α, IL-β, IL-33, IL-36α, IL-36β, and / or IL-36γ signaling by at least 10%, 20%, 30%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, 98%, 99% relative to signaling in the absence of the antibody or antigen-binding fragment of the invention.

[0235] Those skilled in the art will understand that in some situations, complete inhibition is desirable, where a process, e.g., a disease process, dependent on IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and / or IL-36γ signaling should be completely and rapidly affected to alleviate or treat the disease. In other situations, partial inhibition is desirable, where a process, e.g., a process, dependent on IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and / or IL-36γ signaling, e.g., a disease process, is not completely affected, but should be modified, e.g., to avoid the side effects of complete inhibition. Those skilled in the art will further understand that biological systems are complex, involving various known and unknown feedback loops, making it difficult to assess whether a process has been completely inhibited. Moreover, this assessment also depends on the sensitivity of the technique for measuring a process, such as signaling. Those skilled in the art will know which methods and cut-off values ​​are established and accepted in the art to use for assessing the completeness or substantial completeness of inhibition of signaling, or for assessing the degree of partial inhibition of signaling.

[0236] As detailed and illustrated in the Examples below, antibodies can be modified to optimize particular properties.

[0237] In some embodiments, the antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP is humanized.

[0238] In some embodiments, the antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP is a human antibody.

[0239] In some embodiments, the antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP is deimmunized.

[0240] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP is humanized and deimmunized.

[0241] In some embodiments, the antibody or antigen-binding fragment thereof is monoclonal.

[0242] Antibody characteristics The antibodies of the present invention were identified after extensive screening of a large number of anti-IL1RAP antibodies on the basis that they exhibit particularly suitable properties as diagnostic and therapeutic agents for inflammatory, fibrotic and / or neoplastic diseases or disorders.

[0243] Thus, in some embodiments, antibodies or antigen-binding fragments thereof that have binding specificity for IL1RAP exhibit one or more of the following properties: a)K D A binding affinity (K) for IL1RAP characterized by a value of 3 nM or less D ); b) binds to domain 2 of IL1RAP, preferably to the H2 region of domain 2, wherein the H2 region is the amino acid sequence of SEQ ID NO:39; c) cross-reactivity with IL1RAP from cynomolgus monkey or pig; d) Inhibitory effect of IL-1α signal transduction; e) Inhibitory effect of IL-1β signaling; f) Inhibitory effect of IL-33 signal transduction; g) Inhibitory effect of IL-36α signal transduction; h) Inhibitory effect of IL-36β signaling; i) Inhibitory effect of IL-36γ signaling; j) Internalization by IL1RAP expressing cells.

[0244] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP exhibits one of the above named properties.

[0245] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP exhibits two of the above-named properties.

[0246] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP exhibits three of the above-named properties.

[0247] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP exhibits four of the above-named properties.

[0248] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP exhibits five of the above-named properties.

[0249] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP exhibits six of the above-named properties.

[0250] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP exhibits seven of the above-named properties.

[0251] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP exhibits eight of the above-named properties.

[0252] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP exhibits nine of the above-named properties.

[0253] Advantageously, in some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP exhibits all of the above-named properties.

[0254] In other embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP exhibits all of the following properties: a)K D A binding affinity (K) for IL1RAP characterized by a value of 3 nM or less D ); b) binds to domain 2 of IL1RAP, preferably to the H2 region of domain 2, wherein the H2 region is the amino acid sequence of SEQ ID NO:39; c) cross-reactivity with IL1RAP from cynomolgus monkey or pig; d) Inhibitory effect of IL-1α signal transduction; e) Inhibitory effect of IL-1β signaling; f) Inhibitory effect of IL-33 signal transduction; g) Inhibitory effect of IL-36α signal transduction; h) Inhibitory effect of IL-36β signaling; i) Inhibitory effect of IL-36γ signaling; j) Internalization by IL1RAP expressing cells.

[0255] The H2 region corresponds to amino acids 174-191 based on the IL1RAP amino acid sequence of Uniprot ID Q9NPH3.

[0256] In some embodiments, the binding affinity (K D ) is 3nM or less, such as 2.75nM, for example 2.5nM, such as 2nM, for example 1.75nM, such as 1.5nM, for example 1.25nM, such as 1nM, for example 0.75nM, such as 0.5nM, or for example 0.5nM.

[0257] In some embodiments, the binding affinity (K D ) is 3000pM or less, such as 2750pM, for example 2500pM, 2000pM, for example 1750pM, 1500pM, for example 1250pM, 1000pM, for example 750pM, 700pM, for example 650pM, 600pM, for example 550pM, 500pM, for example 450pM, 400pM, for example 350pM, 300pM, for example 250pM, 200pM, for example 150pM, 100pM, for example 50pM.

[0258] One of skill in the art will understand that determining the binding affinity of an antibody or antigen-binding fragment will depend on the method used to determine the binding affinity.

[0259] One skilled in the art will appreciate that IL1RAP antibodies, apart from being able to inhibit cytokine signaling (e.g., of IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and / or IL-36γ), may exhibit one or more additional functions. These functions may be conveyed by the constant region or the Fc region of the antibody, for example, antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cellular phagocytosis (ADCP), thereby killing target cells, such as IL1RAP-expressing tumor cells.

[0260] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP is capable of inducing ADCC of cells expressing IL1RAP.

[0261] In some embodiments, antibodies or antigen-binding fragments thereof that have binding specificity for IL1RAP are unable to induce ADCC of cells expressing IL1RAP.

[0262] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP is capable of inducing ADCP of cells expressing IL1RAP.

[0263] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP is unable to induce ADCP of cells that express IL1RAP.

[0264] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP is capable of inducing ADCC and ADCP of cells expressing IL1RAP.

[0265] Antibody Modifications In some embodiments, the antibody, or antigen-binding fragment thereof, that has binding specificity for IL1RAP further comprises a moiety to increase the in vivo half-life of the agent.

[0266] In some embodiments, the antibody or antigen-binding fragment thereof with binding specificity for IL1RAP further comprises a moiety to increase the in vivo half-life of the agent, wherein the moiety to increase the in vivo half-life is selected from the group consisting of polyethylene glycol (PEG), human serum albumin, glycosylation groups, fatty acids, and dextran.

[0267] In some embodiments, the antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP is PEGylated.

[0268] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for an IL1RAP fragment is covalently linked, directly or indirectly (e.g., via a chelator), to a functional moiety, such as a cytotoxic or detectable moiety.

[0269] In some embodiments, the antibody, or antigen-binding fragment thereof, that has binding specificity for IL1RAP comprises a cytotoxic moiety.

[0270] In some embodiments, the antibody, or antigen-binding fragment thereof, that has binding specificity for IL1RAP comprises a cytotoxic moiety, where said cytotoxic moiety comprises or consists of a radioisotope.

[0271] In some embodiments, an antibody or antigen-binding fragment thereof with binding specificity for IL1RAP comprises a cytotoxic moiety that comprises or consists of a radioisotope, where the radioisotope is selected from the group consisting of a β-emitter, an Auger emitter, a conversion electron-emitter, an α-emitter, and a low photon energy-emitter.

[0272] In some embodiments, an antibody or antigen-binding fragment thereof with binding specificity for IL1RAP comprises a cytotoxic moiety that comprises or consists of a radioisotope, where the radioisotope has a localized absorbed energy emission pattern that results in high dose absorbance in the vicinity of the agent.

[0273] In some embodiments, the antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP comprises a cytotoxic moiety that comprises or consists of a radioisotope, where the radioisotope is 90 Y, 32 P, 186 Re / 186 long-range beta emitters such as Re; 166 Ho, 76 As / 77 As, 153 Sm;131 I, 177 Lu, 67 Cu, 161 intermediate-range β-emitters such as Tb; 45 Ca, 35 S, or 14 low-energy β-emitters such as C; 51 Cr, 67 Ga, 99 Tc m , 111 In, 123 I, 125 I, 201 A conversion or Auger emitter such as Tl; and 212 Bi, 213 Bi, 223 Ac, and 221 At is selected from the group consisting of α-emitters such as At.

[0274] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP comprises a cytotoxic moiety that comprises or consists of a radioisotope, where the radioisotope is 177 This is Lu.

[0275] In some embodiments, the antibody, or antigen-binding fragment thereof, that has binding specificity for IL1RAP comprises a cytotoxic moiety, where the cytotoxic moiety comprises or consists of a cytotoxic agent.

[0276] In some embodiments, an antibody or antigen-binding fragment thereof with binding specificity for IL1RAP comprises a cytotoxic moiety that comprises or consists of a cytotoxic agent, where the cytotoxic agent is selected from the group consisting of cytoactivators; antiandrogens; cortisone and its derivatives; phosphonates; testosterone-5-α-reductase inhibitors; boron additives; cytokines; thapsigargin and its metabolites; toxins (such as saporin or calicheamicin); chemotherapeutic agents (such as antimetabolites); or other cytotoxic agents useful in the treatment of neoplastic disorders.

[0277] In some embodiments, an antibody or antigen-binding fragment thereof with binding specificity for IL1RAP comprises a cytotoxic moiety that comprises or consists of a cytotoxic agent, wherein the cytotoxic agent is suitable for use in activation therapy, such as photon activation therapy, neutron activation therapy, neutron induced Auger electron therapy, synchrotron radiation therapy, or low energy X-ray photon activation therapy.

[0278] In some embodiments, the antibody, or antigen-binding fragment thereof, that has binding specificity for IL1RAP comprises a detectable moiety.

[0279] In some embodiments, the antibody, or antigen-binding fragment thereof, that has binding specificity for IL1RAP comprises a detectable moiety, where said detectable moiety comprises or consists of a radioisotope.

[0280] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP comprises a detectable moiety that comprises or consists of a radioisotope, where said radioisotope is 99m Tc, 111 In, 67 Ga, 68 Ga, 72 As, 89 Zr, 123 I, and 201 Tl.

[0281] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP comprises a detectable moiety that comprises or consists of a radioisotope, where the radioisotope is 89 Zr.

[0282] In some embodiments, the antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP comprises: 86 Y / 90 Y, or 124 I / 211It contains pairs of detectable, cytotoxic radioisotopes such as At.

[0283] In some embodiments, antibodies or antigen-binding fragments thereof with binding specificity for IL1RAP comprise radioisotypes that can act simultaneously in a multimodal manner as a detectable moiety and as a cytotoxic agent.

[0284] In some embodiments, an antibody, or antigen-binding fragment thereof, with binding specificity for IL1RAP comprises a detectable moiety that comprises or consists of a paramagnetic isotope.

[0285] In some embodiments, an antibody or antigen-binding fragment thereof with binding specificity for IL1RAP comprises a detectable moiety that comprises or consists of a paramagnetic isotope, where the paramagnetic isotope is 157 Gd, 55 Mn, 162 Dy, 52 Cr, and 56 The metal is selected from the group consisting of Fe.

[0286] In some embodiments, the antibody or antigen-binding fragment thereof with binding specificity for IL1RAP comprises a detectable moiety, where the detectable moiety is detectable by imaging techniques such as SPECT, PET, MRI, optical, or ultrasound imaging.

[0287] In some embodiments, the antibody or antigen-binding fragment thereof with binding specificity for IL1RAP comprises a cytotoxic moiety and / or a detectable moiety, wherein the cytotoxic moiety and / or the detectable moiety is indirectly conjugated to the antibody or antigen-binding fragment thereof via a linking moiety.

[0288] In some embodiments, the antibody or antigen-binding fragment thereof with binding specificity for IL1RAP comprises a cytotoxic moiety and / or a detectable moiety, wherein the cytotoxic moiety and / or detectable moiety is indirectly attached to the antibody or antigen-binding fragment thereof via a linking moiety, wherein the linking moiety is a chelator.

[0289] In some embodiments, the antibody or antigen-binding fragment thereof with binding specificity for IL1RAP comprises a cytotoxic moiety and / or a detectable moiety, wherein the cytotoxic moiety and / or the detectable moiety is indirectly attached to the antibody or antigen-binding fragment thereof via a linking moiety, wherein the linking moiety is a chelator selected from the group consisting of derivatives of 1,4,7,10-tetraazacyclododecane-1,4,7,10,tetraacetic acid (DOTA), deferoxamine (DFO), derivatives of diethylenetriaminepentaacetic acid (DTPA), derivatives of S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), and derivatives of 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA).

[0290] In some embodiments, the antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP does not comprise a cytotoxic moiety or a detectable moiety.

[0291] The present invention further includes an antibody or antigen-binding fragment thereof having binding specificity for human interleukin-1 receptor accessory protein (IL1RAP), wherein said antibody or antigen-binding fragment is capable of inhibiting the binding of antibody 48D2 disclosed herein to IL1RAP.

[0292] As used herein, the term "capable of inhibiting the binding of antibody 48D2 to IL1RAP" means that the presence of another antibody inhibits, in whole or in part, the binding of 48D2 to IL1RAP. Such competitive binding inhibition can be determined using assays and methods well known in the art, for example, using a BIAcore chip with immobilized IL1RAP and incubating with 48D2 with or without the antibody to be tested.

[0293] Alternatively, a pairwise mapping approach can be used in which antibody 48D2 is immobilized on the surface of a BIAcore chip, IL1RAP antigen is allowed to bind to the immobilized antibody, and then a second antibody is simultaneously tested for IL1RAP binding ability (see 'BIAcore Assay Handbook', GE Healthcare Life Sciences, 29-0194-00 AA 05 / 2012; the disclosure of which is incorporated herein by reference).

[0294] As a further alternative, competitive binding inhibition can be determined using flow cytometry. For example, to test whether a test antibody can inhibit the binding of 48D2 antibody to a cell surface antigen, cells expressing the antigen can be pre-incubated with the test antibody for 20 minutes, then the cells are washed and incubated with fluorochrome-conjugated 48D2 antibody and detected by flow cytometry. If pre-incubation with the test antibody reduces the detection of 48D2 antibody in flow cytometry, the test antibody inhibits the binding of the reference antibody to the cell surface antigen. If the tested antibody shows high affinity for IL1RAP, a shorter period of pre-incubation is used (or no pre-incubation at all).

[0295] Antibody production A second aspect of the invention relates to a polynucleotide encoding an antibody or antigen-binding fragment of the first aspect of the invention, or its component polypeptide chains. As used herein, the term "polynucleotide" includes DNA (eg, genomic or complementary DNA) and mRNA molecules, which can be single-stranded or double-stranded.

[0296] In some embodiments, the polynucleotide is an isolated polynucleotide.

[0297] In some embodiments, the polynucleotide is a cDNA molecule.

[0298] It will be understood by those of skill in the art that polynucleotides can be codon optimized for expression of an antibody or antigen-binding fragment in a particular host cell, e.g., expression in a human cell (see, e.g., Angov, 2011, Biotechnol. J. 6(6):650-659, the disclosure of which is incorporated herein by reference).

[0299] In some embodiments, a polynucleotide encoding an antibody or antigen-binding fragment of the invention encodes an antibody light chain or a variable region thereof.

[0300] In some embodiments, a polynucleotide encoding an antibody or antigen-binding fragment of the invention encodes an antibody heavy chain or a variable region thereof.

[0301] A third aspect of the present invention relates to a vector comprising a polynucleotide according to the second aspect of the present invention.

[0302] In some embodiments, the vector is an expression vector.

[0303] The term "expression vector" is defined herein as a DNA molecule, e.g., linear or circular, that contains a polynucleotide encoding a polypeptide (antibody or antigen-binding fragment thereof) of the invention, operably linked with additional nucleotides that provide for its expression. The terms "plasmid", "expression vector", and "vector" are used interchangeably, since plasmids are currently the most commonly used form of vector. However, the present invention is intended to include such other forms of expression vectors that perform equivalent functions known in the art. As used herein, "expression vector" or "vector" refers to a DNA construct that contains a DNA sequence operably linked to suitable control sequences that can effect expression of the DNA in a suitable host. Such control sequences can include, for example, a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding a suitable mRNA ribosomal binding site, and sequences that control the termination of transcription and translation. A vector can be, for example, a plasmid, a phage, or simply a potential genomic insert. Once transformed into a suitable host, the vector can, for example, replicate and function independently of the host genome, or, in some cases, can be integrated into the genome itself. The expression vector is designed, for example, as described in Li et al. (Construction strategies for developing expression vectors for recombinant monoclonal antibody production in CHO cells, Mol Biol Rep. 2018 Dec; 45(6):2907-2912).

[0304] A fourth aspect of the invention relates to a recombinant host cell comprising a polynucleotide according to the second aspect of the invention or a vector according to the third aspect of the invention.

[0305] In some embodiments, the recombinant host cell is a bacterial cell.

[0306] In some embodiments, the recombinant host cell is a yeast cell.

[0307] In some embodiments, the recombinant host cell is a mammalian cell.

[0308] In some embodiments, the recombinant host cell is a human cell.

[0309] A fifth aspect of the invention relates to a method of producing an antibody or antigen-binding fragment thereof according to the first aspect of the invention, said method comprising culturing a host cell according to the fourth aspect of the invention comprising a polynucleotide according to the second aspect of the invention or a vector according to the third aspect of the invention under conditions allowing expression of the encoded antibody or antigen-binding fragment thereof.

[0310] Pharmaceutical Compositions A sixth aspect of the present invention is a method for producing a composition comprising the steps of: An antibody or antigen-binding fragment according to the first aspect of the invention, A polynucleotide according to the second aspect of the present invention, A vector according to the third aspect of the invention, and / or A host cell according to the fourth aspect of the invention, in a pharmaceutical composition, wherein said composition further comprises a pharma- ceutically acceptable diluent, carrier, or excipient.

[0311] In some embodiments, the composition comprises: An antibody or antigen-binding fragment according to the first aspect of the invention, A polynucleotide according to the second aspect of the present invention, A vector according to the third aspect of the invention, and / or The host cell of the fourth aspect of the invention. It will be appreciated by those skilled in the art that the pharmaceutical compositions may also include additional compounds, including chelating agents such as EDTA, citrate, EGTA, or glutathione. Pharmaceutical compositions can be prepared by methods known in the art that are sufficiently storage stable and suitable for administration to humans and animals. For example, pharmaceutical compositions can be lyophilized by freeze drying, spray drying, spray cooling, or by using particle formation from supercritical particle formation.

[0312] The term "pharmacologically acceptable" is intended to mean a non-toxic material that does not reduce the effectiveness of the IL1RAP binding activity of the antibody or antigen-binding fragment of the invention. Pharmaceutically acceptable buffers, carriers, diluents, excipients, and the like are, for example, well known in the art.

[0313] The term "buffer" is intended to mean an aqueous solution containing an acid-base mixture for the purpose of stabilizing the pH. Examples of buffer solutions are Trizma, Bicine, Tricine, MOPS, MOPSO, MOBS, Tris, Hepes, HEPBS, MES, phosphate, carbonate, acetate, citrate, glycolate, lactate, borate, ACES, ADA, tartaric acid, AMP, AMPD, AMPSO, BES, CABS, cacodylate, CHES, DIPSO, EPPS, ethanolamine, glycine, HEPPSO, imidazole, imidazole lactate, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO, and TES.

[0314] The term "diluent" is intended to mean an aqueous or non-aqueous solution intended to dilute the antibody or antigen-binding fragment in the pharmaceutical formulation. The diluent may be one or more selected from saline, water, polyethylene glycol, propylene glycol, ethanol, oils (such as safflower oil, corn oil, peanut oil, cottonseed oil, or sesame oil).

[0315] The term "adjuvant" is intended to mean any compound added to a formulation to enhance the biological effect of the antibody or antigen-binding fragment of the present invention. The adjuvant may be, for example, but is not limited to, one or more of zinc, copper, or silver salts with different anions, such as fluoride, chloride, bromide, iodide, thiocyanate, sulfite, hydroxide, phosphate, carbonate, lactate, sugar, citrate, borate, tartrate, and acetates with different acyl compositions. The adjuvant may also be a cationized polypeptide, such as a cationic polypeptide, such as a cationic cellulose ether, a cationic cellulose ester, deacetylated hyaluronic acid, chitosan, a cationic dendrimer, a cationic synthetic polymer, such as poly(vinylimidazole), and polyhistine, polylysine, polyarginine, and peptides containing these amino acids.

[0316] The excipient may be one or more of a carbohydrate, a polymer, a lipid, and a mineral. Examples of carbohydrates include lactose, glucose, sucrose, mannitol, cyclodextrin, etc., which are added to the composition, for example, to facilitate lyophilization. Examples of polymers include starch, cellulose ethers, cellulose carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, ethylhydroxyethylcellulose, alginic acid, carrageenan, hyaluronic acid and its derivatives, polyacrylic acid, polysulfonic acid, polyethylene glycol / polyethylene oxide, polyethylene oxide / polypropylene oxide copolymers, polyvinyl alcohol / polyvinyl acetate with different degrees of hydrolysis, and polyvinylpyrrolidone, all with different molecular weights, for example, which are added to the composition to adjust viscosity, bioadhesion, or protect lipids from chemical and proteolytic degradation. Examples of lipids include fatty acids, phospholipids, mono-, di-, and triglycerides, ceramides, sphingolipids, and glycolipids, all with different acyl chain lengths and degrees of saturation, egg lecithin, soy lecithin, hydrogenated egg and soy lecithin, etc., are added to the composition for reasons similar to those of polymers. Examples of minerals include talc, magnesium oxide, zinc oxide, and titanium dioxide, which are added to the composition for benefits such as reduced pooling and favorable pigment properties.

[0317] The antibodies or antigen-binding fragments of the invention can be formulated into any type of pharmaceutical composition known in the art suitable for their delivery.

[0318] In some embodiments, the pharmaceutical composition of the present invention may be in the form of a liposome, in which the antibody or antigen-binding fragment is combined with an amphiphilic agent, such as a lipid, which exists in an aggregated form as a micelle, an insoluble monolayer, and a liquid crystal, in addition to other pharma- ceutically acceptable carriers. Suitable lipids for liposomal formulations include, but are not limited to, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Suitable lipids also include the above lipids modified with poly(ethylene glycol) in the polar head group to extend bloodstream circulation time. Preparation of such liposomal formulations can be found, for example, in US 4,235,871, the disclosure of which is incorporated herein by reference.

[0319] The pharmaceutical composition of the present invention may also be in the form of biodegradable microparticles.Aliphatic polyesters such as polylactic acid (PLA), polyglycolic acid (PGA), copolymers of PLA and PGA (PLGA), or polycaprolactone (PCL), and polyanhydrides are widely used as biodegradable polymers for the manufacture of microparticles.The preparation of such microparticles can be found in US5,851,451 and EP0213303, the disclosures of which are incorporated herein by reference.

[0320] In a further embodiment, the pharmaceutical composition of the present invention is provided in the form of a polymer gel, where polymers such as starch, cellulose ethers, cellulose carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, ethylhydroxyethylcellulose, alginic acid, carrageenan, hyaluronic acid and its derivatives, polyacrylic acid, polyvinylimidazole, polysulfonic acid, polyethylene glycol / polyethylene oxide, polyethylene oxide / polypropylene oxide copolymers, polyvinyl alcohol / polyvinyl acetate with different degrees of hydrolysis, polyvinylpyrrolidone are used to thicken the drug-containing solution. The polymer may also include gelatin and collagen.

[0321] Alternatively, the antibody or antigen-binding fragment may simply be dissolved in saline, water, polyethylene glycol, propylene glycol, ethanol or oil (such as safflower oil, corn oil, peanut oil, cottonseed oil, or sesame oil), tragacanth gum, and / or various buffers.

[0322] It will be understood that the pharmaceutical compositions of the present invention may contain ions and a defined pH for enhancing the action of the active antibody or antigen-binding fragment. Additionally, the compositions may be subjected to conventional pharmaceutical operations such as sterilization and / or contain conventional adjuvants such as preservatives, stabilizers, wetting agents, emulsifiers, buffers, fillers, etc.

[0323] The pharmaceutical compositions according to the invention can be administered by any suitable route known to those skilled in the art. Thus, routes of administration include parenteral (intravenous, subcutaneous, intramuscular), topical, ocular, nasal, pulmonary, buccal, oral, parenteral, vaginal, and rectal. Administration via implants is also possible.

[0324] In a preferred embodiment, the pharmaceutical compositions are administered parenterally, for example, intravenously, intraventricularly, intraarticularly, intraarterially, intraperitoneally, intrathecally, intraventricularly, intrathoracically, intracranially, intramuscularly, or subcutaneously, or they may be administered by infusion techniques. Preferably, the pharmaceutical compositions are administered intravenously. They are conveniently used in the form of a sterile aqueous solution which may contain sufficient salts and other substances, such as glucose, to make them isotonic with blood. The aqueous solution should be suitably buffered (preferably to a pH of 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.

[0325] Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may also contain suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules or vials, and may be stored in a lyophilized (lyophilized) condition requiring only the addition of a sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets of the kind previously described.

[0326] Thus, the pharmaceutical compositions of the present invention are particularly suitable for parenteral administration, for example intravenous administration.

[0327] Alternatively, the pharmaceutical composition may be administered intranasally or by inhalation (e.g., in the form of an aerosol spray) from a pressurized container, pump, spray, or nebulizer using a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, 1,1,1,2-tetrafluoroethane (HFA134A3 or a hydrofluoroalkane such as 1,1,1,2,3,3,3-heptafluoropropane (HFA227EA3), carbon dioxide or other suitable gas). In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurized container, pump, spray, or nebulizer uses a solution or suspension of the active antibody or antigen-binding fragment, for example, a mixture of ethanol and a propellant as a solvent, and may further contain a lubricant, for example, sorbitan trioleate. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of a compound of the invention and a suitable powder base such as lactose or starch.

[0328] Aerosol or dry powder formulations are preferably prepared so that each metered dose or "puff" contains at least 1 mg of a compound of the invention for delivery to the patient. It will be appreciated that the total daily dosage administered by aerosol will vary from patient to patient and can be administered in a single dose or, more usually, in divided doses throughout the day.

[0329] Alternatively, the antibodies or antigen-binding fragments of the invention can be administered in the form of a suppository or pessary, which can be applied topically in the form of a lotion, solution, cream, ointment, or dusting powder. The compounds of the invention can also be administered transdermally, for example, by the use of a skin patch. They can also be administered via the ocular route. For ophthalmic use, the antibodies or antigen-binding fragments of the invention can be formulated as a micronized suspension in isotonic, pH-adjusted, sterile saline or, preferably, as a solution in isotonic, pH-adjusted, sterile saline, optionally combined with a preservative such as benzylalkonium chloride. Alternatively, they can be formulated in an ointment such as petrolatum.

[0330] For topical application to the skin, the antibodies or antigen-binding fragments of the invention can be formulated in a suitable ointment in which the active compound is suspended or dissolved in a mixture of, for example, one or more of mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compounds, emulsifying wax, and water, or alternatively, in a suitable lotion or cream in which the active compound is suspended or dissolved in a mixture of, for example, one or more of mineral oil, sorbitan monostearate, polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water.

[0331] The pharmaceutical composition will be administered to the patient in a pharma- ceutical effective dose. "Therapeutically effective amount", or "effective amount", or "therapeutically effective", as used herein, refers to that amount that provides a therapeutic effect for a given condition and administration regimen. It is a predetermined amount of active material calculated to produce the desired therapeutic effect in association with necessary additives and diluents, i.e., carriers or administration vehicles. It is further intended to mean an amount sufficient to reduce, and most preferably prevent, clinically significant deficits in the activity, function, and response of the host. Alternatively, a therapeutically effective amount is an amount sufficient to cause an improvement of a clinically significant condition in the host. As will be appreciated by those skilled in the art, the amount of a compound may vary depending on its specific activity. A suitable dosage may include a predetermined amount of the active composition calculated to produce the desired therapeutic effect in association with necessary diluents. In the methods of making and using the compositions of the present invention, a therapeutically effective amount of the active ingredient is provided. The therapeutically effective amount can be determined by an ordinarily skilled medical or veterinary practitioner based on patient characteristics such as age, weight, sex, condition, comorbidities, other diseases, etc., as is well known in the art. The administration of a pharma- ceutically effective dose can be carried out both by single administration in the form of individual dose units or several smaller dose units, and by multiple administration of subdivided doses at specific intervals. Alternatively, the dose can be provided as a continuous infusion over a long period of time.

[0332] In the context of diagnostic uses of the antibodies or antigen-binding fragments of the invention, a "pharmacologically effective amount," or an "effective amount," or a "diagnostically effective amount," as used herein, refers to an amount that provides a detectable signal for diagnostic, e.g., in vivo imaging purposes.

[0333] The antibody or antigen-binding fragment can be formulated at various concentrations depending on the efficacy / toxicity of the antibody or antigen-binding fragment used. For example, the formulation may contain the active antibody or antigen-binding fragment at a concentration between 0.1 μM and 1 mM, more preferably between 1 μM and 500 μM, between 500 μM and 1 mM, between 300 μM and 700 μM, between 1 μM and 100 μM, between 100 μM and 200 μM, between 200 μM and 300 μM, between 300 μM and 400 μM, between 400 μM and 500 μM, between 500 μM and 600 μM, between 600 μM and 700 μM, between 800 μM and 900 μM, or between 900 μM and 1 mM. Typically, the formulation contains the active antibody or antigen-binding fragment at a concentration between 300 μM and 700 μM.

[0334] Typically, a therapeutic dose of an antibody or antigen-binding fragment (with or without a therapeutic moiety) in a human patient will be in the range of 100 μg to 1 g per administration (based on a body weight of 70 kg, e.g., between 300 μg to 700 mg per administration). For example, the maximum therapeutic amount may be in the range of 0.1 to 10 mg / kg per administration, e.g., between 0.1 to 5 mg / kg, or between 1 to 5 mg / kg, or between 0.1 to 2 mg / kg. In some embodiments, the therapeutic dose may be 5, 20, or 50 mg / kg, or any range formed from these values. For example, the therapeutic dose may be 5 to 50 mg / kg, 5 to 20 mg / kg, or 20 to 50 mg / kg. It will be understood that such doses may be administered at different intervals as determined by the oncologist / physician, e.g., doses may be administered daily, twice weekly, weekly, biweekly, or monthly.

[0335] It will be appreciated by those skilled in the art that the pharmaceutical compositions of the present invention may be administered alone or in combination with other therapeutic agents used in the treatment of inflammatory, fibrotic and / or neoplastic disorders or diseases.

[0336] In some embodiments, the composition is adapted for parenteral delivery.

[0337] In some embodiments, the composition is adapted for intravenous delivery.

[0338] In some embodiments, the composition is adapted for local delivery.

[0339] In some embodiments, the composition is adapted for subcutaneous delivery.

[0340] In some embodiments, the composition is adapted for intramuscular delivery.

[0341] It will be further appreciated by those skilled in the art that the antibodies or antigen-binding fragments and pharmaceutical compositions or formulations of the present invention are useful in both human and veterinary medicine. Thus, the methods of the present invention can be used in the treatment of both human and non-human animals, such as horses, dogs, and cats. Preferably, however, the patient is a human.

[0342] Antibody Indication and Use A seventh aspect of the present invention is a method for producing a composition comprising the steps of: For medical use, An antibody or antigen-binding fragment according to the first aspect of the invention, A polynucleotide according to the second aspect of the present invention, A vector according to the third aspect of the present invention, A host cell according to the fourth aspect of the invention, and / or The sixth aspect of the present invention relates to a composition.

[0343] An eighth aspect of the invention provides an antibody or antigen-binding fragment of the first aspect of the invention, A polynucleotide according to the second aspect of the present invention, A vector according to the third aspect of the present invention, A host cell according to the fourth aspect of the invention, and / or A composition according to a sixth aspect of the present invention, for use in the prevention and / or treatment and / or alleviation and / or detection and / or diagnosis of a disease or disorder susceptible to treatment with an inhibitor of IL-1α, IL-1β, IL-33, IL-36α, IL-36β and / or IL-36γ signaling, and / or wherein said disease or disorder is associated with cells expressing IL1RAP.

[0344] In an alternative embodiment to the eighth aspect of the invention, the present invention provides a method for producing a composition comprising the steps of: An antibody or antigen-binding fragment according to the first aspect of the invention, A polynucleotide according to the second aspect of the present invention, A vector according to the third aspect of the present invention, A host cell according to the fourth aspect of the invention, and / or A composition according to a sixth aspect of the present invention, For use in the prevention and / or treatment and / or alleviation and / or detection and / or diagnosis of diseases or disorders susceptible to treatment with an inhibitor of IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and / or IL-36γ signaling.

[0345] In another alternative embodiment to the eighth aspect of the invention, the present invention provides a method for producing a composition comprising the steps of: An antibody or antigen-binding fragment according to the first aspect of the invention, A polynucleotide according to the second aspect of the present invention, A vector according to the third aspect of the present invention, A host cell according to the fourth aspect of the invention, and / or A composition according to a sixth aspect of the present invention, For use in the prevention and / or treatment and / or alleviation and / or detection and / or diagnosis of a disease or disorder, which disease or disorder is associated with cells expressing IL1RAP.

[0346] In some embodiments, the disease or disorder is associated with IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and / or IL-36γ signaling.

[0347] In some embodiments, the disease or disorder is associated with cells that express IL1RAP.

[0348] "Treatment" includes both treatment of a patient and prophylactic or preventative treatment. The term "preventive" or "prophylactic" is used to encompass the use of an antibody or antigen-binding fragment thereof, or a formulation thereof, described herein, to prevent or reduce the likelihood of an inflammatory disease or disorder, a fibrotic disease or disorder, or a neoplastic disease or disorder, and is also intended to include the prevention or reduction of neoplastic cell spread, diffusion, or metastasis in a patient or subject. The term "prophylactic" also encompasses the use of an antibody or antigen-binding fragment thereof, or a formulation thereof, described herein, to prevent the recurrence of an inflammatory, fibrotic, and / or neoplastic disease or disorder in a patient who has previously been treated for any of these diseases or disorders.

[0349] "Diagnosis" or "detection" includes detecting cells associated with an inflammatory, fibrotic, and / or neoplastic disease or disorder, either in vivo (i.e., within a patient's body) or ex vivo (i.e., in a tissue or cell sample removed from a patient's body).

[0350] "Amelioration" means, without limitation, a decrease in the symptoms or processes associated with a disease or disorder, ie, resulting in the remission of the disease or disorder.

[0351] "Inflammatory, fibrotic, neoplastic disease or disorder associated with cells expressing IL1RAP" includes such diseases or disorders in which pathological cells that directly or indirectly cause the disorder express IL1RAP on the cell surface. It will be understood that the cells expressing IL1RAP can be immune cells, cells of connective tissue such as fibroblasts, or neoplastic cells such as tumor cells (cancer cells), e.g., tumor cells themselves. Furthermore, such cells also include pathological stem cells (i.e., cancer stem cells, or CSCs) and progenitor cells that are directly or indirectly involved in the development of inflammatory, fibrotic, and / or neoplastic diseases or disorders in an individual. Examples of CSCs are disclosed in Visvader & Lindeman, 2008, Nat Rev Cancer 8:755-768, the disclosure of which is incorporated herein by reference.

[0352] Alternatively, or in addition, cells expressing IL1RAP may be indirectly associated with an inflammatory, fibrotic, and / or neoplastic disease or disorder, e.g., they may mediate cellular processes necessary for the cells to survive. The antibodies or antigen-binding fragments thereof of the invention may in this case target cells essential for the maintenance of inflammatory and / or fibrotic processes, or cells that inhibit, for example, the blood supply (angiogenesis) of a tumor, or beneficial immune responses directed against malignant cells (e.g., suppressive macrophages or T cells).

[0353] Depending on whether it is therapeutically desirable to kill target cells expressing IL1RAP, for example in the case of neoplastic cells, an antibody or antigen-binding fragment according to the first aspect of the invention capable of inducing ADCC may be used. For example, if the target cells expressing IL1RAP are cancer cells (CML, AML, ALL, melanoma, lung cancer cells, etc.), it may be advantageous for the antibody or antigen-binding fragment to be able to induce ADCC in order to eliminate such cells. However, it will be appreciated that an antibody or antigen-binding fragment lacking ADCC activity may still be used to provide a therapeutic effect, for example through inhibition of IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and / or IL-36γ signaling leading to reduced angiogenesis in the vicinity of the cancer or tumor. Similarly, in the case of inflammatory and / or fibrotic diseases and disorders, it may be beneficial to affect cell behavior but not kill the cells.

[0354] Conditions or disease states that are susceptible to treatment with inhibitors of IL-1 and / or that may constitute inflammatory, fibrotic, and / or neoplastic diseases or disorders associated with cells expressing IL1RAP are well known in the art (see Dinarello et al., 2012, Nature Reviews 11:633-652 and Dinarello, 2014, Mol. Med. 20(suppl. 1):S43-S58, the disclosures of which are incorporated herein by reference), and include, but are not limited to, the following:

[0355] Rheumatoid arthritis, general arthritis, psoriatic arthritis, general juvenile arthritis including systemic-onset juvenile idiopathic arthritis (SOJIA), osteoarthritis, familial cold autoinflammatory syndrome (FCAS), Muckle-Wells disease, neonatal-onset multisystem inflammatory disease (NOMID), familial Mediterranean fever (FMF), septic arthritis, pyoderma gangrenosum, and acne (PAPA) syndrome, adult-onset Still's disease, hyper-IgD syndrome, type 2 diabetes, macrophage activation syndrome, and TNF receptor-related Periodontal syndrome, Blau's disease, ankylosing spondylitis, Sweets' disease, lupus arthritis, Alzheimer's disease, psoriasis, asthma, allergies, arteriosclerosis, sarcoidosis, atopic dermatitis, systemic lupus erythematosus, bullous pemphigoid, type I diabetes, chronic obstructive pulmonary disease, Helicobacter pylori gastritis, inflammatory bowel disease (including ulcerative colitis and Crohn's disease), hepatitis, hepatitis C, ischemia-reperfusion injury, multiple sclerosis, neonatal or pneumococcal meningitis, tuberculosis, Behcet's disease syndrome, septic shock, graft-versus-host disease, adult T-cell leukemia, multiple myeloma, periodontitis, obesity and obesity-related diseases (e.g., metabolic syndrome, cardiac hypertrophy, congestive heart failure, myocardial infarction, varicose veins, polycystic ovarian syndrome, etc., gastroesophageal reflux disease (GERD), fatty liver, colon cancer, breast cancer, uterine cancer, chronic renal failure, stroke, and hyperuricemia), intervertebral disc disease, irritable bowel syndrome, Schnitzler syndrome, allergy / atopic dermatitis, acne, Chet's disease, cardiac fibrosis, cardiovascular disease, cryopin-associated periodic syndrome, cystic fibrosis, Goodpasture's syndrome, Guillain-Barre syndrome, renal fibrosis, liver fibrosis, pulmonary fibrosis, dermatofibrosis, myocarditis, autoimmune myocarditis, organ dysfunction following organ transplantation, pancreatitis, peritonitis, uveitis, vasculitis, pneumonia, pulmonary hypertension, sclerosing skin chronic graft-versus-host disease, sepsis, Sjogren's syndrome, systemic sclerosis, Takayasu's arteritis, and gout.

[0356] Blockade of IL-1 signaling may also be effective in treating myocardial infarction. Large-scale clinical trials (CANTOS trail; see Ridker et al., 2011, Am Heart Journal 162(4):597-605, the disclosure of which is incorporated herein by reference) are required to confirm the efficacy of anti-IL-1β antibody (canakinumab) blockade after myocardial infarction.

[0357] It will be appreciated that for such indications, antibodies or antigen-binding fragments that bind to IL1RAP and thereby block IL-1 and / or IL-33 and / or IL-36 signaling associated with immune cells may also be used for therapeutic benefit. Such antibodies may also be modified to lack ADCC activity.

[0358] In some embodiments, the disease or disorder is an inflammatory and / or fibrotic and / or neoplastic disease or disorder.

[0359] In relation to the therapeutic and prophylactic aspects of the invention, it will be appreciated by those skilled in the art that the antibodies or antigen-binding fragments thereof may bind to IL1RAP present on the surface of cells associated with an inflammatory, fibrotic, and / or neoplastic disease or disorder, thereby modulating (i.e., increasing or decreasing) the biological activity of IL1RAP. However, such a modulating effect is not required, and for example, the antibodies or antigen-binding fragments thereof of the invention may simply elicit a therapeutic and prophylactic effect by binding to IL1RAP on the surface of cells associated with a disease or disorder, which may trigger the immune system to induce processes such as cell death (e.g., by ADCC and / or by the presence within the agent of cytotoxic / radioactive moieties).

[0360] In some embodiments, the antibody or antigen-binding fragment thereof that has binding specificity for IL1RAP inhibits a biological activity of IL1RAP. "Biological activity of IL1RAP" includes any interaction or signaling event involving IL1RAP on cells associated with inflammatory, fibrotic, and / or neoplastic diseases or disorders. For example, in one embodiment, the antibody or antigen-binding fragment thereof can block binding of one or more co-receptors to IL1RAP (such as IL1R1, ST2, C-KIT, and / or IL1RL2). Furthermore, as detailed above, in some embodiments, the antibody or antigen-binding fragment thereof having binding specificity for IL1RAP inhibits signaling of IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and / or IL-36γ.

[0361] One of skill in the art will understand that "inhibiting cytokine signaling" (inhibition of signaling of IL-1 family cytokines, e.g., IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and / or IL-36γ) results in inhibition of the biological activity of IL1RAP.

[0362] Such inhibition of the biological activity of IL1RAP by the antibodies or antigen-binding fragments thereof of the invention may be total or partial. For example, the antibodies or antigen-binding fragments thereof may inhibit the biological activity of IL1RAP by at least 10%, preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, and most preferably 100%, as compared to the biological activity of IL1RAP in cells associated with an inflammatory, fibrotic, and / or neoplastic disease or disorder that have not been exposed to the antibody or antigen-binding fragment thereof. In a preferred embodiment, the antibodies or antigen-binding fragments thereof are capable of inhibiting the biological activity of IL1RAP by 50% or more as compared to the biological activity of IL1RAP in cells associated with an inflammatory, fibrotic, and / or neoplastic disease or disorder that have not been exposed to the antibody or antigen-binding fragment thereof.

[0363] Similarly, it will be understood that the inhibition of growth and / or proliferation of cells associated with a neoplastic disease or disorder may be total or partial. For example, an antibody or antigen-binding fragment thereof may be capable of inhibiting the growth and / or proliferation of cells associated with a neoplastic disease or disorder by at least 10%, preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, and most preferably 100%, compared to the growth and / or proliferation of cells associated with a neoplastic disease or disorder not exposed to the antibody or antigen-binding fragment thereof.

[0364] IL-1, IL-33, and IL-36 in Disease IL-1 has been implicated in a wide range of diseases and conditions ranging from gout to cancer (for reviews, see Dinarello et al., 2012, Nature Reviews 11:633-652 and Dinarello, 2014, Mol. Med. 20(suppl. 1):S43-S58; the disclosures of which are incorporated herein by reference): · joint, bone and muscle diseases such as rheumatoid arthritis and osteoarthritis; · Hereditary systemic autoinflammatory diseases such as familial Mediterranean fever; Systemic autoinflammatory diseases such as systemic juvenile idiopathic arthritis and adult-onset Still's disease; · Common inflammatory diseases such as gout and type 2 diabetes; Acute onset ischemic disease, such as myocardial infarction; and Including cancer.

[0365] Many therapeutic approaches to block IL-1 activity are approved and under development. IL-1-targeted therapy began in 1993 with the release of Anakinra (Kineret; Amgen), a recombinant form of the naturally occurring IL-1 receptor antagonist (IL-1Ra or IL1RA) that blocks both IL-1α and IL-1β activity; this therapy has since been used to demonstrate the role of IL-1 in many diseases (see above). Anakinra currently dominates the field of IL-1 therapeutics due to its favorable safety profile, short half-life, and multiple routes of administration. Neutralizing IL-1 with antibodies or soluble receptors is also effective, and the soluble decoy receptor rilonacept (Arcalyst; Regeneron) and the anti-IL-1β neutralizing monoclonal antibody canakineumab (Ilaris; Novartis) have been approved. Other therapeutic approaches, such as neutralization of IL-1α, therapeutic vaccines targeting IL-1β, and chimeric IL-1Ra, are in early clinical trials. In addition, orally active small-molecule inhibitors of IL-1 production, such as caspase-1 inhibitors, have been developed and are being tested.

[0366] Similarly, emerging evidence implicates IL-33 in disease, as detailed above. As detailed above, IL-33 has been implicated in, for example, asthma, allergic diseases, inflammatory bowel disease, and dermatitis. Importantly, IL-33 is involved in fibrosis and inflammation (Kotsiou et al. 2018, IL-33 / ST2 Axis in Organ Fibrosis, Front Immunol. 2018 Oct 24;9:2432). IL-33 can potently stimulate a variety of cells, and its pleiotropic nature is reflected in the role of IL-33 in tissue and metabolic homeostasis, infection, inflammation, cancer, and diseases of the central nervous system.

[0367] Similarly, as mentioned above, new evidence is emerging implicating IL-36 in disease, but the field is still developing and knowledge related to IL-36 is still limited compared to IL-1. As detailed above, emerging evidence indicates that IL-36 signaling is involved in the activation of innate and adaptive immune responses (Ding L,IL-36 cytokines in autoimmunity and inflammatory disease,Oncotarget,Vol.9,(No.2),pp:2895-2901(2018)). In addition to its role in inflammatory skin diseases such as psoriasis and atopic dermatitis, emerging evidence also suggests aberrant IL-36 activity in pulmonary, renal, and intestinal inflammatory diseases, indicating the potential of IL-36 as a therapeutic target for inflammatory diseases.

[0368] One of skill in the art will appreciate that the antibodies, or antigen-binding fragments thereof, of the present invention, by their ability to inhibit, for example, IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and / or IL-36γ signaling, may or can be expected to simultaneously target multiple IL-dependent pathways and have profound effects on inflammatory and / or fibrotic diseases.

[0369] In some embodiments, the disease or disorder is an inflammatory and / or fibrotic disease or disorder.

[0370] In some embodiments, the disease or disorder is an inflammatory and / or fibrotic disease or disorder; In this regard, the inflammatory and / or fibrotic disease or disorder may be rheumatoid arthritis, all types of arthritis, psoriatic arthritis, all types of juvenile arthritis including systemic onset juvenile idiopathic arthritis (SOJIA), osteoarthritis, familial cold autoinflammatory syndrome (FCAS), Muckle-Wells disease, neonatal onset multisystem inflammatory disease (NOMID), familial Mediterranean fever (FMF), septic arthritis, pyoderma gangrenosum and acne (PAPA) syndrome, adult onset Still's disease, hyper IgD syndrome, type 2 osteoarthritis, osteoporosis ... Diabetes, macrophage activation syndrome, TNF receptor-associated periodontitis, Blau's disease, ankylosing spondylitis, Sweets' disease, articular lupus, Alzheimer's disease, psoriasis, asthma, allergy, arteriosclerosis, sarcoidosis, atopic dermatitis, systemic lupus erythematosus, bullous pemphigoid, type I diabetes, chronic obstructive pulmonary disease, Helicobacter pylori gastritis, inflammatory bowel disease (including ulcerative colitis and Crohn's disease), hepatitis, hepatitis C, ischemia-reperfusion injury, multiple sclerosis, neonatal or pneumococcal Meningitis, tuberculosis, Behcet's syndrome, septic shock, graft-versus-host disease, adult T-cell leukemia, multiple myeloma, periodontitis, obesity and obesity-related diseases (e.g., metabolic syndrome, cardiac hypertrophy, congestive heart failure, myocardial infarction, varicose veins, polycystic ovary syndrome, gastroesophageal reflux disease (GERD), fatty liver, colon cancer, breast cancer, uterine cancer, chronic renal failure, stroke, and hyperuricemia), intervertebral disc disease, irritable bowel syndrome, Schnitzler's syndrome, allergy / atopic dermatitis, acne, Behcet's syndrome, disease, cardiac fibrosis, cardiovascular disease, cryopin-associated periodic syndrome, cystic fibrosis, Goodpasture's syndrome, Guillain-Barre syndrome, renal fibrosis, liver fibrosis, pulmonary fibrosis, dermatofibrosis, myocarditis, autoimmune myocarditis, organ dysfunction associated with organ transplantation, pancreatitis, peritonitis, uveitis, vasculitis, pneumonia, pulmonary hypertension, sclerosing skin chronic graft-versus-host disease, sepsis, Sjogren's syndrome, systemic sclerosis, Takayasu's arteritis, and gout.

[0371] In some embodiments, the disease or disorder is systemic sclerosis, also called scleroderma or systemic scleroderma.

[0372] In some embodiments, the disease or disorder is peritonitis, such as acute peritonitis.

[0373] In some embodiments, the disease or disorder is psoriasis. In some embodiments, the disease or disorder is psoriatic arthritis. In one embodiment, the disease or disorder is psoriasis and psoriatic arthritis.

[0374] In some embodiments, the disease or disorder is atherosclerosis. In some embodiments, the use of the inventions disclosed herein in preventing, treating, mitigating, detecting, and / or diagnosing atherosclerosis includes reducing atherosclerosis, e.g., reducing clinical signs, symptoms, or pathophysiological correlates of atherosclerosis, e.g., reducing atherosclerotic plaque inflammation, e.g., aortic plaque inflammation, and / or reducing plaque size (e.g., plaque volume and / or plaque area).

[0375] Plaque inflammation can be assessed based on CD45+ leukocyte cell counts by flow cytometry analysis of aortas (e.g., as shown in Example 4). A higher number of CD45+ leukocytes in the test aorta (e.g., a suspected or diagnosed atherosclerotic aorta) compared to healthy control aortas indicates atherosclerotic plaque inflammation. In some embodiments, leukocyte counts can be assessed based on myeloid CD11b+ cells (including Ly6G+ neutrophils) and / or TCR-β+ cells (including CD4+ and / or CD8+ cells).

[0376] Plaque size can be assessed based on plaque volume and / or plaque area. Increased plaque size (e.g., plaque volume and / or plaque area) in a test aorta (e.g., an aorta suspected or diagnosed as atherosclerotic aorta) compared to a healthy control aorta (which may be free of plaque) indicates the presence of atherosclerotic plaque.

[0377] In some embodiments, the disease or disorder is pulmonary fibrosis, also known as pulmonary fibrosis.

[0378] Fibrosis is a hallmark of many diseases. In some cases, fibrosis is an integral part of the disease, meaning that fibrosis may be the result of abnormal or pathological cellular behavior that results in pathological deposition of extracellular matrix proteins. In other cases, fibrosis may be a secondary event that occurs in diseased tissues in parallel with the primary disease or during the healing process. For example, fibrosis may be scar formation. Any type of fibrosis may be targeted and, for example, treated or prevented by antibodies as disclosed herein. Fibrosis may be classified as uncontrolled or unregulated scar tissue formation caused by excessive accumulation of extracellular matrix components such as collagen. Thus, the use of the invention disclosed herein in preventing, treating, mitigating, detecting, and / or diagnosing fibrosis is considered to be upstream of the prevention, treatment, mitigating, detecting, and / or diagnosing of a disease characterized by fibrosis. Alternatively or additionally, the use of the invention disclosed herein in preventing, treating, mitigating, detecting, and / or diagnosing fibrosis may be downstream of a disease in which fibrosis is a secondary event in said disease.

[0379] In some embodiments, the disease or disorder is myocarditis.

[0380] In some embodiments, the disease or disorder is autoimmune myocarditis.

[0381] In some embodiments, the use of the inventions disclosed herein in the prevention, treatment, mitigation, detection, and / or diagnosis of myocarditis, such as autoimmune myocarditis, includes reducing myocarditis, such as autoimmune myocarditis, e.g., reducing clinical signs, symptoms, or pathophysiological correlates of myocarditis, such as autoimmune myocarditis, e.g., reducing deterioration of cardiac function, reducing inflammation, and / or reducing fibrosis.

[0382] In some embodiments, the disease or disorder is graft-versus-host disease, including chronic graft-versus-host disease and sclerocutaneous chronic graft-versus-host disease. In one embodiment, the disease or disorder is dermal fibrosis and / or pulmonary fibrosis in graft-versus-host disease. In certain embodiments, the graft-versus-host disease is caused by syngeneic transplantation. In alternative embodiments, the graft-versus-host disease is caused by allogeneic transplantation.

[0383] In some embodiments, the disease or disorder is an acute inflammatory disease or disorder.

[0384] In some embodiments, the disease or disorder is a chronic inflammatory disease or disorder. Those skilled in the art will appreciate that diseases or disorders may be grouped in various ways: a disease or disorder may not be explicitly labeled as an inflammatory disease or a fibrotic disease, but may still have inflammatory and / or fibrotic components, e.g., inflammatory and / or fibrotic processes that form part of or contribute to the disease or disorder.

[0385] In some embodiments, the disease or disorder has an inflammatory and / or fibrotic component.

[0386] In some embodiments, the disease or disorder is an autoimmune disease or disorder.

[0387] In some embodiments, the antibody or antigen-binding fragment of the first aspect of the invention inhibits an inflammatory process.

[0388] In some embodiments, the antibody or antigen-binding fragment of the first aspect of the invention inhibits the fibrotic process.

[0389] In some embodiments, the antibody or antigen-binding fragment of the first aspect of the invention inhibits a proliferative process.

[0390] IL1RAP as a biomarker for neoplastic diseases or disorders A neoplasm refers to a neoplastic disease or disorder (i.e., cancer), which is a type of abnormal and excessive growth (also called abnormal proliferation) of tissues or cells, e.g.

[0391] Tumor biomarkers or neoplastic disease or disorder biomarkers are endogenous proteins or metabolites whose amounts or modifications indicate tumor status, progression characteristics, and responsiveness to treatment. They are present in tumor tissues and body fluids and encompass a wide variety of molecules, including transcription factors, cell surface receptors, and secreted proteins. Effective tumor markers are in high demand because they have the potential to reduce cancer mortality by early diagnosis and personalized treatment. In the past decade, our understanding of carcinogenesis and tumor progression has improved and many potential tumor markers have been identified. With the application of current technologies, including tissue microarrays, antibody arrays, and mass spectrometry, it is expected that many more will be discovered in the near future.

[0392] Interleukin-1 receptor accessory protein (IL1RAP) has previously been identified as a cell surface biomarker associated with hematological neoplastic disorders such as chronic myeloid leukemia (CML), acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) (see, e.g., WO2011 / 021014 to Cantargia AB, Jaras et al., 2010, Proc Natl Acad Sci USA 107(37):16280-5, Askmyr et al., 2013, Blood. 121(18):3709-13 and Barreyro et al., 2012, Blood 120(6):1290-8, the disclosures of which are incorporated herein by reference). More recently, the utility of IL1RAP as a diagnostic and therapeutic biomarker for solid tumors such as melanoma has also been demonstrated (see WO2012 / 098407 to Cantargia AB, the disclosure of which is incorporated herein by reference).

[0393] Thus, in some embodiments of the above aspects of the invention, the disease or disorder is a neoplastic disease or disorder.

[0394] One of skill in the art will appreciate that the antibodies of the present invention may have profound effects on neoplastic diseases or disorders due to their ability to inhibit, for example, IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and IL-36γ signaling, thereby simultaneously targeting multiple IL-dependent pathways.

[0395] In some embodiments, the disease or disorder is a neoplastic disease or disorder, and the neoplastic disease or disorder is a hematological disease or disorder or a solid tumor.

[0396] In some embodiments, the neoplastic disease or disorder is a hematological disease, and the neoplastic hematological disease or disorder is selected from the group consisting of chronic myeloid leukemia (CML), myeloproliferative disorder (MPD), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL), and acute myeloid leukemia (AML).

[0397] In some embodiments, the neoplastic disease or disorder is a solid tumor, wherein the solid tumor is selected from the group consisting of prostate cancer, breast cancer, lung cancer, colon cancer, colorectal cancer, melanoma, bladder cancer, brain / CNS cancer, cancer of the urinary system, biliary tract cancer (also known as cholangiocarcinoma), cervical cancer, esophageal cancer, gastric cancer, head / neck cancer, kidney cancer, liver cancer, lymphoma, ovarian cancer, pancreatic cancer, sarcoma, skin cancer, and uterine cancer.

[0398] Those skilled in the art will appreciate that the antibodies of the present invention can simultaneously target multiple IL-dependent pathways by inhibiting signaling of IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and / or IL-36γ, and therefore can be expected to have profound effects on, for example, neoplastic diseases or disorders.

[0399] One of skill in the art will appreciate, for example, in light of the evidence above, that the antibodies of the present invention can be used to prevent, treat, alleviate, detect, and / or diagnose any disease or disorder involving the activity of IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and / or IL-36γ.

[0400] A ninth aspect of the invention provides an antibody or antigen-binding fragment of the first aspect of the invention, A polynucleotide according to the second aspect of the present invention, A vector according to the third aspect of the present invention, A host cell according to the fourth aspect of the invention, and / or A composition according to a sixth aspect of the present invention, For use in inducing cell death and / or inhibiting the growth and / or proliferation of pathological cells associated with a neoplastic disease of a subject, or their stem or progenitor cells, wherein said cells express IL1RAP.

[0401] A tenth aspect of the present invention is a method for producing a composition comprising the steps of: An antibody or antigen-binding fragment according to the first aspect of the invention, A polynucleotide according to the second aspect of the present invention, A vector according to the third aspect of the present invention, A host cell according to the fourth aspect of the invention, and / or A composition according to a sixth aspect of the present invention, in the preparation of a medicament for the prevention, treatment, mitigation, detection, and / or diagnosis of a disease or disorder susceptible to treatment with an inhibitor of IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and / or IL-36γ signaling, and / or said disease or disorder is associated with cells expressing IL1RAP.

[0402] An eleventh aspect of the present invention is a method for producing a composition comprising the steps of: An antibody or antigen-binding fragment according to the first aspect of the invention, A polynucleotide according to the second aspect of the present invention, A vector according to the third aspect of the present invention, A host cell according to the fourth aspect of the invention, and / or A composition according to a sixth aspect of the present invention, It is used in the preparation of a medicament for the detection and / or diagnosis of a disease or disorder associated with cells expressing IL1RAP.

[0403] A twelfth aspect of the present invention relates to a method for the prevention and / or treatment and / or alleviation and / or detection and / or diagnosis of a disease or disorder susceptible to treatment with an inhibitor of IL-1α, IL-1β, IL-33, IL-36α, IL-36β and / or IL-36γ signaling, and / or said disease or disorder relates to IL1RAP expressing cells in a subject, an antibody or antigen-binding fragment according to the first aspect of the invention, a polynucleotide according to the second aspect of the invention, - a vector according to the third aspect of the invention, a host cell according to the fourth aspect of the invention, and / or - administering an effective amount of a composition according to the sixth aspect of the invention.

[0404] A thirteenth aspect of the present invention relates to an in vitro method for detecting cells expressing IL1RAP in a subject, said method comprising: (a) providing a sample of cells from a subject to be tested, such as a tissue biopsy or blood sample; (b) optionally extracting and / or purifying cells present in the sample; (c) contacting the antibody or antigen-binding fragment of the first aspect of the invention with cells present in said sample; (d) determining whether the antibody or antigen-binding fragment thereof binds to the cell; wherein said binding of said antibody or antigen-binding fragment thereof to a cell indicates the presence of a disease or disorder associated with cells expressing IL1RAP in said tissue of the subject.

[0405] A fourteenth aspect of the invention relates to an in vitro method for identifying a patient having a disease or disorder associated with cells expressing IL1RAP, who would benefit from treatment with an antibody or antigen-binding fragment of the first aspect of the invention, comprising: (a) providing a cell sample, such as a tissue biopsy or blood sample, from a patient to be tested; (b) optionally extracting and / or purifying cells present in the sample; (c) contacting the antibody or antigen-binding fragment of the first aspect of the invention with cells present in said sample; (d) determining whether the antibody or antigen-binding fragment thereof binds to the cell; Here, binding of the antibody or antigen-binding fragment thereof to cells expressing IL1RAP is indicative of a patient that would benefit from treatment with the antibody or antigen-binding fragment of the first aspect of the invention.

[0406] A fifteenth aspect of the invention relates to a method for treating a patient having a disease or disorder associated with expression of IL1RAP in a cell, said method comprising: a) selecting a patient identified as having a disease or disorder associated with cells expressing IL1RAP using a method according to the fourteenth aspect of the invention; and b) administering to said patient a therapeutic agent effective to treat said disease or disorder.

[0407] A sixteenth aspect of the present invention relates to a method for detecting cells expressing IL1RAP, said method comprising: (a) contacting an antibody or antigen-binding fragment thereof according to the first aspect with a cell to be analyzed for expression of IL1RAP; (b) determining whether the antibody or antigen-binding fragment thereof binds to the cell; wherein said binding of said antibody or antigen-binding fragment thereof to a cell indicates the presence of a disease or disorder associated with cells expressing IL1RAP in said tissue of the subject.

[0408] In some embodiments, the method of the sixteenth aspect is an in vitro method.

[0409] In some embodiments, the method of the sixteenth aspect is an in vivo method.

[0410] One skilled in the art will appreciate that when the method of the sixteenth aspect is applied, an antibody or antigen-binding fragment having binding specificity for IL1RAP is administered to the subject, for example as described herein.

[0411] A seventeenth aspect of the present invention relates to a method for reducing inflammation in a subject having peritonitis, said method comprising: an antibody or antigen-binding fragment according to the first aspect of the invention, a polynucleotide according to the second aspect of the invention, - a vector according to the third aspect of the invention, a host cell according to the fourth aspect of the invention, and / or - administering an effective amount of a composition according to the sixth aspect of the invention.

[0412] In some embodiments, the method for reducing inflammation in a subject with peritonitis comprises reducing the infiltration of immune cells, such as neutrophils and monocytes, into the peritoneum.

[0413] In some embodiments, the method for reducing inflammation in a subject with peritonitis includes reducing the levels of cytokines, for example, reducing the levels of eotaxin, G-CSF, IL-5, MCP-1, MIP-1β, IL-6, and / or KC (also known as CXCL1).

[0414] An eighteenth aspect of the invention relates to a method for reducing disease severity in a subject having psoriasis or psoriatic arthritis, said method comprising: an antibody or antigen-binding fragment according to the first aspect of the invention, a polynucleotide according to the second aspect of the invention, - a vector according to the third aspect of the invention, a host cell according to the fourth aspect of the invention, and / or - administering an effective amount of a composition according to the sixth aspect of the invention.

[0415] In some embodiments, the method for reducing disease severity in a subject with psoriasis or psoriatic arthritis comprises reducing skin inflammation and / or skin erythema.

[0416] In some embodiments, the methods for reducing disease severity in a subject with psoriasis or psoriatic arthritis include reducing the level of a cytokine, for example, reducing the level of IL-17.

[0417] A nineteenth aspect of the present invention relates to a method of reducing inflammation of atherosclerotic plaque in a subject having atherosclerosis, said method comprising: an antibody or antigen-binding fragment according to the first aspect of the invention, a polynucleotide according to the second aspect of the invention, - a vector according to the third aspect of the invention, a host cell according to the fourth aspect of the invention, and / or - administering an effective amount of a composition according to the sixth aspect of the invention.

[0418] In some embodiments, the method for reducing inflammation of atherosclerotic plaques in an atherosclerosis patient comprises reducing the number of CD45+ leukocytes, e.g., myeloid CD11b+ cells, e.g., Ly6G+ neutrophils, and / or reducing the number of TCR-β+ cells, e.g., CD4+ and / or CD8+ cells.

[0419] A twentieth aspect of the present invention relates to a method of reducing atherosclerotic plaque volume and / or atherosclerotic plaque size in a subject having atherosclerosis, said method comprising: an antibody or antigen-binding fragment according to the first aspect of the invention, a polynucleotide according to the second aspect of the invention, - a vector according to the third aspect of the invention, a host cell according to the fourth aspect of the invention, and / or - administering an effective amount of a composition according to the sixth aspect of the invention.

[0420] A twenty-first aspect of the present invention relates to a method for reducing inflammation and / or fibrosis in a subject having myocarditis, said method comprising administering to said subject: an antibody or antigen-binding fragment according to the first aspect of the invention, a polynucleotide according to the second aspect of the invention, - a vector according to the third aspect of the invention, a host cell according to the fourth aspect of the invention, and / or - administering an effective amount of a composition according to the sixth aspect of the invention.

[0421] A twenty-second aspect of the present invention relates to a method for counteracting deterioration of cardiac function in a subject having myocarditis or autoimmune myocarditis, said method comprising administering to said subject: an antibody or antigen-binding fragment according to the first aspect of the invention, a polynucleotide according to the second aspect of the invention, - a vector according to the third aspect of the invention, a host cell according to the fourth aspect of the invention, and / or - administering an effective amount of a composition according to the sixth aspect of the invention.

[0422] A twenty-third aspect of the invention relates to a method for reducing skin fibrosis in a subject having systemic sclerosis, said method comprising administering to said subject: an antibody or antigen-binding fragment according to the first aspect of the invention, a polynucleotide according to the second aspect of the invention, - a vector according to the third aspect of the invention, a host cell according to the fourth aspect of the invention, and / or - administering an effective amount of a composition according to the sixth aspect of the invention.

[0423] In some embodiments, the method for reducing dermal fibrosis in a subject with systemic sclerosis comprises reducing skin thickness, reducing the number of myofibroblasts, and / or reducing the amount of collagen in the skin.

[0424] A twenty-fourth aspect of the present invention relates to a method of reducing pulmonary fibrosis in a subject with systemic sclerosis, said method comprising: an antibody or antigen-binding fragment according to the first aspect of the invention, a polynucleotide according to the second aspect of the invention, - a vector according to the third aspect of the invention, a host cell according to the fourth aspect of the invention, and / or - administering an effective amount of a composition according to the sixth aspect of the invention.

[0425] In some embodiments, the method for reducing pulmonary fibrosis in a subject with systemic sclerosis comprises reducing the Ashcroft score and / or the amount of collagen in the lung.

[0426] The embodiments and explanations according to the eighth aspect shall also apply to the 9th, 10th, 11th, 12th, 13th, 14th, 15th and / or 16th aspects of the invention, e.g. in the context of diseases or disorders susceptible to treatment with inhibitors of IL-1α, IL-1β, IL-33, IL-36α, IL-36β and / or IL-36γ signalling and / or in the context of diseases or disorders associated with cells expressing IL1RAP. They may also be applicable to the 17th, 18th, 19th, 20th, 21st, 22nd, 23rd or 24th aspects of the invention. EXAMPLES

[0427] Example 1: Inhibition of cytokine signaling by the mouse surrogate anti-IL1RAP antibody mCAN10, a specific inhibitor of mouse IL1RAP

[0428] the purpose This example illustrates the characterization of the murine surrogate anti-IL1RAP antibody mCAN10, a specific inhibitor of murine ILRAP, for blocking signaling by IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and IL-36γ.

[0429] Materials and Methods Chickens were immunized with mouse IL1RAP antigen and multiple anti-mouse IL1RAP binders were identified. These selections were cloned into mouse IgG2a spines. One such clone, mCAN10, capable of binding to domain 2 of mouse ILRAP, was evaluated for its ability to inhibit signaling by IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and IL-36γ.

[0430] For this evaluation, HEK-Blue™ cells stably transfected with mouse IL-1R were used. Alternatively, HEK-Blue™ cells were transiently transfected with mouse IL-33 and IL-36 receptors the day before the assay to allow for analysis of mouse IL-33 and IL-36 signaling as well. HEK-Blue™ cells were seeded in 96-well plates and allowed to settle for at least 2 hours before continuing. Cells were then exposed to increasing concentrations of mCAN10 (0, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, and 100 μg / ml) and incubated at 37° C., 5% CO2 for 1 hour before addition of mouse cytokines. Cytokines were added at the following concentrations: 3 pg / ml IL-1α, 30 pg / ml IL-1β, 5 ng / ml IL-33, 4 ng / ml IL-36α, 5 ng / ml IL-36β, and 10 ng / ml IL-36γ. Because mouse IL-1α and IL-1β may cross-react with the human IL-1R endogenously expressed by HEK-Blue™ cells, anti-human IL1RAP antibodies were used to block signaling through IL-1R before stimulating these cells with mouse IL-1α and IL-1β. Cells were cultured at 37°C, 5% CO2 for 16-18 hours and then analyzed for NF-κB activation and subsequent production of SEAP using QUANTI-Blue™ solution and measured at 620 nm using a SpectraMax i3x spectrophotometer.

[0431] result Increasing concentrations of mCAN10 were added to HEK-Blue™ and constant concentrations of mouse IL-1α, IL-1β, IL-33, IL-36α, IL-36β, or IL-36γ were added. A graph of the optical density values ​​at 620 nm was used to calculate the IC50 values ​​shown in Table 2. mCAN10 induced inhibition of IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and IL-36γ signaling.

[0432] [Table 5]

[0433] conclusion mCAN10 is a mouse surrogate anti-mouse IL1RAP inhibitor capable of blocking signaling by IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and IL-36γ. Thus, mCAN10 provides a useful tool to evaluate the therapeutic efficacy of IL1RAP blockade in various mouse disease models. Because anti-human IL1RAP antibodies may lack cross-reactivity to mouse IL1RAP (e.g., the anti-human IL1RAP antibodies described in Examples 9-22), mCAN10 is a suitable surrogate for anti-human IL1RAP antibodies with similar functional properties. In subsequent in vivo experiments (Examples 2-8), an effector function silent mouse IgG2a format of mCAN10 was used. EXAMPLES

[0434] Example 2: The murine surrogate anti-IL1RAP antibody mCAN10, a specific inhibitor of murine IL1RAP, reduces the inflammatory response in a model of acute peritonitis

[0435] the purpose The aim of these experiments was to evaluate how blockade of IL-1α / β, IL-33, and IL-36α / β / γ signaling with the murine surrogate anti-IL1RAP antibody mCAN10 affects the inflammatory response in a mouse model of acute peritonitis. An additional aim was to compare the effect of this antibody with anakinra, a recombinant IL-1R antagonist (IL1RA) protein that blocks only IL-1α / β signaling.

[0436] Materials and Methods Female C57Bl / 6 mice (10-18 weeks old), either wild type (WT) or IL1RAP knockout (KO), were immunized intraperitoneally (ip) with 2.5 mg of monosodium urate (MSU) crystals. WT mice that were not immunized with MSU crystals were included as controls (no MSU). All mice were terminated 6 h after immunization, and peritoneal lavage fluid was collected for quantification of infiltrating cells (B cells, T cells, monocytes, neutrophils, and eosinophils) by flow cytometry (Figure 1A).

[0437] Alternatively, female C57Bl / 6 WT mice (8-9 weeks old) were treated with 20 mg / kg mCAN10 or a molar equivalent dose (2.3 mg / kg) of IL1RA. Mice treated with isotype control antibody or PBS were included as controls. One hour (hr) after treatment, mice were immunized intraperitoneally with 2.5 mg MSU crystals. Mice that did not receive any treatment and were not immunized with MSU crystals were included as controls (no MSU). All mice were terminated 6 hr after immunization and peritoneal lavage fluid was collected for quantification of infiltrating cells by flow cytometry (Figure 1B) or for quantification of various cytokines and chemokines (eotaxin, G-CSF, IL-5, MCP-1, MIP-1β, IL-6, and KC (also known as CXCL1)) by Luminex assay (Figure 1C).

[0438] result After immunization with MSU crystals, IL1RAP KO mice showed a significant reduction in the infiltration of cells into the peritoneal cavity compared to WT mice, with the greatest effect on infiltrating neutrophils and monocytes (Figure 1A). Treatment with mCAN10 and IL1RA significantly reduced the number of infiltrating cells, such as neutrophils and monocytes, in the peritoneal cavity of WT mice immunized with MSU crystals compared to isotype and PBS controls, respectively. However, mCAN10 had a stronger anti-inflammatory effect on monocytes and neutrophils than IL1RA (Figure 1B). mCan10 and IL1RA also significantly reduced G-CSF and IL-6 compared to isotype and PBS controls, respectively. mCAN10 also reduced eotaxin, IL-5, MCP-1, and MIP-1β compared to isotype controls, and showed a significantly greater reduction in IL-6 compared to IL1RA (Figure 1C).

[0439] conclusion mCAN10 (which blocks IL-1α / β, IL-33, and IL-36α / β / γ signaling) reduces inflammatory responses more potently than IL1RA (which blocks only IL-1α / β signaling) in a mouse model of acute peritonitis. EXAMPLES

[0440] Example 3: Mouse surrogate anti-IL1RAP antibody mCAN10, a specific inhibitor of mouse IL1RAP, reduces disease severity in models of psoriasis and psoriatic arthritis

[0441] the purpose The aim of these experiments was to assess how blockade of IL-1α / β, IL-33, and IL-36α / β / γ signaling with the murine surrogate anti-IL1RAP antibody mCAN10 affects disease severity in imiquimod-induced psoriasis and mannan-induced psoriatic arthritis. An additional aim was to compare the effect of this antibody with an anti-IL-1β antibody that blocks only IL-1β signaling.

[0442] Materials and Methods Psoriasis was induced in female BALB / c mice (8-10 weeks old) by topical application of Zyclara cream (3.75% imiquimod; approximately 71.4 mg / day) daily, starting on day 0 and ending on day 7. The cream was applied to the shaved backs of the mice. During this period of disease induction, mice received intraperitoneal treatments on days 0, 3, and 5. Mice were treated with mCAN10 (10 mg / kg), anti-IL-1β (0.5 mg / kg), or isotype controls at the same doses for these antibodies. Mice treated with PBS only (vehicle) or dexamethasone (10 mg / kg) were included as negative and positive controls, respectively. Disease severity was assessed daily throughout the experiment by scoring skin inflammation and erythema on the shaved back area using a score ranging from 0 to 4 (a total of 8 for each mouse) (Figure 2A-B). Alternatively, psoriatic arthritis was induced in male and female B6N.Q.NCF1 mice (8-14 weeks old) that received 20 mg of Saccharomyces cerevisiae-derived mannan intraperitoneally on day 0. These mice were treated in the same manner as above on days 0, 3, and 5. Disease severity was assessed by scoring paw joint inflammation using a macroscopic scoring system of 4 paws ranging from 0 to 15 (total of 60 for each mouse) (Figure 2C-D). At the end of the experiment, blood was collected for analysis of IL-17, a major contributing cytokine in psoriatic arthritis (Figure 2E).

[0443] result mCAN10 reduced disease severity in imiquimod-induced psoriasis, but not anti-IL-1β antibodies (Figure 2A-B). Similarly, mCAN10 reduced disease severity in mannan-induced psoriatic arthritis (Figure 2C-D) and also reduced levels of circulating IL-17 compared to vehicle (Figure 2E), but not anti-IL-1β antibodies.

[0444] conclusion mCAN10 (which blocks IL-1α / β, IL-33 and IL-36α / β / γ signaling), but not anti-IL-1β antibodies (which block only IL-1β signaling), reduces disease severity in both imiquimod-induced psoriasis and mannan-induced psoriatic arthritis. EXAMPLES

[0445] Example 4: Mouse surrogate anti-IL1RAP antibody mCAN10, a specific inhibitor of mouse IL1RAP, reduces atherosclerosis and aortic plaque inflammation in Apoe KO mice

[0446] the purpose The goal of these experiments was to evaluate how blockade of IL-1α / β, IL-33, and IL-36α / β / γ signaling with the murine surrogate anti-IL1RAP antibody mCAN10 affects atherosclerosis in apolipoprotein E (Apoe) KO mice.

[0447] Materials and Methods Female Apoe KO mice (10-12 weeks old) were fed a high cholesterol diet (HCD; 21% fat, 0.21% cholesterol) to develop atherosclerotic lesions. Four weeks after the HCD, mice treatment began, with mice receiving either mCAN10 (20 mg / kg for the first dose; 10 mg / kg for subsequent doses) or the same dose of isotype control antibody intraperitoneally every other week (twice a week) for six weeks. Throughout these six weeks, mice continued to be fed the HCD. To reduce cage-to-cage variability, for each cage, mice were divided between treatment groups. 48 h after the last dose, mice were anesthetized and sacrificed, and then aortas were harvested to study changes in immune cell composition, including myeloid cells (sum of CD11b+ cells and Ly6G+ neutrophil subpopulations) (Figure 3A-C) and T lymphocytes (sum of TCR-β+ cells, and CD4+ and CD8+ subpopulations) (Figure 3D-F), by flow cytometry. Hearts were harvested and sectioned at the aortic root, sections were stained for lipid accumulation by Oil-Red O, and plaque size was compared between groups (Figure 4A-B).

[0448] result Flow cytometry analysis of atherosclerotic aortas shows that mCAN10 reduces plaque inflammation. This is observed by a decrease in the number of CD45+ leukocytes (Figure 3A). Among these, myeloid CD11b+ cells, including Ly6G+ neutrophils, were decreased (Figure 3B-C). The number of TCR-β+ cells, including both CD4+ and CD8+ cells, was also decreased (Figure 3D-F). Additionally, mCAN10 significantly reduced aortic plaque volume and area in HCD-fed Apoe KO mice compared to isotype controls (Figure 4A-B).

[0449] conclusion Blockade of IL1RAP with mCAN10 reduces atherosclerosis and limits plaque inflammation in Apoe KO mice. EXAMPLES

[0450] Example 5: Mouse surrogate anti-IL1RAP antibody mCAN10, a specific inhibitor of mouse IL1RAP, reduces inflammation and fibrosis in experimental autoimmune myocarditis

[0451] the purpose The aim of these experiments was to evaluate how blockade of IL-1α / β, IL-33, and IL-36α / β / γ signaling by the murine surrogate anti-IL1RAP antibody mCAN10 affects the development of inflammation and fibrosis in experimental autoimmune myocarditis (EAM).

[0452] Materials and Methods EAM was induced in male BALB / c mice (7–8 weeks old) by subcutaneously immunizing mice on days 0 and 7 with 100 μg of mouse α-myosin heavy chain (αMHC) peptide emulsified in complete Freund's adjuvant. Mice were treated intraperitoneally every other week for 4 weeks with mCAN10 (20 mg / kg for the first dose; 10 mg / kg for subsequent doses) or the same dose of isotype control antibody. Mice treated with PBS were included as controls. Treatment began on day 14, 1 week after the final immunization with αMHC. At the end of the experiment, on day 42, mice were sacrificed and hearts were harvested. Hearts were fixed in 10% buffered formalin, embedded in paraffin, and sectioned longitudinally. One central section per heart was stained with hematoxylin and eosin (H&E) to assess the degree of inflammation in the left ventricle (LV) by grading the area infiltrated by hematopoietic cells using a 0-5 range score system (Figure 5A-B). Alternatively, one central section per heart was stained with Masson's trichrome to detect collagen deposition as a measure of fibrotic tissue. Fibrotic blue area and total area were measured using computerized planimetry (ImageJ). Fibrotic area is presented as a percentage of total area (Figure 5C-D).

[0453] result mCAN10 reduced infiltrating inflammatory cells in the myocardium as assessed by scoring of H&E stained sections of hearts from EAM-induced mice (Figure 5A-B). Additionally, mCAN10 also reduced cardiac fibrosis as determined by analysis of Masson's Trichrome stained cardiac sections (Figure 5C-D).

[0454] conclusion mCAN10 can reduce the occurrence of inflammation and fibrosis in EAM. EXAMPLES

[0455] Example 6: Mouse surrogate anti-IL1RAP antibody mCAN10, a specific inhibitor of mouse IL1RAP, reverses the deterioration of cardiac function in experimental autoimmune myocarditis

[0456] the purpose The aim of these experiments was to evaluate how blockade of IL-1α / β, IL-33, and IL-36α / β / γ signaling by the murine surrogate anti-IL1RAP antibody mCAN10 affects cardiac function in experimental autoimmune myocarditis (EAM). A further aim was to compare the effect of this antibody with an anti-IL-1β antibody that blocks only IL-1β signaling, anakinra, a recombinant IL-1R antagonist (IL1RA) protein that blocks only IL-1α / β signaling, and prednisone, an anti-inflammatory glucocorticoid.

[0457] Materials and Methods EAM was induced in male BALB / c mice (7-8 weeks old) by immunizing mice subcutaneously on days 0 and 7 with 100 μg of mouse α-myosin heavy chain (αMHC) peptide emulsified in complete Freund's adjuvant. Treatment began on day 7, the same day as the final immunization with αMHC. Mice were treated intraperitoneally every other week for 5 weeks with mCAN10 (first dose 20 mg / kg; subsequent doses 10 mg / kg), anti-IL-1β antibody (0.5 mg / kg), or the same dose of isotype control antibody. Alternatively, mice were treated daily for 5 weeks with 25 mg / kg IL1RA subcutaneously, 5 mg / kg prednisone by oral gavage, or the relevant vehicle control. Mice treated with PBS were included as controls. To assess cardiac function, transthoracic echocardiography was performed on mice at the start of the study and on days 28 and 42 for measurement of left ventricular ejection fraction (LVEF) (Figure 6A-C).

[0458] result mCAN10 significantly preserved cardiac function as assessed by LVEF measurement in EAM-induced mice compared to isotype and anti-IL-1β antibodies (Figure 6A). However, IL1RA and prednisone showed no significant effect on cardiac function compared to their respective control groups (Figure 6B). Additionally, mCAN10 also preserved cardiac function when treatment was initiated on day 14 instead of day 7 (Figure 6C).

[0459] conclusion mCAN10 has a therapeutic effect on cardiac function in EAM-induced mice, whereas anti-IL-1β antibody, IL1RA and prednisone do not. EXAMPLES

[0460] Example 7: Murine surrogate anti-IL1RAP antibody mCAN10, a specific inhibitor of murine IL1RAP, ameliorates dermal and pulmonary fibrosis in a model of sclerosant chronic graft-versus-host disease

[0461] the purpose The purpose of these experiments was to evaluate how blockade of IL-1α / β, IL-33, and IL-36α / β / γ signaling with the murine surrogate anti-IL1RAP antibody mCAN10 affects fibrosis in a mouse model of sclerosant chronic graft-versus-host disease (scl cGvHD).

[0462] Materials and Methods Sclerocutaneous chronic graft-versus-host disease Recipient female BALB / c mice (H-2 d ; 8 weeks old; n = 10 / group) were subjected to total body irradiation at 700 cGy. Six hours after irradiation, all BALB / c (H-2 d The recipients were syngeneic female BALB / c mice (H-2 d ) or in an allogeneic transplantation fashion in male B10.D2 mice (H-2 d ) from donor mice. Due to MHC mismatch, allogeneic transplanted mice develop scl cGvHD with fibrosis of the skin and multiple internal organs. For transplantation, 5x10 6 spleen cells and 2x10 6 The bone marrow cells were resuspended in PBS and injected via the tail vein. Treatment began 21 days after bone marrow transplantation. Mice receiving allogeneic transplants were treated intraperitoneally with mCAN10 (first dose 20 mg / kg; subsequent doses 10 mg / kg) or the same dose of isotype control antibody every other week for 4 weeks. Alternatively, these mice were orally administered 50 mg / kg of the small molecule tyrosine kinase inhibitor nintedanib daily for 4 weeks. Nintedanib is approved for the treatment of systemic sclerosis patients with interstitial pneumonia and is used herein as a positive control. Mice receiving syngeneic transplants were treated with isotype control antibody alone. At the end of the experiment, on day 49, the mice were sacrificed to collect skin samples and lungs.

[0463] Histological evaluation of dermal and pulmonary fibrosis 1cm of upper back 2Skin samples of defined areas were fixed in 4% formalin for 6 hours and embedded in paraffin. Sections were cut at 5 μm and stained with hematoxylin and eosin (H&E). Dermal thickness was quantified for H&E stained sections by manually measuring the distance between the epidermal-dermal junction and the dermal-subcutaneous fat junction in four sections per mouse using images captured with a light microscope (Nikon Eclipse 80i) at 100x magnification. The right lobe of the lung samples was similarly fixed, paraffin embedded, sectioned, and subsequently stained with trichrome and Sirius Red. Histological readout included assessment of the stained (fibrotic) area as a percentage of the total lung area in Sirius Red stained sections (two sections per mouse) and quantification of lung changes by the Ashcroft score, a numerical scale for determining the degree of fibrosis in lung specimens (four sections per mouse).

[0464] Hydroxyproline quantification The amount of collagen protein in skin and lung samples was determined by quantification of hydroxyproline. After digestion in 6 M hydrogen chloride at 120°C for 3 h, the pH of the samples was adjusted to 6 with 6 M sodium hydroxide. 0.06 M chloramine T was then added and the samples were incubated at room temperature for 20 min. 3.15 M perchloric acid and 20% p-dimethylaminobenzaldehyde were then added and the samples were incubated at 60°C for 20 min. Absorbance was determined at 557 nm on a Spectra MAX 190 microplate spectrophotometer. Absolute values ​​were determined using a standard curve generated with collagen type I.

[0465] Myofibroblast detection Myofibroblasts for α-SMA (smooth muscle actin) were detected by incubation with monoclonal anti-α-SMA antibody (clone 1A4). Expression was visualized using horseradish peroxidase-labeled secondary antibody and 3,3-diaminobenzidine tetrahydrochloride (DAB). Monoclonal mouse IgG antibody was used as a control. Images of sections stained for α-SMA were captured with a light microscope (Nikon Eclipse 80i). Images were manually evaluated in four different areas at 200x magnification. Myofibroblasts were defined as single spindle-shaped α-SMA positive cells in the dermis.

[0466] result Allogeneic transplants induced marked dermal fibrosis with increased dermal thickness, myofibroblast numbers, and hydroxyproline content compared to syngeneic transplants (Figure 7A-C). Mice treated with mCAN10 showed significantly reduced dermal thickening (Figure 7A) and myofibroblast numbers (Figure 7B), and reduced hydroxyproline, a measure of collagen content (Figure 7C), compared to allogeneic transplanted mice treated with an isotype control antibody. The effects of mCAN10 on these parameters were within the range of those observed with nintedanib (Figure 7A-C). Allogeneic bone marrow transplantation induced moderate pulmonary fibrosis with increased Ashcroft score, Sirius Red stained area, and hydroxyproline content (Figure 8A-C). Treatment with mCAN10 significantly reduced Ashcroft score (Figure 8A), Sirius Red staining (Figure 8B), and hydroxyproline content (Figure 8C) compared to allogeneic transplanted mice treated with isotype control antibody. The effects of mCAN10 on these parameters were again within the range of those observed with nintedanib (Figure 8A-C). mCAN10 was well tolerated, with no signs of toxicity on clinical examination, biopsy, or histology. Additionally, treatment with mCAN10 ameliorated scl cGvHD-induced weight loss more effectively than nintedanib (Figure 9).

[0467] conclusion IL1RAP blockade by mCAN10 is an efficient strategy to reduce scl cGvHD-induced dermal and pulmonary fibrosis. EXAMPLES

[0468] Example 8: Mouse surrogate anti-IL1RAP antibody mCAN10, a specific inhibitor of mouse IL1RAP, alters gene expression profiles associated with inflammatory processes

[0469] the purpose The aim of this study was to evaluate molecular pathways affected by IL1RAP blockade using the murine surrogate anti-IL1RAP antibody mCAN10 in a mouse model of sclerotic chronic graft-versus-host disease (scl-cGvHD). The aim was also to identify disease-associated differentially expressed genes (DEGs) affected by IL1RAP inhibition in skin from scl-cGvHD mice and compare these to disease-associated genes in skin biopsies from systemic sclerosis (SSc) patients.

[0470] Materials and Methods Mouse samples As reported in Example 7, samples were obtained from mice that underwent syngeneic or allogeneic transplants with or without treatment with mCAN10.

[0471] RNASeq preprocessing and analysis Raw paired-end reads were aligned to the GRCm39 reference genome and mapped reads were counted using Rsubread (v2.6.4) in R (v4.1.1). Principal component analysis (PCA) was performed to determine potential outliers among samples in each batch. After PCA, three samples (one per condition) were excluded. For each group, four samples were selected that resulted in tight group-specific clustering. Median ratio normalization and differential expression analysis were performed using the DESeq2 package (v1.32.0). Significant DEG lists were considered with adjusted p-values ​​≤ 0.05 and |log2FC| ≥ 1.5 for comparison. Transcriptome profiles of SSc patients were retrieved from the North American SSc Patient Cohort (NCBI / GEO / GSE130955), which consists of 143 SSc patients and 22 healthy individuals. For comparison between mouse and human samples and detection of mutually expressed genes, human orthologs were mapped to mouse gene symbols using the r package "biomaRt" (v2.48.3).

[0472] Treatment Response Signature Due to the size of the sample, the selection of features for gene combination treatment response signature was performed using logistic regression modeling by applying the R statistical package. Logistic regression model allows classification between two groups (binary outcome system: 1 [= e.g. treatment], 0 [= e.g. healthy]), where stepwise feature selection identifies the best gene set combination for group classification. MASS and ROCR packages were used to select gene combination models based on Akaike information criterion (AIC) and area under the curve (AUC) values. The AIC value is a quality indicator of how well a statistical gene combination model describes the data considering the number of features used, while AUC describes model performance.

[0473] result Analysis of RNASeq data showed a separation between allografted mice (Allo), syngeneic transplanted mice (Syn), and allografted mice treated with mCAN10 (Treatment), where the Treated group was closer to the Syn group. Comparison between Allo vs. Syn identified 2308 DEGs (1023 upregulated, 1285 downregulated; adjusted p-value ≦0.05, |log2FC|≧1.5), where deregulation of processes and pathways related to inflammation and fibrosis was observed. Meanwhile, comparison between Treated vs. Allo identified 495 DEGs (398 upregulated, 106 downregulated; adjusted p-value ≦0.05, |log2FC|≧1.5), where deregulation of several processes related to inflammation was observed upon IL1RAP inhibition by mCAN10. Interestingly, mCAN10 altered gene expression of multiple IL-1 family genes such that the profile was more similar to the Syn group than the Allo group (Figure 10). Analysis of overlap between Allo DEGs (Allo vs. Syn), treated DEGs (treated vs. Allo) and SSc DEGs (SSc vs. Healthy; information retrieved from previously published dataset NCBI / GEO / GSE130955) identified 449 overlapping genes between the mouse model (Allo vs. Syn) and human dataset (SSc vs. Healthy). Of these, 177 (39.4%) genes overlapped between all three groups of DEGs (Figure 11), of which 58 genes (20 upregulated and 38 downregulated; adjusted p-value ≦0.05 and |log2FC| ≧0.25) were mutually regulated between treated DEGs and SSc DEGs.

[0474] conclusion mCAN10 affects inflammation-related processes and pathways in scl GvHD mice, supporting the observed effect of mCAN10 on disease manifestations in this model. A significant number of genes affected by mCAN10 in scl GvHD mice were differentially expressed in SSc patients compared to healthy controls, suggesting that IL1RAP inhibition may also impact human disease. EXAMPLES

[0475] Example 9: Immunization with IL1RAP to produce anti-IL1RAP antibodies

[0476] the purpose This example illustrates how anti-IL1RAP antibodies were isolated from rabbits immunized with IL1RAP.

[0477] Materials and Methods Rabbits were immunized four times and then boosted with a mixture of recombinantly expressed ectodomains of human and mouse IL1RAP, 0.2 mg of antigen per injection. Immunizations were performed by subscapular injection after every 3 weeks. The first immunization was with Freund's complete adjuvant, and the second to fourth immunizations were with Freund's incomplete adjuvant. For the booster, half of the antigen amount was injected subscapularly with Freund's incomplete adjuvant, and the other half was injected intravenously. The immune response of each animal was measured by ELISA using serum approximately 10 days after the third immunization. Specific antibody titers were measured by ELISA, and plates were coated with human and mouse IL1RAP separately for immune response comparison. Two to three weeks after the fourth immunization, animals were boosted once, and spleens were harvested 4 to 5 days after the booster. Spleens of test animals were homogenized, frozen, and stored in liquid nitrogen. Monoclonal antibody sequences were isolated from immunized rabbits using the approach described by Kivi et al. (Kivi G, HybriFree: a robust and rapid method for the development of monoclonal antibodies from different host species. BMC Biotechnology 16:2 (2016)). Streptavidin-coated 96-well plates were coated with 5 μg / ml biotinylated human or mouse IL1RAP or a mixture of both. Ten thousand spleen cells were used for each panning. A total of 48 panning reactions were performed. After 45 min of incubation, the wells were washed with PBS to remove unbound cells. RNA was isolated from bound cells, VH and VL cDNA were synthesized and used to construct a combinatorial VH-VL library in a human IgG expression plasmid format. Plasmid DNA was purified and transfected into CHO cells for the production of chimeric rabbit / human IgG1 pools. Antibody pool supernatants were analyzed using ELISA 48 h after transfection to identify positive pools. Bacteria containing plasmid DNA from the positive pools were plated on LB-ampicillin solid medium, and single colonies were isolated and grown in liquid medium. Plasmid DNA was purified from the liquid cultures and transfected into CHO cells for transient antibody production. Supernatants were analyzed by ELISA 48-72 hours after transfection.

[0478] result Antibodies that bind to human and / or mouse IL1RAP were identified. Chimeric rabbit / human IgG1 ELISA IL1RAP positive clones were further analyzed by sequencing. Chimeric antibodies were further analyzed for their properties, such as the ability to inhibit IL-1, IL-33 or IL-36 signaling. Chimeric antibody 48D2 was selected for further characterization and optimization (see Examples below). The light chain variable region of this antibody comprises a light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO:19. The heavy chain variable region of this antibody comprises a heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO:20. To generate the whole antibody, the variable regions were combined with a light chain constant region comprising or consisting of the amino acid sequence of SEQ ID NO:35 and an IgG1 heavy chain constant region comprising or consisting of the amino acid sequence of SEQ ID NO:36 or SEQ ID NO:2.

[0479] conclusion A monoclonal chimeric rabbit / human IgG1 antibody against IL1RAP was isolated. EXAMPLES

[0480] Example 10: Identification and characterization of antibodies that bind to IL1RAP and block IL-1, IL-33, and IL-36 signaling

[0481] the purpose This example illustrates the characterization of a selected antibody, 48D2 chimeric (ch) rabbit / human anti-human IL1RAP, generated as described in Example 9. The goal was to identify an antibody that has high affinity for both cynomolgus and human IL1RAP and completely blocks all IL1RAP-regulated signaling pathways.

[0482] Materials and Methods Antibody binding to IL1RAP-expressing cells The malignant melanoma (or malignant melanoma) cell line SKMEL-5, which expresses IL1RAP on the cell surface, was used for the analysis. SKMEL-5 cells were cultured according to standard procedures. 5x10 4 SKMEL-5 cells were blocked with human Fc block and then stained with eight serial dilutions (100 μg / ml to 0.03 μg / ml) of chimeric 48D2 or isotype control antibody. After secondary staining with Alexa488-conjugated anti-human IgG antibody, 10,000 cells / sample were analyzed on a CytoFlex (Beckman Coulter). HTB183 cells, which express low levels of surface IL1RAP, were used as a negative control.

[0483] Inhibition of cytokine signaling For analysis of IL-1 and IL-33 inhibition, HEK-Blue™ (IL-33 / IL-1) cells were used as provided. For IL-36 analysis, HEK-Blue™ (IL-33 / IL-1) cells were either transiently transfected with IL-36 receptor the day before the assay or a HEK-Blue™ clone (generated in-house) stably expressing the IL-36 receptor was used. Briefly, HEK-Blue™ cells were seeded in 384-well plates and allowed to settle for a minimum of 2 hours before continuing. Cells were then exposed to increasing concentrations of chimeric 48D2 (as indicated in the figures) and incubated at 37° C., 5% CO2 for 1 hour before the addition of cytokines. Cytokines were added at the following concentrations: 2 ng / ml IL-1α, 0.1 ng / ml IL-1β, 0.2 ng / ml IL-33, 1 ng / ml IL-36α, 3 ng / ml IL-36β, and 0.2 ng / ml IL-36γ. Cells were cultured at 37°C, 5% CO2 for 16-18 hours and then analyzed for NF-κB activation and subsequent SEAP production using QUANTI-Blue™ solution and measured at 620 nm using a SpectraMax i3x spectrophotometer.

[0484] Expression and purification of chimeric 48D2 Chimeric 48D2 was transiently expressed in CHO cells and purified using Protein A affinity chromatography and gel filtration.

[0485] Affinity measurement by biolayer interferometry Affinity measurements were performed with ForteBio's BLItz™ Label-Free Protein Analysis System using the Basic Kinetics Module of Molecular Devices' BLItz Pro 1.3.1.3 software. Protein A biosensors were neutralized with blocking buffer (PBS, 0.5% BSA, 0.05% Tween 20, pH 7.4) for a minimum of 10 minutes at room temperature before coating. Protein A biosensors were coated by immersing the biosensors in 200 μl of antibody solution (10 μg / ml in blocking buffer) for 30 minutes at room temperature. The sensors were then incubated in 200 μl of blocking buffer for at least 10 minutes before use. Each antibody was analyzed at four different concentrations of hIL1RAP(21-367) in blocking buffer; 0.8 nM (0.032 μg / ml), 4 nM (0.16 μg / ml), 20 nM (0.8 μg / ml) and 100 nM (4 μg / ml). A baseline was generated in blocking buffer with a duration of 30 seconds. Association in hIL1RAP was followed by dissociation in blocking buffer for 120 seconds.

[0486] Domain Mapping The IL1RAP ectodomain consists of three domains (domain 1, domain 2, and domain 3). To understand where 48D2 binds to IL1RAP, a series of IL1RAP constructs corresponding to the different domains were generated and binding to these was tested in an indirect ELISA assay using duplicate samples. Constructs were made using the amino acid sequence of IL1RAP shown in Uniprot, ID Q9NPH3. Microtiter plates were coated with 100 ng of recombinant hIL1RAP domains 1+2+3 (composed of domains 1, 2, and 3) (aa21-367) (positive control), recombinant hIL1RAP domains 1+2 (composed of domains 1 and 2) (aa21-234), or domain 1 (aa21-134), or recombinant hIL1RAP domain 3 (aa235-367), (100 μl / well) diluted in 0.01 M PBS, pH 7.4, and incubated overnight at 4° C. After washing the plates with ELISA washing buffer (0.01 M PBS, 0.05% Tween 20, pH 7.4), a blocking step was performed using 150 μl / well of ELISA blocking solution (PBS, 0.5% BSA, 0.05% Tween 20, pH 7.4). After incubation at room temperature for 1 h with agitation, the plate was washed again with ELISA wash buffer. A series of 48D2 dilutions in ELISA blocking solution were prepared (ranging from 1 to 10000 ng / ml) and then transferred to the wells at 100 μl / well. The plate was incubated at room temperature for 1 h with agitation and then washed with ELISA wash solution. Goat anti-human IgG conjugated to alkaline phosphatase was added at 100 μl / well and incubated at room temperature for 1 h with agitation. After washing the plate with ELISA wash solution, substrate (4-nitrophenyl phosphate disodium salt hexahydrate, Sigma-Aldrich, 1 mg / ml) was added at 100 μl / well. The plate was then incubated at room temperature with agitation and the absorbance at 405 nm was measured continuously for 30 min. The absorbance at 0 min was taken as background signal. Polyclonal anti-hIL1RAP antibody KMT-1 was used as a control.

[0487] Epitope mapping Based on the domain mapping and cross-reactivity data, two regions in domain 2 of IL1RAP were identified as potential interacting parts of 48D2 (humanized and deimmunized 48D2 variant VH5.GL:VL4 (see Example 13)), corresponding to aa 141-157 (designated as H1) and 174-191 (designated as H2) (based on human IL1RAP amino acid sequence from Uniprot, ID Q9NPH3). Two chimeric constructs of mouse / human IL1RAP were generated, in which the human sequences of the two regions of interest were grafted into mouse IL1RAP: chimeric IL1RAP.H1 and chimeric IL1RAP.H2. Microtiter plates were coated with the chimeric IL1RAP constructs and ELISA measurements were performed as described above for domain mapping. Polyclonal anti-hIL1RAP antibodies KMT-2 (affinity-purified rabbit polyclonal antibody against human IL1RAP) and KMT-3 (affinity-purified rabbit polyclonal antibody against mouse IL1RAP) served as positive controls.

[0488] Cross-reactivity Microtiter plate wells were coated with IL1RAP orthologues (Mus musculus, Rattus norvegicus, Macaca fascicularis (Mf, synonymous with cynomolgus monkey), Oryctolagus cuniculus, Canis lupus familiaris, Sus scrofa) and incubated for 1 h at 37°C. The plates were washed followed by a blocking step. After 1 h incubation at room temperature with agitation, the plates were washed again. Antibodies were diluted in 4-fold serial dilutions ranging from 4000 ng / ml to 0.24 ng / ml in ELISA blocking solution and then transferred to the ELISA plate. The plates were incubated for 30 min at 37°C with agitation and then washed. Goat anti-human antibody conjugated to alkaline phosphatase was added and incubated for 30 min at 37°C with agitation. The plates were washed followed by the addition of substrate (4-nitrophenyl phosphate disodium salt hexahydrate). The plate was then incubated at room temperature with stirring and the absorbance at 405 nm was measured continuously for 50 min, with the absorbance at 0 min being taken as the background signal.

[0489] result Antibody binding to IL1RAP-expressing cells Flow cytometry analysis of IL1RAP-expressing SKMEL-5 cells stained with chimeric 48D2 or isotype control revealed higher mean fluorescence intensity (MFI) for 48D2 compared to the isotype control antibody (Figure 12). Increasing concentrations of 48D2 and isotype control were added to the cells. No binding was observed to HTB183 cells, which have low surface expression of IL1RAP (data not shown).

[0490] Inhibition of cytokine signaling FIG. 13 shows the inhibitory activity of ch48D2 on IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and IL-36γ signaling in HEK cells. Antibodies were added in increasing concentrations followed by the indicated cytokines at constant concentrations. The graph shows the optical density (OD) at 620 nm, with higher OD indicating signaling downstream of the cytokine receptor. Chimeric antibody 48D2 induced significant inhibition of IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and IL-36γ signaling, up to complete inhibition (blockage) of these cytokine signaling. IC50 values ​​are shown in Table 3.

[0491] [Table 6]

[0492] Affinity measurement The affinity measurements for chimeric 48D2, and the humanized 48D2 variant VH5:VL4 (see Example 11), humanized and deimmunized 48D2 variant VH5.GL:VL4 (Example 13) are shown in Table 4. a is the association rate constant for characterizing antibody binding to the target, and k d is the dissociation rate constant for characterizing antibody dissociation from the target, and K D is the equilibrium dissociation constant between the antibody and its antigen, and k d / k a It is the ratio of K D corresponds to the antibody concentration at which 50% of the antigen-binding sites are occupied at equilibrium. D The lower the value (lower the concentration), the higher the affinity. The 48D2 variants showed fast binding and slow dissociation to human (h)IL1RAP, resulting in affinities in the nM range. No loss of affinity for human IL1RAP was observed for the humanized or humanized and deimmunized variants compared to chimeric 48D2.

[0493] [Table 7]

[0494] Domain Mapping The domain mapping data is summarized below in Table 5. The control polyclonal antibody KMT-1 bound to all domains, whereas 48D2 bound only to IL1RAP constructs containing domain 2, suggesting that the antibody binds to this domain.

[0495] [Table 8]

[0496] Epitope mapping Epitope mapping results using mouse IL1RAP with grafted human sequences for regions H1 and H2 are shown in Table 6. Binding of h48D2 VH5.GL:VL4 (see Example 13) to mouse IL1RAP was restored when the human sequence of the H2 region was introduced into mouse IL1RAP, suggesting that H2 is involved in the interaction with 48D2 (compare Tables 5 and 6). Positive control polyclonal antibodies KMT-2 and KMT-3 bound to both chimeric versions of IL1RAP.

[0497] [Table 9]

[0498] Cross-reactivity Figure 14 shows the cross-reactivity of chimeric 48D2 to IL1RAP from different species. Increasing concentrations of antibodies are added and absorbance at 405 nm is used to detect binding of the antibodies to IL1RAP orthologues. 48D2 is cross-reactive to IL1RAP from human, cynomolgus monkey (cyno), and pig, but not to IL1RAP from mouse, rat, rabbit, or dog (Figure 14 and Table 7).

[0499] [Table 10]

[0500] conclusion Chimeric 48D2 was found to bind to domain 2 of hIL1RAP, had high affinity for both cynomolgus monkey and human IL1RAP, selectively bound to IL1RAP on the cell membrane, and completely inhibited IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and IL-36γ signaling, and was therefore selected for humanization. Chimeric 48D2 showed superior properties to other antibodies generated, as described in Example 9. EXAMPLES

[0501] Example 11: Humanization of chimeric 48D2

[0502] the purpose This example illustrates how a humanized variant of 48D2 was obtained.

[0503] Materials and Methods In-silico humanization The antibody light and heavy chain variable region sequences of 48D2 were searched against databases containing mature human antibody sequences and human germline sequences. CDRs were defined according to the IMGT and Kabat numbering systems, and amino acid residues were included as a combination of both (see SEQ ID NO: 1 and CDR-L2 with sequences 3-18 and KAS). The selection of receptor frameworks for humanization was based on sequence homology and subsequent ab initio structural predictions made using the software tool Maestro. Frameworks were selected to maintain important structural framework residues. Five sequences each for VL and VH were obtained and 25 different humanized antibody variants were generated and further characterized (VL1-VL5 for combination with VH1-VH5). For receptor frameworks based on human germline antibody sequences, back mutations were introduced to ensure the structure and function of the antibody. Part of the humanization was also the prediction of immunogenicity (MHC class II binding affinity) and manufacturability. MHC class II binding affinity predictions were made using the NetMHCII server.

[0504] result Five in silico humanized variants of VL and five of VH were generated and confirmed to be humanized according to the WHO definition of a humanized antibody. Immunogenicity was evaluated to avoid the introduction of sequences with potential MHC class II binding affinity. Manufacturability was evaluated to avoid the introduction of glycosylation motifs or sequences susceptible to deamidation, isomerization, or fragmentation. The sequences are shown in Table 8.

[0505] [Table 11]

[0506] conclusion Five in silico humanized variants of the VL and five of the VH were generated and synthesized in frame with the IgG1 constant domain for the production and characterization of 25 humanized antibody variants (Example 12). EXAMPLES

[0507] Example 12: Properties of humanized 48D2

[0508] the purpose This example describes how humanized 48D2 variants (see Example 11) were expressed and characterized, with the goal of finding a humanized antibody that retains affinity, cytokine inhibition, and optimized biochemical properties.

[0509] Materials and Methods Expression, purification, and characterization of humanized variants The heavy and light variable domains of 25 humanized variants of 48D2 (see Table 8) were subcloned into a vector containing the human constant domain: - The kappa constant domain (Km3 allotype) is associated with the VL domain forming the entire light chain (SEQ ID NO: 35). - The heavy chain constant domain of IgG1za (za allotype) combines with the VH domain to form the entire heavy chain. To avoid antibody effector functions via the Fc-γ-receptor, the heavy chain constant domain also contains the LALA mutation (SEQ ID NO:2) (corresponding to, for example, those described in Xu et al., 2000, Cell. Immuno. 200:16-26). Humanized antibody (h48D2) was transiently expressed in HEK-293 cells and Protein A was purified from harvested media of 50 ml cultures and buffer exchanged into PBS pH 7.4. The antibodies were characterized for A) binding (ELISA analysis, human and cynomolgus IL1RAP), B) inhibition of cytokine signaling (HEK-Blue™ assay), C) affinity (Biolayer interferometer), D) size heterogeneity (SE-HPLC), and E) yield (concentration, A280). Binding ELISAs were performed in a similar manner to the indirect ELISAs shown for domain mapping in Example 10, using recombinant hIL1RAP aa21-367 or recombinant mfIL1RAP aa21-367 as coating reagent. SE-HPLC was performed using PBS as running buffer, using standard procedures. Affinity and cytokine inhibition were measured as previously described in Example 10. All 25 humanized clones were analyzed for blocking activity against IL-1α, IL-1β, and IL-33 signals, and only those clones that retained similar inhibitory activity as chimeric 48D2 were analyzed for IL-36 inhibition.

[0510] result A) Binding of humanized antibody clones to IL1RAP (ELISA method) The IC50 values ​​obtained by ELISA are shown in Table 9. Using the binding ELISA, only minor differences in binding could be seen between the different clones, and each clone had similar binding affinity to human and cynomolgus monkey (cyno) IL1RAP.

[0511] [Table 12]

[0512] [Table 13]

[0513] B) Inhibition of cytokine signaling Figures 15A-O show the inhibitory activity of humanized 48D2 antibody clones against IL-1α, IL-1β, and IL-33 signaling in HEK cells. Antibodies are added at increasing concentrations followed by constant concentrations of the depicted cytokines. The graphs show optical density (OD) at 620 nm, with higher OD indicating signaling downstream of the cytokine receptor. Antibody clones carrying VH5 variants induced complete inhibition of IL-1α, IL-1β, and IL-33 signaling with similar potency as chimeric 48D2 antibody (Figures 15E, 15J, 15O). Antibody clones carrying variants VH1-VH4 inhibited IL-1α, IL-1β, and IL-33 signaling with various potencies compared to chimeric 48D2. These variants achieved partial inhibition of IL-1α (Fig. 15A-D) and IL-33 (Fig. 15K-N), and complete inhibition of IL-1β (Fig. 15F-I). Antibody clones carrying VH1-VH4 variants were not analyzed for IL-36 inhibition. The respective IC50 values ​​are shown in Table 10. Fig. 15P-R shows that the antibody clone carrying the VH5 variant also induced complete inhibition of IL-36α, IL-36β, and IL-36γ signals with a potency similar to that of the chimeric 48D2 antibody.

[0514] [Table 14]

[0515] [Table 15]

[0516] [Table 16]

[0517] C) Affinity measurement Affinity KD values ​​for human IL1RAP are shown in Table 11 for the humanized 48D2 variants. Affinities were measured by Biolayer Interferometry. All antibody variants showed affinities in the nanomolar range. The highest affinity was shown by the VH5 series with KD values ​​in the range of 1-4 nM. See also Table 4 for a direct comparison of chimeric 48D2, humanized 48D2 variant VH5:VL4 and humanized deimmunized 48D2 variant VH5.GL:VL4.

[0518] [Table 17]

[0519] [Table 18]

[0520] D) size heterogeneity Size heterogeneity was determined using size exclusion HPLC (SE-HPLC) (Table 12). Intact monomeric antibodies are composed of two identical heavy chains and two identical light chains covalently linked by disulfide bonds. High molecular weight (HMW) species may include, for example, dimeric soluble antibody aggregates. Low molecular weight (LMW) species may include, for example, antibodies with cleaved peptide bonds, i.e., fragmented antibodies. Nearly all variants were highly monomeric (≥97%), except for h48D2 VH4:VL3, which contained >5% high molecular weight (HMW) species. No low molecular weight (LMW) species were detected in any analyzed variants.

[0521] [Table 19]

[0522] [Table 20]

[0523] E) Yield For the humanized 48D2 variants, protein concentration was measured by measuring absorbance at 280 nm, and yields of 50 ml cultures were determined (Table 13). The VL influenced the yield, with the highest yields being obtained with the VL4 variant and the lowest with the VL3 variant. Thus, the yield was partially dependent on the VL sequence.

[0524] [Table 21]

[0525] [Table 22]

[0526] conclusion The humanized antibody variants carrying the VH5 variant retained the ability to completely inhibit IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and IL-36γ signaling with potency similar to that of the chimeric 48D2 antibody. The antibody variants with variants VH1-VH4 had varying potencies compared to chimeric 48D2, completely inhibiting IL-1β signaling and partially inhibiting IL-1α and IL-33 signaling. The affinity for human IL1RAP was highest in the VH5 series, and among this series, variants h48D2 VH5:VL4 and h48D2 VH5:VL5 gave good yields and high monomer content by SE-HPLC. Therefore, further studies were carried out using these variants. EXAMPLES

[0527] Example 13: Deimmunization of two of the humanized 48D2 clones

[0528] the purpose This example shows that sequences predicted to have affinity for MHC class II molecules in VL5 and VH5 of h48D2 were deimmunized by germline division.

[0529] Materials and Methods Removal of sequences predicted to have affinity for MHC class II molecules The humanized 48D2 VL5 and VH5 sequences were generated using the germline antibody sequences. For these two sequences, a number of back mutations were introduced into the original rabbit sequence to ensure retention of the antibody structure and function (see Example 11). These back mutations introduced generated sequences with predicted affinity for MHC class II molecules. In VH5, two back mutations in framework region 2 conferred immunogenic potential, and in VL5, six back mutations in framework region 1. For deimmunization of VH5, the back mutations were reverted to the germline sequence as single mutations and mutations of both residues (resulting in three sequence variants). For VL5, all six back mutations were reverted to the germline sequence as one new sequence (resulting in one sequence variant). This resulted in the new sequence of the deimmunized VH5 being: h48D2 VH5.AP (alanine to proline) (SEQ ID NO:32), h48D2 VH5.SK (serine to lysine) (SEQ ID NO:33), h48D2.VH5.GL ("germline", both residues reverted to germline sequences) (SEQ ID NO:34); And for the deimmunized VL5, the new sequence is: h48D2 VL5.GL ("germline", all six residues are reverted to the germline sequence) (SEQ ID NO:31).

[0530] Results and Conclusions Deimmunization was performed on VL5 and VH5. The new VL5 and VH5 were combined with previously obtained sequences for h48D2 VL4, h48D2 VL5, and h48D2 VH5 to generate 10 additional variants for expression and characterization: h48D2 VH5:VL5.GL h48D2 VH5.AP:VL4 h48D2 VH5.AP:VL5 h48D2 VH5.AP:VL5.GL h48D2 VH5.SK:VL4 h48D2 VH5.SK:VL5 h48D2 VH5.SK:VL5.GL h48D2 VH5.GL:VL4 h48D2 VH5.GL:VL5 h48D2 VH5.GL:VL5.GL EXAMPLES

[0531] Example 14: Characterization of humanized deimmunized 48D2 clones

[0532] the purpose This example shows how humanized and deimmunized 48D2 variants were expressed and characterized, with the goal of finding humanized and deimmunized antibodies that retain affinity, cytokine inhibition, and optimized biochemical properties.

[0533] Materials and Methods Expression, purification, and characterization of deimmunized h48D2 Expression, purification, and characterization were performed as previously described for chimeric 48D2 and the humanized variants of Examples 10 and 12.

[0534] result Binding of humanized and deimmunized antibody clones to IL1RAP (ELISA) IC50 values ​​obtained in ELISA measurements are shown for the deimmunized h48D variants in Table 14. Using the binding ELISA, only small differences in binding can be seen between the different variants. For each clone, the binding affinity to human and cynomolgus monkey (cyno) IL1RAP was roughly similar.

[0535] [Table 23]

[0536] Binding of humanized and deimmunized antibody clones to IL1RAP Flow cytometry analysis of IL1RAP-expressing SKMEL-5 cells stained with h48D2 VH5.GL:VL4 and h48D2 VH5.GL:VL5.GL or isotype control revealed higher mean fluorescence intensity (MFI) for VH5.GL:VL4 and VH5.GL:VL5.GL compared to the isotype control antibody when antibody was added at high concentrations (Figure 16).

[0537] Inhibition of cytokine signaling Figure 17 shows the inhibitory activity of humanized and deimmunized 48D2 antibody clones against IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and IL-36γ signaling in HEK cells. Antibodies were added at increasing concentrations and depicted cytokines were added at constant concentrations. Humanized variants h48D2 VH5:VL4 and h48D2 VH5:VL5 were included as controls. Graphs show OD values ​​at 620 nm, with higher OD representing signaling downstream of cytokine receptors. All variants retained inhibitory effects against IL-1α (A), IL-1β (B), IL-33 (C), IL-36α (D), IL-36β (E), and IL-36γ (F). IC50 values ​​are shown in Table 15.

[0538] [Table 24]

[0539] In the experimental setup used to obtain the results in Figures 17A-F, HEK-Blue™ (IL-33 / IL-1) cells were transiently transfected with IL-36 receptor the previous day. Further evaluation of h48D2 VH5.GL:VL4 inhibition of IL-33 and IL-36 cytokine signaling was then performed using HEK-Blue™ cells stably transfected with IL-36 receptor. This evaluation showed inhibitory effects on IL-33 (Figure 17G), IL-36α (Figure 17H), IL-36β (Figure 17I) and IL-36γ (Figure 17J). IC50 values ​​are shown in Table 16.

[0540] [Table 25]

[0541] Affinity measurement The KD values ​​of affinity for human IL1RAP are shown for the deimmunized 48D2 variants in Table 17. Affinities were measured by Biolayer Interferometry. Affinities were similar for all deimmunized variants with KD values ​​of 1-2 nM. See also Table 4 for a direct comparison of 48D2, humanized 48D2, and deimmunized h48D2.

[0542] [Table 26]

[0543] Size heterogeneity Size heterogeneity was determined using size exclusion HPLC (SE-HPLC) (Table 18). Nearly all variants were highly monomeric (>= 98%), with the exception of the h48D2 variant with VL5.GL, which contained several HMW species. No LMW species were detected in any of the variants analyzed.

[0544] [Table 27]

[0545] yield Protein concentration was measured by absorbance measurement at 280 nm and yields of 50 ml cultures were determined for the deimmunized h48D2 variants (Table 19). The antibody variant with the VL variant VL5.GL showed lower obtainable yields compared to the other variants.

[0546] [Table 28]

[0547] [Table 29]

[0548] Cross-reactivity Figure 18 shows the cross-reactivity of h48D2 VH5.GL:VL4 and h48D2 VH5.GL:VL5.GL to IL1RAP from different species. Increasing concentrations of antibodies are added and binding to IL1RAP is detected by absorbance at 405 nm. For chimeric 48D2, the humanized and deimmunized variants h48D2 VH5.GL:VL4 and h48D2 VH5.GL:VL5.GL cross-reacted with cynomolgus monkey (cyno) and pig IL1RAP, but not with mouse, rat, rabbit, or dog IL1RAP (Figure 18 and Table 20).

[0549] [Table 30]

[0550] conclusion All deimmunized variants were K D The variant h48D2 VH5.GL:VL4 showed high affinity with affinity values ​​of 1-3 nM and retained biological function. The variant h48D2 VH5.GL:VL4 excelled in SE-HPLC with good yield and high monomer content. EXAMPLES

[0551] Example 15: ADCC effect of chimeric 48D2

[0552] the purpose In this example, we aimed to investigate the antibody-dependent cellular cytotoxicity (ADCC) effect of chimeric 48D2 expressed in hIgG1-wild type (WT) or effector function silent IgG1-LALA format.

[0553] Materials and Methods In vitro ADCC assays were performed using the melanoma cell line SKMEL-5, which expresses IL1RAP on the cell surface, as a target. Target cells were seeded at a density of 10,000 cells / well in 96-well plates. Then, 48D2-WT, 48D2-LALA or isotype control antibodies were added to the wells at different concentrations and incubated for 30 min, after which 100,000 NK effector cells were added to each well. NK cells were extracted from leukocyte concentrates using an NK cell negative cell isolation kit (Miltenyi Biotech, Bergisch Gladbach, Germany) according to the manufacturer's instructions. Nonspecific human IgG1-WT and IgG1-LALA antibodies were used as controls in the experiments. The extent of cell death was assessed by detection of DAPI-positive cells after 18 h of culture using a FACS LSR Fortessa flow cytometer (BD).

[0554] result In vitro ADCC assays show that chimeric 48D2 in hIgG1 format induces NK cells to kill SKMEL-5 cells in an antibody-specific, dose-dependent manner (Figure 19). After treatment with chimeric 48D2 expressed in hIgG1-LALA format, there was no increase in the percentage of dead SKMEL-5 cells compared to isotype control and untreated cells (untreated cells correspond to 0 ng / ml antibody). The effect was shown to be dose-dependent. When chimeric 48D2 is expressed in hIgG1-LALA format, the ADCC effect is abolished. It can be assumed that antibody variants of the 48D2 antibody (antibodies described in the examples above, e.g., humanized antibodies, or humanized and deimmunized antibody variants) will have a similar effect when expressed in hIgG1-WT or hIgG1 LALA format. Indeed, Example 19 below demonstrates that the antibody variant VH5.GL:VL4 in hIgG1 LALA format does not induce Fc-mediated immune activation in a blood loop assay.

[0555] conclusion This experiment shows that 48D2 can induce NK cells to specifically kill melanoma cell lines expressing IL1RAP on their surface, and that the cytotoxicity induced by 48D2 is dose-dependent. Furthermore, the ADCC effect can be abolished by expressing 48D2 in an effector-function-silent hIgG1-LALA format. EXAMPLES

[0556] Example 16: Inhibition of cytokine signaling in human fibroblasts by chimeric 48D2

[0557] the purpose The purpose of this example is to investigate the inhibitory activity of chimeric 48D2 on IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and IL-36γ-stimulated IL-6 gene expression in human fibroblasts in vitro.

[0558] Materials and Methods Fibroblasts from healthy human skin were commercially obtained and grown in complete fibroblast growth medium. 75000 cells per well were seeded in 24-well plates. Cells at 80% confluence were serum starved overnight in fibroblast medium containing only 1% FBS. The medium was changed to fresh fibroblast medium with 1% FBS and no growth factors, and 20ug / ml chimeric 48D2 was added to the wells 1 hour before stimulation with different concentrations of cytokines (as depicted in Figure 20). After 24 hours of stimulation of cells, RNA was isolated according to the manufacturer's instructions. IL-6 mRNA levels were analyzed using SYBR Green and data were analyzed by the ΔΔCT method, showing changes of 2% compared to untreated controls. -ΔΔCT As shown in.

[0559] result IL-1α, IL-1β, IL-36α, IL-36β, and IL-36γ induced IL-6 gene expression in dermal fibroblasts in a dose-dependent manner (FIG. 20). In this experiment, chimeric 48D2 inhibited the increase in gene expression, lowering it to a level similar to that of the unstimulated control. IL-33 did not affect the expression of IL-6 in these cells (data not shown). It can be assumed that antibody variants of the 48D2 antibody (such as the antibodies described in the above examples, e.g., humanized antibodies, or humanized and deimmunized antibody variants) will have a similar effect. Indeed, Example 17 demonstrates that the antibody variant VH5.GL:VL4 inhibits IL-1β signaling in human whole blood.

[0560] conclusion 48D2 can block cytokine-induced increases in IL-6 gene expression in human fibroblasts. EXAMPLES

[0561] Example 17: Inhibition of IL-1β signaling in human whole blood by 48D2 variant VH5.GL:VL4

[0562] the purpose The aim of this study was to investigate how blocking IL-1α / β, IL-33, and IL-36α / β / γ signaling by preincubating human whole blood with 48D2 variant VH5.GL:VL4 affects the release of various downstream cytokines and chemokines in response to IL-1β stimulation.

[0563] Materials and Methods Human blood was collected in heparinized tubes from two different donors. Blood was transferred to wells of a 96-well plate and incubated with 150 μg / ml VH5.GL:VL4 at 37°C, 5% CO2 for 30 min with agitation. IL-1β or LPS, both at 1 ng / ml, was then added to the wells and the blood was incubated for 20 h at 37°C, 5% CO2 for 30 min with agitation. Wells in which blood samples were incubated with formulation buffer alone were included as negative controls (Ctrl). The plate was centrifuged at 1500 rpm to separate plasma and blood cells. Plasma was diluted 1:2 and the levels of 71 different cytokines and chemokines (6CKine, BCA-1, CTACK, EGF, ENA-78, eotaxin, eotaxin-2, eotaxin-3, FGF-2, Flt3L, fractalkine, G-CSF, GM-CSF, GROα / CXCL1, I-309, IFNα2, IFNγ, IL-1α, IL-1β, IL-1RA, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12p40, IL-12p70, IL-13, IL-15, IL-16, IL-17, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, IL-40, IL-41, IL-42, IL-43, IL-44, IL-45, IL-46, IL-47, IL-48, IL-49, IL-50, IL-51, IL-52, IL-53, IL-54, IL-55, IL-56, IL-57, IL-58, IL-59, IL-60, IL-61, IL-62, IL-63, IL-64, IL-65, IL-66, Cytokine / chemokine 7A, IL-17E / IL-25, IL-17F, IL-18, IL-20, IL-21, IL-22, IL-23, IL-27, IL-28, IL-33, IP-10, LIF, MCP-1, MCP-2, MCP-3, MCP-4, M-CSF, MDC, MIG, MIP-1α, MIP-1β, MIP-1δ, PDGF-AA, PDGF-AB / BB, RANTES, sCD40L, SCF, SDF-1α+β, TARC, TGFα, TNFα, TNFβ, TPO, TRAIL, TSLP, VEGF-A) were measured in plasma samples using the Cytokine / Chemokine 71-Plex Discovery Assay Array (Eve Technologies; HD71) according to the manufacturer's instructions. The results for four of these, G-CSF, GROα / CXCL1, IL-17A, and TNF-α, are described below.

[0564] result Blockade of IL-1α / β, IL-33, and IL-36α / β / γ signals by VH5.GL:VL4 reduced the release of G-CSF, GROα / CXCL1, IL-17A, and TNF-α in human whole blood in response to stimulation with IL-1β (Figure 21A-D). Similar effects were observed when blood was stimulated with LPS, where VH5.GL:VL4 significantly reduced the levels of G-CSF, GROα / CXCL1, and TNF-α, and only slightly reduced the levels of IL-17A (Figure 21A-D).

[0565] conclusion Blockade of IL1RAP by VH5.GL:VL4 affects the downstream effects of IL-1β- and LPS-mediated IL-1R signaling, reducing the release of multiple cytokines and chemokines. EXAMPLES

[0566] Example 18: IL-1β signaling inhibition in a blood loop system by 48D2 variant VH5.GL:VL4

[0567] the purpose The aim of this study was to investigate how blocking IL-1α / β, IL-33, and IL-36α / β / γ signaling by preincubating human whole blood with 48D2 variant VH5.GL:VL4 affects the release of IL-6 and IL-8 in response to stimulation with IL-1β in a blood loop system mimicking the human blood circulation.

[0568] Materials and Methods Fresh whole blood was collected from 10 healthy donors and low amounts of soluble heparin were added. Blood was immediately transferred to pre-coated plastic tubes to form loops in a blood loop system. The loops were placed on a rotating wheel and samples were run in parallel. VH5.GL:VL4 was administered to samples at 32 μg / ml and IL-1β was added at 1 ng / ml 15 min later. PBS was used as a control. Each loop was sampled after 4 h and EDTA was added to each sample at a concentration of 10 mM to stop the reaction at the sampling time point. Plasma samples were prepared by centrifugation, aliquoted, and stored at ≦-60°C until further cytokine analysis. Cytokines IL-6 and IL-8 were measured using Meso Scale Discovery's MULTI-ARRAY® technology. All samples were diluted 1:4 and run in duplicate according to the manufacturer's instructions.

[0569] result Blockade of IL-1α / β, IL-33, and IL-36α / β / γ signaling by VH5.GL:VL4 reduced the release of IL-6 and IL-8 in response to stimulation with IL-1β in human whole blood circulating in a blood loop system (Figures 22A-B).

[0570] conclusion In a blood loop system mimicking human blood circulation, IL1RAP blockade with VH5.GL:VL4 was confirmed to affect the downstream effects of IL-1β-mediated IL-1R signaling, reducing the release of cytokines IL-6 and IL-8. EXAMPLES

[0571] Example 19: Internalization of 48D2 variant VH5.GL:VL4 by IL1RAP expressing cells

[0572] the purpose The aim of this experiment was to investigate membrane binding and potential internalization of fluorescently labeled 48D2 variant VH5.GL:VL4 by cells expressing IL1RAP.

[0573] Materials and Methods WT and IL1RAP KO SKMEL human melanoma cells were grown to 60-80% confluence and then transferred to 8-well Ibidi-treat microscope chamber slides at 12500 cells per well. Cells were incubated with 3 μg / ml AlexaFluor647 (AF647)-conjugated VH5.GL:VL4 for 1, 2, or 4 hours at 37°C. Control cells were incubated with AF647-conjugated isotype control antibody. Alternatively, WT SKMEL cells incubated with A647-conjugated VH5.GL:VL4 for 2 hours were further incubated with 5 μg / ml rabbit anti-EEA1 antibody and 10 μg / ml mouse anti-Lamp1 antibody for 3 hours to stain for endosomal and lysosomal markers, respectively. Cells were washed and incubated with AlexaFluor488 (AF488)-conjugated anti-rabbit antibody and rhodamine (RX)-conjugated anti-mouse antibody for 30 minutes. After incubation, cells were washed and fixed with 2% paraformaldehyde. Cells were washed again and then labeled with DAPI to stain the cell nuclei. Cells were analyzed using a Zeiss LSM800 confocal microscope at 63x oil immersion. Detection levels were set from control cells not incubated with A647-conjugated VH5.GL:VL4, or anti-EEA1, anti-Lamp1, and the same settings were used throughout the analysis. Interactive visual analysis was performed by scanning 500 nm optical sections within the cells and taking representative images.

[0574] result In WT SKMEL cells expressing IL1RAP, initial membrane binding and cellular internalization of A647-conjugated VH5.GL:VL4 was detected at 1 hour of incubation (Figure 23; top left image; arrows indicate membrane binding and internalization). Maximal internalization was noted approximately 2 hours after incubation (Figure 23; top middle image). The lack of binding and internalization of A647-conjugated VH5.GL:VL4 by IL1RAP KO cells indicates that the effects observed in WT cells are mediated by interaction with IL1RAP (Figure 23; middle panel; only nuclear staining with DAPI observed). Furthermore, no binding or internalization of the isotype control antibody was observed in WT cells (Figure 23; bottom panel; only nuclear staining with DAPI observed). Additional co-staining with EEA1 and Lamp1, markers of endosomes and lysosomes, shows overlap with the signal of A647-labeled VH5.GL:VL4 (Figure 24). This indicates that VH5.GL:VL4 localizes to these compartments upon internalization.

[0575] conclusion VH5.GL:VL4 is internalized upon binding to IL1RAP-expressing SKMEL cells, and this internalization is dependent on the binding of VH5.GL:VL4 to IL1RAP. EXAMPLES

[0576] Example 20: Fc-mediated immune activation in a blood loop system by 48D2 variant VH5.GL:VL4

[0577] the purpose The aim of this study is to investigate whether and to what extent 48D2 variant VH5.GL:VL4 expressed in an effector function-silenced hIgG1-LALA format can induce immune activation in the absence of other stimuli in a blood loop system mimicking the human circulation, as assessed by the release of inflammatory cytokines and complement activation in circulating human whole blood.

[0578] Materials and Methods Fresh whole blood was collected from 10 healthy donors and low amounts of soluble heparin were added to allow the analysis of the effects of drugs on the complement system. The blood was immediately transferred to precoated plastic tubes and loops were formed in the blood loop system before adding 0.0125 mg / ml, 0.125 mg / ml, or 1.25 mg / ml of VH5.GL:VL4. Samples spiked with the anti-CD52 antibody alemtuzumab at 3 μg / ml or formulation buffer alone (vehicle) were used as positive and negative controls, respectively. The loops were placed on a rotating wheel and samples were run in parallel. Each loop was sampled after 15 minutes and 4 hours, and EDTA was added to each sample at a concentration of 10 mM to stop the reaction at the sampling time points. The sample taken after 15 minutes was processed into plasma for complement analysis, and the blood sample taken after 4 hours was processed into plasma for cytokine analysis. Plasma samples were prepared by centrifugation, aliquoted, and stored at ≦-60°C until analysis. The cytokines IFNγ, IL-6, IL-8, and TNFα were measured using Meso Scale Discovery's MULTI-ARRAY® technology. All samples were diluted 1:4 and run in duplicate according to the manufacturer's instructions. Complement activation was analyzed by measuring the complement split products C3a and C5a using ELISA kits (RayBio® Human C3a ELISA kit and RayBio® Human C5a ELISA kit). Plasma samples were diluted 1:500 in sample diluent for C3a analysis and 1:50 in sample diluent for C5a analysis and run in duplicate according to the manufacturer's instructions.

[0579] result VH5.GL:VL4 had no effect on the release of IFNγ, IL-6, IL-8, TNFα (FIGS. 25A-D), or complement activation as measured by C3a and C5a levels (FIGS. 26A-B) in the blood loop system at any of the three concentrations evaluated. In contrast, the anti-CD52 antibody alemtuzumab induced complement activation concomitant with cytokine release.

[0580] conclusion VH5.GL:VL4, which has an effector-function-silent Fc region, does not induce Fc-mediated immune activation. EXAMPLES

[0581] Example 21: Initial safety and pharmaco-toxicokinetic properties of intravenously administered 48D2 variant VH5.GL:VL4

[0582] the purpose The objective of this study was to determine the pharmacokinetics and potential toxicity of VH5.GL:VL4 following a single intravenous dose administered to male and female cynomolgus monkeys, followed by three escalating doses up to the maximum tolerated dose.

[0583] Materials and Methods VH5.GL:VL4 was administered intravenously via a peripheral vein as a single dose (bolus) of 5, 20, or 50 mg / kg to one male or female cynomolgus monkey for each dose level. Animals were observed daily for 2 weeks after dosing for mortality, clinical signs, and food intake. Hematology and serum chemistry were performed pre-study and on study day 8. Body weights were recorded pre-study and on days 8 and 15. Sampling for bioanalytical and toxicokinetic evaluations was performed from the femoral vein pre-dose and at 0.083, 1, 3, 6, 24, 48, 96, 168, 264, and 336 hours post-dose (at least 0.6 ml blood for each sample). Blood samples were allowed to clot for 30 minutes at room temperature in serum separator tubes. Clots were spun down by centrifugation at 1200 g for 10 minutes at 4°C. Resulting serum was stored at -80°C until analysis.

[0584] Biotinylated human IL1RAP was added to a streptavidin-precoated Meso Scale Discovery (MSD) plate and incubated at room temperature. After washing, serum samples were added to the plate and incubated at room temperature. The plate was washed and anti-human IgG conjugated to an electrochemiluminescent label (MSD SULFO-TAG) was added to the plate. After a final incubation and wash, lead buffer was added. SULFO-TAG emits light when a voltage is applied to the plate electrodes in the MSD instrument. The instrument quantitatively measures VH5.GL:VL4 in the sample by measuring the luminescence intensity. Serum toxicokinetic analysis was performed following a standard noncompartmental approach using the WinNonlin package (v.8.1, Pharsight Inc, a Certara Company, USA).

[0585] result There were no abnormal clinical signs and no relevant toxicological changes in body weight and clinical pathology. No toxicologically relevant changes were observed in serum chemistry and hematology. Dose-normalized maximum concentrations of the compound were similar across the dose range investigated. Serum toxicokinetic parameters of VH5.GL:VL4 in males and females at each dose level were similar. Terminal half-lives of VH5.GL:VL4 averaged 98 hours (4 days) after the 5 mg / kg dose and 220 hours (9 days) after the 20 mg / kg and 50 mg / kg dose parameters, two-fold higher than the 5 mg / kg dose (Table 21). VH5.GL:VL4 was found to decline biexponentially (Figure 27). At all doses, serum clearance of VH5.GL:VL4 was low, with terminal volumes of distribution found to be on the same order as total body water in monkeys (≈700 ml / kg).

[0586] [Table 31]

[0587] conclusion After a single intravenous dose of VH5.GL:VL4 to cynomolgus monkeys, the pharmacokinetics of the antibody was characterized by low clearance, low volume of distribution, and long half-life. Over the dose range studied, the maximum concentration of the compound increased directly proportional to the dose, whereas the AUC 最終 tended to increase rather than increase in direct proportion to dose. EXAMPLES

[0588] Example 22: Pharmacokinetic characterization of 48D2 variant VH5.GL:VL4 following subcutaneous administration

[0589] the purpose The objective of this study was to determine the pharmacokinetics and bioavailability of VH5.GL:VL4 antibody following a single subcutaneous dose in female cynomolgus monkeys.

[0590] Materials and Methods VH5.GL:VL4 was administered intravenously or subcutaneously in the dorsum of two female cynomolgus monkeys at a single dose of 10 mg / kg per route of administration. Sampling for pharmacokinetic evaluation was taken pre-dose and 1, 3, 6, 24, 48, 96, 168, 264, 336, 480, and 672 hours post-dose (at least 0.6 ml of blood per sample). Additionally, the first dose was taken 0.083 hours after intravenous dosing and 0.5 hours after subcutaneous dosing. Blood samples were allowed to clot for 30 minutes at room temperature in serum separator tubes. Clots were spun down by centrifugation at 1200 g for 10 minutes at 4°C. Resulting serum was stored at -80°C until analysis. Serum was analyzed by MSD as described in Example 21. Serum pharmacokinetic analysis of VH5.GL:VL4 was performed using the Phoenix-WinNonlin package (Certara Company, USA).

[0591] result Following a single intravenous dose of 10 mg / kg VH5.GL:VL4 in female cynomolgus monkeys, serum concentrations of VH5.GL:VL4 declined over a mean time of 86.9 hours (Figure 28 and Table 22). Both serum clearance and volume of distribution of VH5.GL:VL4 were low; the volume of distribution accounted for approximately one-tenth of the total body water in monkeys (Table 22). Following subcutaneous administration of 10 mg / kg VH5.GL:VL4, absorption of the compound was slow, with a T max The mean time to treatment was 48 to 96 hours after administration (Figure 28 and Table 23). max After reaching 100 mg / kg / day, serum concentrations of the compound declined with a mean terminal half-life of 112 hours (Table 23). Subcutaneous bioavailability was high at 93%.

[0592] [Table 32]

[0593] [Table 33]

[0594] conclusion The pharmacokinetics of VH5.GL:VL4 after a single intravenous dose of 10 mg / kg in cynomolgus monkeys was characterized by low clearance and volume of distribution, as well as a long half-life. Following subcutaneous administration of VH5.GL:VL4 at 10 mg / kg, the compound was slowly absorbed. max After reaching a concentration of 100 mg / kg / day, serum compound concentrations declined with a mean terminal half-life similar to that of the intravenous dose. Subcutaneous bioavailability was high. [Sequence List Free Text]

[0595] array CDR sequences (defined according to IMGT) SEQ ID NO:1 Variable light chain complementarity determining region 1 (CDR-L1) ESISTA

[0596] Variable light chain complementarity determining region 2 (CDR-L2) KAS

[0597] SEQ ID NO:3 Variable light chain complementarity determining region 3 (CDR-L3) QQGFSSGNVHNA

[0598] SEQ ID NO:4 Variable heavy chain complementarity determining region 1 (CDR-H1) GPSLSHFD

[0599] SEQ ID NO:5 Variable heavy chain complementarity determining region 2 (CDR-H2) ISPGVST

[0600] SEQ ID NO:6 Variable heavy chain complementarity determining region 3 (CDR-H3) ARGGVGSSWKAFDL

[0601] CDR sequences (defined according to Kabat) SEQ ID NO:7 Variable light chain complementarity determining region 1 (CDR-L1) QASESISTALA

[0602] SEQ ID NO:8 Variable light chain complementarity determining region 2 (CDR-L2) KASTLPS

[0603] SEQ ID NO:9 Variable light chain complementarity determining region 3 (CDR-L3) QQGFSSGNVHNA

[0604] SEQ ID NO:10 Variable heavy chain complementarity determining region 1 (CDR-H1) HFDIT

[0605] SEQ ID NO:11 Variable heavy chain complementarity determining region 2 (CDR-H2) TISPGVSTYYASWAKS

[0606] SEQ ID NO:12 Variable heavy chain complementarity determining region 3 (CDR-H3) GGVGSSWKAFDL

[0607] CDR sequences (defined according to a combination of IMGT and Kabat) SEQ ID NO:13 Variable light chain complementarity determining region 1 (CDR-L1) QASESISTALA

[0608] SEQ ID NO:14 Variable light chain complementarity determining region 2 (CDR-L2) KASTLPS

[0609] SEQ ID NO:15 Variable light chain complementarity determining region 3 (CDR-L3) QQGFSSGNVHNA

[0610] SEQ ID NO:16 Variable heavy chain complementarity determining region 1 (CDR-H1) GPSLSHFDIT

[0611] SEQ ID NO:17 Variable heavy chain complementarity determining region 2 (CDR-H2) TISPGVSTYYASWAKS

[0612] SEQ ID NO:18 Variable heavy chain complementarity determining region 3 (CDR-H3) ARGGVGSSWKAFDL

[0613] It is important to note that within each CDR definition category (i) Kabat, ii) IMGT or iii) a combination of IMGT and Kabat, the CDR sequences (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, or CDR-H3, respectively) are the same for chimeric antibody 48D2 and all its optimized antibody variants, e.g., humanized antibody variants or humanized / deimmunized antibody variants (see those shown in the light chain variable region and heavy chain variable region sequences below). - CDR residues highlighted in bold are identified by the IMGT numbering system, - underline The CDR residues highlighted in are identified using the Kabat numbering system. - CDR residues are defined by a combination of the IMGT and Kabat numbering systems (combination of bold and underlined sequences).

[0614] Same variable chain region as chimeric 48D2 antibody (Non-humanized, non-deimmunized) (e.g., in Examples 9 and 10) [ka]

[0615] Humanized variable chain region (See, for example, Examples 11 and 12) [ka] [ka]

[0616] Humanized and deimmunized variable chain regions (For example, Examples 13 and 14) Amino acid residues in bold and italics indicate residues that are restored during the deimmunization process. [ka]

[0617] Constant region SEQ ID NO:35 Immunoglobulin kappa constant light chain (light chain constant region) (Km3 allotype) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0618] The bold and underlined amino acid residues indicate the residues that are changed when the LALA mutation is introduced. vinegar.

[0619] SEQ ID NO:36 Immunoglobulin IgG1 constant heavy chain (heavy chain constant region) (za allotype) [ka]

[0620] SEQ ID NO:2 Immunoglobulin IgG1 constant heavy chain (heavy chain constant region) with "LALA" mutation (za allotype) [ka]

[0621] Human IL1RAP SEQ ID NO:37 Human IL1RAP full length MTLLWCVVSLYFYGILQSDASERCDDWGLDTMRQIQVFEDEPARIKCPLFEHFLKFNYSTAHSAGLTLIWYWTRQDRDLEEPINFRLPENRISKEKDVLWFRPTLLNDTGNYTCMLRNTTYCSKVAFPLEVVQKDSCFNSPMKLPVHKLYIEYGIQRITCPNVDGYFPSSVKPTITWY MGCYKIQNFNNVIPEGMNLSFLIALISNNGNYTCVVTYPENGRTFHLTRTLTVKVVGSPKNAVPPVIHSPNDHVVYEKEPGEELLIPCTVYFSFLMDSRNEVWWTIDGKKPDDITIDVTINESISHSRTEDETRTQILSIKKVTSEDLKRSYVCHARSAKGEVAKAAKVKQKGNRCGQ

[0622] SEQ ID NO:38 Domain 2 of IL1RAP KDSCFNSPMKLPVHKLYIEYGIQRITCPNVDGYFPSSVKPTITWYMGCYKIQNFNNVIPEGMNLSFLIALISNNGNYTCVVTYPENGRTFHLTRTLTVKVV

[0623] SEQ ID NO:39 H2 region of domain 2 of human IL1RAP TITWYMGCYKIQNFNNVI

Claims

1. An antibody or antigen-binding fragment thereof having binding specificity to interleukin-1 receptor accessory protein (IL1RAP), wherein the antibody or antigen-binding fragment is Light chain variable region, a) CDR-L1 comprising or consisting of an amino acid sequence selected from the group consisting of ESISTA (SEQ ID NO: 1) and QASESISTALA (SEQ ID NO: 7), b) CDR-L2 comprising or consisting of an amino acid sequence selected from the group consisting of KAS and KASTLPS (SEQ ID NO: 8), c) The light chain variable region comprising CDR-L3 comprising or consisting of the amino acid sequence of QQGFSSGNVHNA (SEQ ID NO: 3); and Heavy chain variable region, d) CDR-H1 comprising or consisting of an amino acid sequence selected from the group consisting of GPSLSHFD (SEQ ID NO: 4), HFDIT (SEQ ID NO: 10), and GPSLSHFFDIT (SEQ ID NO: 16), e) CDR-H2 comprising or consisting of an amino acid sequence selected from the group consisting of ISPGVST (SEQ ID NO: 5) and TISPGVSTYYASWAKS (SEQ ID NO: 11), f) The heavy chain variable region comprising CDR-H3 comprising or consisting of an amino acid sequence selected from the group consisting of ARGGVGSSWKAFDL (SEQ ID NO: 6) and GGVGSSWKAFDL (SEQ ID NO: 12), An antibody or its antigen-binding fragment, including the above.

2. (a) The IL1RAP is a human IL1RAP; (b) The IL1RAP is expressed on the cell surface; and / or (c) The antibody or antigen-binding fragment according to claim 1, wherein the antibody or antigen-binding fragment is bound to domain 2 of IL1RAP.

3. The antibody or its antigen-binding fragment (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 31, or an amino acid sequence having at least 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 31, or a light chain variable region consisting of such; and / or (b) The antibody or antigen-binding fragment according to claim 1 or 2, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34, or comprising an amino acid sequence having at least 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 20, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 34, or comprising a heavy chain variable region comprising such amino acid sequence.

4. (a) The antibody or its antigen-binding fragment comprises the light chain constant region or a portion thereof; (b) The antibody or its antigen-binding fragment contains the amino acid sequence of SEQ ID NO: 35, or an amino acid sequence having at least 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 35, or contains a light chain constant region consisting of the same; (c) The antibody or its antigen-binding fragment comprises a heavy chain constant region or a portion thereof; (d) The antibody or its antigen-binding fragment comprises or consists of a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 36 or SEQ ID NO: 2, or an amino acid sequence having at least 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 36 or SEQ ID NO: 2; (e) At least one amino acid in the light chain constant region and / or the heavy chain constant region is replaced with another amino acid, provided that no more than five amino acids are replaced in this way; (f) The antibody or its antigen-binding fragment includes an Fc region; and / or (g) The antibody contains an Fc region in which the IgG constant region has been mutated. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

5. (a) intact antibodies; and / or (b) Antigen-binding fragment selected from the group consisting of Fv fragments, Fab-like fragments, and domain antibodies. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, comprising or consisting of the above.

6. The antibody or its antigen-binding fragment (a) It can inhibit the signaling of interleukin-1 (IL-1) family cytokine ligands and / or receptors; and / or (b) It is possible to inhibit the signaling of at least one cytokine selected from the group consisting of IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and IL-36γ, or combinations thereof. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

7. The antibody or its antigen-binding fragment is as follows: a) Binding affinity (KD) to IL1RAP characterized by a KD value of 3 nM or less; b) Binding of IL1RAP to domain 2; c) The binding to the H2 region of domain 2 of IL1RAP, wherein the H2 region contains or consists of the amino acid of SEQ ID NO: 39; d) Cross-reactivity with IL1RAP derived from cynomolgus monkeys or pigs; e) Inhibitory effect on IL-1α signaling; f) Inhibitory effect on IL-1β signaling; g) Inhibition of IL-33 signaling; h) Inhibitory effect on IL-36α signaling; i) Inhibitory effect on IL-36β signaling; j) Inhibitory effect on IL-36γ signaling; k) Internalization by IL1RAP-expressing cells; l) Ability to induce ADCC in cells expressing IL1RAP, or inability to induce ADCC in cells expressing IL1RAP. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, exhibiting one or more of the characteristics of the antibody.

8. The antibody or its antigen-binding fragment (a) further comprising a site for increasing the in vivo half-life of the drug; (b) PEG-encoded; and / or (c) The antibody or antigen-binding fragment according to any one of claims 1 to 7, which is directly or indirectly covalently bound to a functional site.

9. A polynucleotide encoding an antibody or its antigen-binding fragment or a constituent polypeptide chain according to any one of claims 1 to 8.

10. A vector comprising the polynucleotide described in claim 9.

11. Recombinant host cells comprising the polynucleotide described in claim 9 or the vector described in claim 10.

12. A method for producing an antibody or antigen-binding fragment according to any one of claims 1 to 8, the method comprising the step of culturing a host cell according to claim 11, which contains a polynucleotide according to claim 9 or a vector according to claim 10, under conditions that enable the expression of the encoded antibody or the antigen-binding fragment.

13. A pharmaceutical composition, An antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, The polynucleotide according to claim 9, The vector according to claim 10, and / or A pharmaceutical composition comprising the recombinant host cells described in claim 11, wherein the composition further comprises a pharmaceutically acceptable diluent, carrier, or excipient.

14. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, a polynucleotide according to claim 9, a vector according to claim 10, a recombinant host cell according to claim 11, and / or a composition according to claim 13, for use in pharmaceuticals.

15. For use in pharmaceutical use, an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, an antibody according to claim 9, a vector according to claim 10, a recombinant host cell according to claim 11, and / or a composition according to claim 13, for use in the prevention and / or treatment and / or mitigation and / or detection and / or diagnosis of a disease or disorder that is sensitive to treatment with inhibitors of IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and / or IL-36γ signaling, and / or wherein the disease or disorder is associated with cells expressing IL1RAP.

16. The aforementioned disease or disorder (a) an inflammatory and / or fibrous disease or disorder; (b) Having inflammatory and / or fibrous components; and / or (c) an autoimmune disease or disorder, An antibody, an antigen-binding fragment thereof, a polynucleotide, a vector, a host cell, or a composition for use according to claim 15.

17. The inflammatory and / or fibrous disease or disorder is myocarditis, systemic sclerosis, psoriasis, psoriatic arthritis, atherosclerosis, rheumatoid arthritis, all types of arthritis, all types of juvenile arthritis, osteoarthritis, familial cold autoinflammatory syndrome (FCAS), Macklewell's disease, neonatal onset multiorgan inflammatory disease (NOMID), familial Mediterranean fever (FMF), pyoderma gangrenosum and acne (PAPA) syndrome, adult Human-onset Still's disease, hyper-IgD syndrome, type 2 diabetes, macrophage activation syndrome, TNF receptor-associated periodic syndrome, Blau's disease, ankylosing spondylitis, Sweet's disease, lupus arthritis, Alzheimer's disease, asthma, allergy, sarcoidosis, atopic dermatitis, systemic lupus erythematosus, bullous pemphigoid, type 1 diabetes, chronic obstructive pulmonary disease, Helicobacter pylori gastritis, inflammatory bowel disease, hepatitis, hepatitis C, ischemia-reperfusion injury, multiple An antibody, its antigen-binding fragment, polynucleotide, vector, host cell, or composition for use according to claim 16, selected from the group consisting of sclerosis, Neisseria or pneumococcal meningitis, tuberculosis, Behçet's syndrome, septic shock, graft-versus-host disease, adult T-cell leukemia, multiple myeloma, periodontitis, obesity and obesity-related diseases, intervertebral disc disease, irritable bowel syndrome, Schnitzler syndrome, allergic / atopic dermatitis, hidradenitis suppurativa, Behçet's disease, cardiac fibrosis, cardiovascular disease, cryopin-associated periodic syndromes, cystic fibrosis, Goodpasture syndrome, Guillain-Barré syndrome, renal fibrosis, hepatic fibrosis, pulmonary fibrosis, dermatofibrosis, autoimmune myocarditis, organ dysfunction associated with organ transplantation, pancreatitis, peritonitis, uveitis, vasculitis, pneumonia, pulmonary hypertension, chronic graft-versus-host disease of sclerosing skin, sepsis, Sjögren's syndrome, Takayasu's arteritis, and gout.

18. An antibody, an antigen-binding fragment thereof, a polynucleotide, a vector, a host cell, or a composition for use according to claim 15, wherein the disease or disorder is a neoplastic disease or disorder.

19. The neoplastic disease or disorder is (a) a blood disorder or condition, or a solid tumor; (b) selected from the group consisting of chronic myeloid leukemia (CML), myeloproliferative disorder (MPD), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL), and acute myeloid leukemia (AML); and / or (c) A solid tumor selected from the group consisting of prostate cancer, breast cancer, lung cancer, colon cancer, colorectal cancer, melanoma, bladder cancer, brain / CNS cancer, urinary tract cancer, biliary tract cancer, cervical cancer, esophageal cancer, stomach cancer, head / neck cancer, kidney cancer, liver cancer, lymphoma, ovarian cancer, pancreatic cancer, sarcoma, skin cancer, and uterine cancer. An antibody, its antigen-binding fragment, polynucleotide, vector, host cell, or composition for use according to claim 18.

20. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, a polynucleotide according to claim 9, a vector according to claim 10, a recombinant host cell according to claim 11, and / or a composition according to claim 13, used to induce cell death and / or inhibit the growth and / or proliferation of diseased cells, or stem cells, or progenitor cells associated with a target neoplastic disorder, wherein the cells express IL1RAP.

21. An in vitro method for detecting cells expressing IL1RAP in a target, wherein the method is: (a) A step of providing a cell sample from the subject to be tested, (b) A step of contacting an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 with cells present in the sample, (c) A step of determining whether the antibody or its antigen-binding fragment binds to the cell, Herein, the binding of the antibody or its antigen-binding fragment to cells indicates the presence of a disease or disorder related to cells expressing IL1RAP in the target tissue.

22. An in vitro method for identifying patients having a disease or disorder related to cells expressing IL1RAP who would benefit from treatment with an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 8, wherein the method is: (a) A step of providing a cell sample from the patient to be tested, (b) A step of contacting an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 with cells present in the sample, (c) A step of determining whether the antibody or its antigen-binding fragment binds to the cell, Herein, the binding of an antibody or its antigen-binding fragment to cells expressing IL1RAP indicates a patient who would benefit from treatment with the antibody or its antigen-binding fragment according to any one of claims 1 to 8.

Images